Cannabis compound can help cells
February 19, 2009
Neurones which have been labelled with a fluorescent marker.
(PhysOrg.com) -- Cannabis has been used recreationally
and for medicinal purposes for centuries, yet its 60 plus
active components are only partly understood. Now
scientists have discovered how a compound in cannabis
can help cells to function in our bodies, and aid recovery
after a damaging event.
In a paper published in the Journal of Neuroscience, the researchers report on their studies into
cannabidiol - a naturally occurring molecule found in cannabis.
Also known as CBD, it is not the constituent that gives the high - that compound is called
tetrahydrocannabinol or THC - and so may be more acceptable as a drug treatment.
Both compounds are currently used in a pharmaceutical medicine to help patients relieve
symptoms of Multiple Sclerosis.
pain
and other
Now researchers have discovered how CBD actually works within brain cells.
By interacting with mitochondria - which are the power generators of all cells - it can help maintain normal
levels of calcium allowing cells to function properly and providing a greater resistance to damage.
Disturbance of calcium levels has long been associated with a number of brain disorders. So the finding
could have implications for the development of new treatments for disorders related to malfunctioning
mitochondria.
Dr Bettina Platt, from the University's School of Medical Sciences, said: "Scientists have known for a long
time that cannabidiol can help with pain relief but we never really knew how it worked.
"However we have discovered what it actually does at the cellular level.
"We are hoping that our findings can instruct the development of cannabidiol based treatments for
disorders related to mitochondrial dysfunction such as Parkinson's disease or Huntington's disease."
Nevertheless, Dr Platt warned that smoking cannabis would not necessarily have the same effect.
"There are different strains of cannabis out there and many no longer contain cannabidiol. In fact, these
have been deliberately bred out to enhance the THC content," she said.
"As a result, smoking cannabis would not necessarily have the same beneficial effect, and could even
exacerbate neuronal damage."
Provided by University of Aberdeen
Cannabis derivatives safe to use in schizophrenia, Parkinson's?
Nupur Shridhar
Researchers from the University of Aberdeen, UK, have confirmed the anti-convulsant and neuroprotectant
properties of cannabidiol (CBD), a cannabinoid found in the plant Cannabis satvia, by demonstrating its
ability to regulate intracellular Ca2+ levels within mitochondria.
The team, led by Dr Bettina Platt, exposed hippocampal cultures to CBD and observed the resulting effects
on neuronal activity. Their results, published in the 18 February edition of The Journal of Neuroscience,
suggest that CBD-mediated Ca2+ regulation is bidirectional: while cells of normal excitability experienced
only a slight increase in Ca2+ levels, highly excitable cells-those with high levels of K+experienced a
marked decrease in Ca2+ levels that successfully prevented oscillations. Since neuronal excitability is
responsible for the random, unstimulated electric impulses that trigger seizures, CBD's ability to prevent
these firings might make it invaluable as consumer demands for newer, more efficient anti-convulsant and
anti-epileptic drugs increase.
Imaging the hippocampal cultures further revealed that CBD targets receptors on the mitochondria, not on
the endoplasmic reticulum (ER) as was previously hypothesised. Mitochondria are especially important in
neurons, since they produce the high levels of energy cells require. Their ATP-producing pathways are, in
turn, controlled by large-yet consistent-Ca2+ fluctuations. Any imbalances in Ca2+ levels therefore
decrease neuronal energy levels, and several age-related diseases, including Alzheimer's, are associated
with such energy deficiencies. These disorders might respond well to CBD-induced homeostasis.
The team also exposed human neuroblastoma cell lines (SH-SY5Y) to various mitochondrial toxins and
then treated them with CBD. Their results confirmed the compound's neuroprotective properties: CBD
offered 53 percent protection against the uncoupler FCCP and 15 and percent protection against hydrogen
peroxide- and oligomysin-mediated cell death, respectively. This echoes the results of a 2005 study that
demonstrated both in vitro and in vivo neuroprotection and went on to suggest that CBD might also help
treat Parkinson's.
Additionally, researchers around the world have also been exploring CBD's anxiolytic, anti-inflammatory,
and anti-psychotic properties. A 2006 study published in the Brazilian Journal of Medical and Biological
Research compared the efficacy of CBD to that of haloperidol and clozapine, two well-known
antipsychotics, and concluded that CBD “has a pharmacological profile similar to that of [other] atypical
antipsychotic drugs," suggesting that it might be a "safe and well-tolerated" treatment for schizophrenia.
In 2007, researchers at the California Pacific Medical Center further contributed to the compound's
impressive track record: they found that it suppresses a gene that allows aggressive breast cancer cells to
metastasise. Furthermore, it targets cancer cells with a specificity that traditional chemotherapeutic agents
lack. Clearly, CBD is a drug well-worth attention and careful study, and pharmaceuticals would do well to
invest some of their intellectual and financial resources in further exploring its beneficial properties.
Yet funding for CBD research is limited, even in India. Though the Indian government allows the
cultivation and processing of Cannabis for medical and scientific purposes CBD remains controversial. The
drug may have inherited its reputation from its sister-compound, tetrahydrocannabinol (THC)-the chemical
sought out by recreational Cannabis users. Though THC itself has some beneficial properties-primarily as
an analgesic and appetite stimulant in terminally-ill patients-its psychoactive and potentially addictive
nature make it a less-than-ideal drug. Unlike THC, however, CBD is not psychoactive, does not seem to
create dependency, and, most importantly, appears to treat a variety of conditions without pronounced
adverse effects.
Despite this, many countries, including the US, list cannabidiol as a Schedule I drug, which means that the
US government believes that CBD has a high potential for abuse and absolutely no medical application.
Essentially, these laws are preventing their researchers from fully exploring CBD's potential-and this is an
exciting opportunity for Indian pharma companies whose experiments are not directly restricted by
Western policy. Researching CBD's beneficial properties would allow India to penetrate the growing anticonvulsant, antipsychotic, and chemotherapeutic markets that have, thus far, been dominated by US and
European pharma companies.
No. of
Patients
Worldwide
Epilepsy
50 Million1
Schizophrenia 24 Million5
No. of
Patients
in India
Annual
Worldwide
Sales
Adverse Side
Effects
10 Million2 (Pred.
2010) $14
Billion3
Neurontin
(Pfizer);
Topamaz (J&J);
Depakote/Valcote
(Abbott)3
4.3 to 8.7
Million6
Zyprexa (Eli
Zyprexa: dizziness,
Lilly); Risperdal
dry mouth,
(J&J); Seroquel
tremours9
(AstraZeneca and
Fujisawa
Pharmaceutical)8
(2004) $12
Billion7
Breast Cancer 101.1/100,000 25/100,000 (2007)
People10
People10
$11.3 to 18
Billion11
References for table:
Popular
Treatments
Neurontin:
dizziness,
drowsiness,
peripheral edema4
Taxotere (Sanofi- Taxotere;
Aventis);
immunosuppression,
Farmorubicin
hair loss, nausea13
(Pfizer);
Herceptin
(Roche)12
1. World Health Organization <http://www.who.int/mediacentre/factsheets/fs999/en/>
2. World Health Organization
<http://www.searo.who.int/LinkFiles/Information_and_Documents_facts.pdf>
3. "New drugs balance genericisation of epilepsy market"
<http://pharmalicensing.com/public/articles/view/1105704251_41e7b53b24a8d/>
4. Pfizer <http://media.pfizer.com-/files/products/uspi_neurontin.pdf>
5. World Health Organization <http://www.who.int /mental_health/management/schizophrenia/en/>
6. "Schizophrenia Facts and Statistics" <http://www.schizophrenia.com/szfacts.htm>
7. "New Schizophrenia Drug Shows Promise in Trials" <http://www.nytimes.com
/2007/09/03/business/03drug.html>
8. "Leading Drugs for Psychosis Come Under New Scrutiny" <http://
www.schizophrenia.com/meds/sgascrutiny.html>
9. Eli Lilly <http://www.zyprexa.com /index.jsp>
10. "Breast Cancer: Statistics on Incidence, Survival, and Screening"
<http://www.imaginis.com/breasthealth/statistics.asp>
11. "Breast Cancer Drug Discoveries"
http://www.piribo.com/publications/drug_discovery/breast_cancer_drug_discoveries_future_holds_2008.ht
ml>
12. "India's Breast Cancer drug market will almost double by 2012"
<http://pharmalicensing.com/public/press/view/1229010085_494134a5af14d/india-s-breast-cancer-drugmarket-will-almost-double-by-2012>
13. sanofi aventis <http:// www.taxotere.com/consumer/taxotere_treatment/side_effects.aspx>
(The writer is a pre-med student at Brown University, Providence, US, who interned with Express Pharma.
She can be contacted at nupur.shridhar@gmail.com)
Mutated human SOD1 causes dysfunction of oxidative
phosphorylation in mitochondria of tran
A growing body of evidence suggests that impaired mitochondrial energy production and increased
oxidative radical damage to the mitochondria could be causally involved in motor neuron death in
amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn
superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired
mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of
patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the
pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and
G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was
localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer
chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the
disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the
other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of
hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93Amutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating
the neurodegenerative process in motor neurons.
www.mitochondrial.net/showabstract.php?pmid=12050154
Cannabinoids act as necrosis-inducing factors in Cannabis
sativa
Yoshinari Shoyama, Chitomi Sugawa, Hiroyuki Tanaka and Satoshi Morimoto
Volume 3, Issue 12
December 2008
Pages 1111 – 1112
Cannabis sativa is well known to produce unique secondary metabolites called cannabinoids. We recently
discovered that Cannabis leaves induce cell death by secreting tetrahydrocannabinolic acid (THCA) into leaf
tissues. Examinations using isolated Cannabis mitochondria demonstrated that THCA causes mitochondrial
permeability transition (MPT) though opening of MPT pores, resulting in mitochondrial dysfunction (the
important feature of necrosis). Although Ca2+ is known to cause opening of animal MPT pores, THCA
directly opened Cannabis MPT pores in the absence of Ca2+. Based on these results, we conclude that
THCA has the ability to induce necrosis though MPT in Cannabis leaves, independently of Ca2+. We
confirmed that other cannabinoids (cannabidiolic acid and cannabigerolic acid) also have MPT-inducing
activity similar to that of THCA. Moreover, mitochondria of plants which do not produce cannabinoids were
shown to induce MPT by THCA treatment, thus suggesting that many higher plants may have systems to
cause THCA-dependent necrosis.
Addendum to: Morimoto S, Tanaka Y, Sasaki K, Tanaka H, Fukamizu T, Shoyama Y, Shoyama Y, Taura F.
Identification and characterization of cannabinoids that induce cell death through mitochondrial
permeability transition in Cannabis leaf cells. J Biol Chem 2007; 282:20739-51.
Authors
Yoshinari Shoyama
Graduate School of Pharmaceutical Sciences; Kyushu University; Japan
Chitomi Sugawa
Graduate School of Pharmaceutical Sciences; Kyushu University; Japan
Hiroyuki Tanaka
Graduate School of Pharmaceutical Sciences; Kyushu University; Japan
Satoshi Morimoto
Graduate School of Pharmaceutical Sciences; Kyushu University; Japan
Cannabidiol Targets Mitochondria to Regulate Intracellular
Ca2+ Levels
Duncan Ryan, Alison J. Drysdale, Carlos Lafourcade, Roger G. Pertwee, and Bettina Platt
School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen
Abstract
Cannabinoids and the endocannabinoid system have attracted considerable interest for therapeutic
applications. Nevertheless, the mechanism of action of one of the main nonpsychoactive
phytocannabinoids, cannabidiol (CBD), remains elusive despite potentially beneficial properties as an anticonvulsant and neuroprotectant. Here, we characterize the mechanisms by which CBD regulates Ca2+
homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal
cultures using fura-2 AM suggested that CBD-mediated Ca2+ regulation is bidirectional, depending on the
excitability of cells. Under physiological K+/Ca2+ levels, CBD caused a subtle rise in [Ca2+]i, whereas CBD
reduced [Ca2+]i and prevented Ca2+ oscillations under high-excitability conditions (high K+ or exposure to
the K+ channel antagonist 4AP). Regulation of [Ca2+]i was not primarily mediated by interactions with
ryanodine or IP3 receptors of the endoplasmic reticulum. Instead, dual-calcium imaging experiments with a
cytosolic (fura-2 AM) and a mitochondrial (Rhod-FF, AM) fluorophore implied that mitochondria act as sinks
and sources for CBD's [Ca2+]i regulation. Application of carbonylcyanide-ptrifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na+/Ca2+ exchange inhibitor, CGP 37157,
but not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBDinduced Ca2+ responses. In established human neuroblastoma cell lines (SH-SY5Y) treated with
mitochondrial toxins, CBD (0.1 and 1 µM) was neuroprotective against the uncoupler FCCP (53%
protection), and modestly protective against hydrogen peroxide- (16%) and oligomycin- (15%) mediated
cell death, a pattern also confirmed in cultured hippocampal neurons. Thus, under pathological conditions
involving mitochondrial dysfunction and Ca2+ dysregulation, CBD may prove beneficial in preventing
apoptotic signaling via a restoration of Ca2+ homeostasis.
Key words: excitotoxicity; hippocampus; cannabinoids; ATP synthase; Na+/Ca2+ exchanger;
neuroprotection
Introduction
Two fundamental determinants of neuronal survival and viability under pathological conditions are Ca2+
homeostasis and metabolic activity, both reliant on mitochondrial function. Neurons have a particularly high
energy demand and correspondingly high metabolic activity, alongside large fluctuations in [Ca2+]i; thus,
mitochondria play a particularly important role in this cell type. Even subtle mitochondrial deficits can have
deleterious effects that can ultimately result in degenerative processes (for review, see Kajta, 2004 ).
Energy deficiencies are also associated with aging (Bowling et al., 1993 ) (for review, see Wiesner et al.,
2006 ) and age-related disorders, e.g., Alzheimer's disease (de la Monte and Wands, 2006 ), indicating a
correlation with mitochondrial dysfunction, as also recently suggested by a corresponding treatment
success in Alzheimer's patients (Doody et al., 2008 ). Mitochondria are preferentially located in areas of
highest [Ca2+]i adjacent to the endoplasmic reticulum, essential for the functional coupling of these two
organelles (Robb-Gaspers et al., 1998 ; Szabadkai et al., 2003 ; Saris and Carafoli, 2005 ). Moreover,
mitochondria determine cellular survival by generation of reactive oxygen species (Lafon-Cazal et al., 1993
) and apoptotic factors (Hong et al., 2004 ). This process involves an increased permeability of
mitochondrial membranes [including opening of the mitochondrial permeability transition pore (mPTP)
(Hunter et al., 1976 )]. Therefore, identification of agents that can restore normal mitochondrial function is
highly desirable.
The plant Cannabis sativa has for many centuries been reputed to possess therapeutically relevant
properties. Its most widely studied and characterized component, 9-tetrahydrocannabinol (THC), is one of
60+ compounds from Cannabis sativa, collectively known as phytocannabinoids. However, THC may have
a limited usefulness due to psychoactivity, dependence, and tolerance (Sim-Selley and Martin, 2002 );
therefore, attention has turned to some of the nonpsychoactive phytocannabinoids, most notably
cannabidiol (CBD). CBD has little agonistic activity at the known cannabinoid receptors (CB1 and CB2)
(Pertwee, 2004 ), and may possess therapeutic potential, e.g., anti-epileptic (Cunha et al., 1980 ),
anxiolytic (Guimarães et al., 1994 ), anti-inflammatory (Carrier et al., 2006 ), and even anti-psychotic
properties (Leweke et al., 2000 ) [for review, see Pertwee (2004) and Drysdale and Platt (2003) ]. In
addition, CBD has shown neuroprotection in a range of in vivo (Lastres-Becker et al., 2005 ) and in vitro
models (Esposito et al., 2006 ), some in association with a reduction in [Ca2+]i (Iuvone et al., 2004 ).
The highly lipophilic nature of cannabinoids grants them access to intracellular sites of action, and a
number of studies have suggested mitochondria as targets for cannabinoids (Bartova and Birmingham,
1976 ; Sarafian et al., 2003 ; Athanasiou et al., 2007 ). Modulation of [Ca2+]i by CBD has also been
observed in a variety of cell types (Ligresti et al., 2006 ; Giudice et al., 2007 ), including our previous work
which demonstrated a CBD-induced non-CB1/TRPV1-receptor-mediated increase in [Ca2+]i in hippocampal
neurons (Drysdale et al., 2006 ). Subsequent studies showed CBD effects to be negatively modulated by
the endocannabinoid system (Ryan et al., 2007 ), but the exact mechanisms remained to be fully
characterized. Therefore, the present study investigated CBD actions upon mitochondria and Ca2+
homeostasis as a potential basis for CBD's neuroprotective properties.
Materials and Methods
Hippocampal culture preparation. Preparation of standard primary hippocampal cultures from Lister–Hooded rat
pups (1–3 d old) was conducted as described previously (Drysdale et al., 2006 ; Ryan et al., 2006 ),
conforming to Home Office and institute regulations. Briefly, pups were killed by cervical dislocation and
the brain removed, and the hippocampi were dissected out and placed in filtered ice-cold HEPES-buffered
solution (HBS, composition in mM: NaCl, 130; KCl, 5.4; CaCl2, 1.8; MgCl2, 1; HEPES, 10; glucose, 25;
compounds from Sigma-Aldrich). Hippocampal tissue was finely chopped and placed in a 1 mg/ml protease
solution (type X and XIV, Sigma-Aldrich) for 40 min. Graded fire-polished glass Pasteur pipettes were used
to triturate the tissue a number of times. Following centrifugations, the tissue pellet was resuspended in
tissue culture medium [90% minimum essential medium (MEM; Invitrogen), 10% fetal bovine serum (FBS)
(Helena Biosciences), and 2 mM L-glutamine (Sigma-Aldrich)], kept in a humidified incubator at 37°C and in
5% CO2, and plated in 35 mm culture dishes (Invitrogen, coated with poly-L-lysine, Sigma-Aldrich). After 1
h, an additional 2 ml of tissue culture medium was gently added to each dish and stored in a humidified
incubator (37°C; 5% CO2). After 2 d of maturation, the MEM was replaced with Neurobasal medium
(Invitrogen) to reduce glial growth [composition of culture by cell-type (2:1, neurons:glia) was in keeping
with that outlined in previous publications (Platt et al., 2007 )], containing 2% B27, 2 mM L-glutamine, and
25 µM L-glutamate (Sigma-Aldrich). Culture dishes were checked for uniform density and deemed suitable
for imaging experiments from 5 to 10 d in vitro based on fully reproducible NMDA responses (variability:
<5%), with control experiments conducted at regular intervals.
Fura-2 AM Ca2+ imaging. For calcium imaging experiments (see also Ryan et al., 2006 ), hippocampal cultures
were washed with HBS (as above) at room temperature and loaded with the cell-permeable fluorescent
calcium indicator fura-2 AM (10 µM, Invitrogen) for 1 h in the dark. To allow the monitoring of postsynaptic
events uncontaminated by spontaneous activity and transmitter release, the sodium channel blocker
tetrodotoxin (TTX, 0.5 µM, Alomone Labs) was added to all perfusion media (except in experiments with
4AP). Cultures were perfused with HBS or low-Mg2+ (0.1 mM) HBS, using a gravity perfusion system at a
flow rate of 1–2 ml/min.
The imaging system, fitted onto an Olympus BX51WI fixed stage microscope, used the Improvision
software package Openlab (version 4.03, Improvision) with a DG-4 illumination system (Sutter
Instruments) and a Hamamatsu Orca-ER CCD camera for ratiometric imaging. After an appropriate field of
cells was identified, a gray-scale transmission image was visualized and captured. Cells were excited with
wavelengths of 340 and 380 nm, and the ratio of fluorescence emitted at 510 nm analyzed after
subtraction of background fluorescence levels. As described in our previous publications, fields of cells and
regions of interest (ROIs) were chosen based on homogenous and equal cell densities, with a neuronal
population of 15–40 cells per field of view. ROIs were placed on all fura-2 AM-loaded neuronal cell bodies
and large, star-shaped glia, confirmed to be astrocytes by GFAP staining, and based on an overlay of a
transmission image (Koss et al., 2007 ). Following this, time courses were created for all cells (neurons
and glia), with frames captured every 5 s.
Mitochondrial Ca2+ imaging. The mitochondrial and cytosolic Ca2+ compartments were visualized simultaneously
by preloading cultures with the mitochondrion-specific Ca2+ sensor Rhod-FF, AM (Invitrogen). Culture
dishes were incubated with Rhod-FF, AM (5 µM, in standard HBS) for 15 min on the day before
experimentation to allow compartmentalization of the marker (specificity of this marker was confirmed by
the abolition of compartmentalization by FCCP application) (see Fig. 4Ci,Cii). HBS was replaced with fresh
Neurobasal medium and returned to the incubator overnight. The following day, cells were loaded with
fura-2 AM as described above. Dual imaging was performed with alternating wavelengths relevant to RhodFF (excitation: 550 nm; emission: 580 nm) and fura-2 AM (as above) delivered at intervals of 3 s. Both
images were background subtracted, and separate graphs were plotted on-line (see Fig. 4). For off-line
analysis of mitochondrial responses, data were imported into the Volocity analysis program (version 4.02,
Improvision). Areas of most intense Rhod-FF mitochondrial fluorescence within a single neuron were
allocated ROIs.
SH-SY5Y cell preparation. The established human neuroblastoma cell line, SHSY-5Y (SH), was grown in 30 ml
flasks in MEM-based medium supplemented with growth factor F12, 10% fetal bovine serum, 2 mM Lglutamine, and 50 µg/ml antibiotic. Cells were maintained at 37°C at 5% CO2. Medium was replaced every
2–4 d, after washing with PBS (1 mM phosphate). Once cells proliferated to 80% confluency they were
passaged or transferred to a 96-well plate (Greiner) for experimental treatment (final volume in medium:
150 µl of cell suspension per well). Plates were used for experimentation when 80% confluency was
achieved (typically taking 4 d).
Each treatment and relevant vehicle controls were run in six samples (wells) per experiment and repeated
at least twice, viability was compared using the nontoxic cell viability marker Alamar Blue (Serotec). This
marker was made up as a 10% solution in MEM and applied to all wells (following the removal of
treatment medium) for 2 h at 37°C, 5% CO2. The plates were then run in a plate reader (either Victor2
1420, Wallac, Perkin-Elmer or Synergy HT, Bio-Tek) and the fluorescence (excitation: 530 nm and
emission: 590 nm) measured.
Cell death in hippocampal cultures. Hippocampal cultures were preincubated with CBD for 1 h before
coapplication of CBD with mitochondrion-acting toxins overnight, following which cell death was quantified
using a Live-Dead staining kit (Sigma) (modified from our previous publications) (Platt et al., 2007 ).
Briefly, 10 µl of solution A and 4 µl of solution B were diluted in 5 ml of HBS (at room temperature). Each
dish was washed in HBS three times and 500 µl of the staining solution added and incubated for 20 min (in
the dark, at room temperature). After a further wash with HBS, live images were capture in HBS with a 40x
phase-contrast water-immersion objective [brightfield, FITC (live cells) and rhodamine filters (dead cells)]
using an Axioskop 2 plus microscope (Carl Zeiss) fitted with an AxioCam HRc camera, with AxioVision
software (version 3.1). Three images were taken from each dish and each experiment performed on at
least two dishes from three different cultures.
MitoCapture. SH-SY5Y cells were grown on 96-well plates and treated with FCCP overnight as described
above. MitoCapture reagent (Calbiochem) was diluted 1:1000 in PBS (at room temperature) before use.
The medium was removed from the wells of the plate and replaced with 50 µl of reagent solution and
placed in an incubator (37°C at 5% CO2) for 15–20 min. The cells were then washed twice with PBS and
run through the plate reader (Synergy HT, Bio-Tek) with two fluorescence channels measured (green
monomers: excitation 488 nm and emission 530 nm; red aggregates: excitation 488 nm and emission 590
nm). Control and toxin groups were run in 6 samples (wells) per experiment and performed three times.
Drugs and stock solutions. CBD, obtained from GW Pharmaceuticals, and AM281 (Tocris Bioscience) were
stored in ethanol (1 mg/ml) at –20°C. For use in experiments, the ethanol was evaporated and the
cannabinoid resuspended in dimethyl sulfoxide (DMSO) at 1 mM (control experiments confirmed that 0.1%
DMSO did not alter basal Ca2+ levels or NMDA-induced Ca2+ responses, data not shown). The toxins tested
in the SH-SY5Y model were as follows: hydrogen peroxide (H2O2, Sigma-Aldrich) at 0.1 and 0.5 mM, 3 h
application; oligomycin (20 µM, Sigma-Aldrich) applied overnight (used in the same manner in hippocampal
culture cell death models also); FCCP (Sigma-Aldrich) was also applied overnight at 20 µM (also applied in
the same concentration and duration in hippocampal culture cell death models). In each case, pilot
experiments were performed to determine suitable concentrations resulting in a degree of cell death that
leaves capacity for either a reduction or increase in cell viability (targeted reduction in cell viability: 40–
70%). The sites of action of these toxins can be seen in Figure 1. Other compounds tested to elucidate
CBD's mechanisms of action were (with final concentrations and stock solvents listed) as follows: catalase
(500 and 1000 U/ml; MEM), cyclosporin A (CsA; 1–20 µM; DMSO), butylated hydroxytoluene (BHT; 3 and
10 µM; DMSO), and dantrolene (10 µM; H2O), all from Sigma-Aldrich. Additionally, 4-aminopyridine (4AP; 50
µM; DMSO), 2-aminoethoxydiphenyl borate (2-APB; 100 µM; DMSO), and CGP 37157 (10 µM; DMSO) were
obtained from Tocris Bioscience. For all compounds tested, drug-only controls were performed.
F/F, where F is the change in
fluorescence, calculated as a percentage of baseline fluorescence (F), which was defined as an average of
five baseline values before drug application (Drysdale et al., 2006 ; Ryan et al., 2007 ). Each group of
experiments consisted of at least three independent replications from different cultures. A change in
fluorescence of 10% of baseline fluorescence was deemed a genuine response to drug applications (with
Data analysis. All fura-2 AM fluorescence values were converted into %
intrinsic Ca2+ fluctuations ± 5%). Data were exported to Excel and GraphPad Prism (version 4, Graph Pad
Software) for preparation of graphs and statistical analysis. Due to the absence of normal distribution,
Kruskal–Wallis nonparametric tests with Dunn's post hoc test were used for multiple-group comparisons,
and a Mann–Whitney U test applied for paired comparisons. For work with SH cells, data generated as
units of fluorescence intensity were transferred to Excel and converted into percentage of within-plate
controls for graphical presentation only. Statistical analysis was performed on raw data using Prism, with
an overall one-way ANOVA performed for multiple-group comparison. For overall p values <0.05, Tukey's
posttest was used for paired comparison. Comparison of two relevant groups was conducted using an
unpaired t test.
Results
CBD regulates Ca2+ homeostasis in hippocampal tissue
Previous studies from our group have strongly suggested a link between CBD signaling and [Ca2+]i
regulation via intracellular Ca2+ stores (Drysdale et al., 2006 ; Ryan et al., 2007 ). To explore and
characterize the underlying mechanisms, experimental conditions were used which enhance excitability and
increase the degree of loading of intracellular Ca2+ stores. It was predicted that such conditions should
increase the CBD response compared with responses in standard HBS (Fig. 2A), as reported for other
store-operated signaling cascades (Irving and Collingridge, 1998 ). Thus, CBD was applied (1 µM; 5 min) in
HBS with doubled K+ concentration (10.8 mM). Surprisingly, under these conditions the effect of CBD
application was to reduce [Ca2+]i in both neurons and glia (Fig. 1B). The neuronal response was –26 ± 2%
F/F (n = 19), with almost identical responses in glia [–26 ± 4% F/F (n = 19)], p values <0.001
compared with CBD controls (Fig. 1C), suggesting that [Ca2+]i regulation by CBD is bidirectional and
depends on excitability.
Figure 1. Mitochondrial components of Ca2+ regulation and targets for
drug action used to assess the action of CBD. Drugs/enzymes used to
induce cell death in SH-SY5Y cells are italic and underlined, and those
applied to identify possible mechanisms of protection are outlined.
SOD2, Superoxide dismutase 2.
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Figure 2. Bidirectional Ca2+ responses to CBD in hippocampal cultures. A, B,
Sample traces for CBD-mediated Ca2+ responses in neurons (black traces) and glia
(gray traces) in normal (A) and high-excitability (B) HBS (double K+
concentration). NMDA applications at the end of each experiment were used to
confirm intact signaling in neurons. C, Mean responses of CBD in normal (ctrl) and
high (high ex)-excitability HBS. Data are presented as % F/F + SEM. ***p <
0.001.
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version (16K):
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In an alternative approach, we induced seizure-like Ca2+ oscillations by applying the K+ channel blocker
4AP, thus also probing previously reported anti-convulsant actions of CBD. Here, 4AP (50 µM) applied to
primary hippocampal cultures (Fig. 3A) induced a sustained rise in [Ca2+]i that continued to cause Ca2+
oscillations a few minutes after wash. When the 4AP application was immediately followed by 1 µM CBD
(Fig. 3B), oscillations were silenced (n = 29; 5 glia, 24 neurons). Alternating the order of application
robustly demonstrated that CBD could also prevent the initiation of epileptiform activity by 4AP. This was
proven to be the case in all neurons (n = 10) and almost all glia (n = 30/31) investigated (Fig. 3C).
Figure 3. CBD effects on epileptiform activity in cultured hippocampal neurons.
A, Application of the K+ channel antagonist 4AP to naive cultures induces
spontaneous Ca2+ oscillations. B, C, The presence of CBD following (B), or
preceding (C), 4AP application dampened Ca2+ oscillations. Data are presented as
% F/F.
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CBD, mitochondria, and [Ca2+]i levels
Previous work from our laboratory indicated a link between CBD-induced Ca2+ responses and intracellular
Ca2+ stores (Drysdale et al., 2006 ), rather than extracellular Ca2+ sources. Thus, we next investigated a
potential role of mitochondria, fundamental players in cellular Ca2+ homeostasis, in CBD's action. To
simultaneously study mitochondrial signaling together with cytosolic Ca2+ responses, cultures were
preloaded with the mitochondrion-specific Ca2+-sensitive fluorescent marker, Rhod-FF, AM, followed by
fura-2 AM loading (Fig. 4). The fluorescence pattern and responses to FCCP (10 µM), an uncoupler of ATP
synthesis due to its action as a protonophore, confirmed the specificity of this protocol, causing leakage of
mitochondrial Ca2+ from mitochondria accompanied by an increased cytosolic Ca2+ concentration (Fig. 4).
Application of CBD (1 µM) resulted in an increase in cytosolic Ca2+, preceded by a response in the Rhod-FF
fluorescence (Fig. 5). Two Rhod-FF response patterns were observed, biphasic (an initial rise followed by a
decrease) or a continuous decline (see sample traces given in Fig. 5A,B). Subsequently, we confirmed that
the pattern observed with CBD in this dual-fluorescence model genuinely represents a release from
mitochondrial Ca2+ stores by preapplication of FCCP (1 µM), applied to dual-loaded cultures (see Fig. 1 for
the sites of action for this and other mitochondrion-acting compounds). At this concentration, FCCP induced
an immediate reduction in Rhod-FF fluorescence in the mitochondrial compartment, and somewhat delayed
in onset and progression, an increase in cytosolic Ca2+ levels was observed. More importantly, no further
responses to CBD could be induced in mitochondria (Fig. 5C), while raised cytosolic Ca2+ levels recovered
partially, in agreement with our previous experiments in high-K+ HBS and 4AP.
Figure 4. Dual-loading of hippocampal cultures with fura-2 AM and RhodFF, AM. A, Typical transmission image shows clearly defined neuronal
appearance. B, C, Rhod-FF fluorescence (B) demonstrates a clear
compartmentalization into mitochondria, a pattern disrupted by FCCP
application (Ci, Cii). The corresponding cytosolic Ca2+ alterations are
monitored using fura-2 AM (D) with responses shown in both compartments
to the mitochondrial uncoupler FCCP and NMDA (B, Di–Div). E, The raw
values (OD, optical density) for each channel are plotted.
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Figure 5. Mitochondrial and cytosolic CBD (1 µM) responses in naive cultures
loaded with Rhod-FF, AM and fura-2 AM. A, B, Sample traces from neurons
showing delayed cytosolic (black trace) and early biphasic mitochondrial (gray
trace) Ca2+ responses. NMDA application was used as an indicator of neuronal
viability and to make a clear distinction between neurons and glia. C, Application
of FCCP (1 µM) led to a drop in mitochondrial Ca2+ levels and prevented a further
Ca2+ rise by CBD. Vertical lines have been added to visualize the order of
responses. All Rhod-FF data are raw fluorescence values (OD, optic density), and
fura-2 AM responses are presented as ratio values.
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Overall, FCCP eradicated CBD responses in neurons [mean: –1 ± 6% F/F (n = 25), p < 0.001 compared
with controls] and significantly reduced responses in glia [reduced by 61 ± 5% (n = 8), p < 0.001]. As
these data strongly suggested a mitochondrial site of action, we aimed to exclude the ER as the primary
source of Ca2+ for CBD responses by applying CBD in the presence of specific antagonists to the receptors
linked to Ca2+ release pathways from the ER (dantrolene and 2-APB, acting as ryanodine and IP3 receptor
antagonists, respectively). The blockade of one of these receptors has been shown to upregulate the
activity of the other, implying that both release mechanisms share a common pool of Ca2+ (White and
McGeown, 2003 ). Thus, both antagonists were coapplied to fully block ER receptor-mediated release.
Such a blockade transiently altered baseline Ca2+ levels, but longer duration of antagonist treatment (10
min) allowed a settled baseline to be established before CBD application. When CBD was coapplied with 2APB and dantrolene, responses did not significantly differ from control values (p > 0.05), with glial
responses increased compared with controls (p < 0.001) (Fig. 6), further confirming that ER receptors are
somewhat modulating, but not mediating CBD-induced responses.
Figure 6. Effects of ER- and mitochondrion-acting drugs on CBD responses. A,
B, The role of mitochondria in CBD responses were confirmed in neurons (A)
and glia (B). The uncoupler FCCP prevented neuronal CBD response and largely
reduce glial responses while blockade of IP3 and ryanodine receptors [by 2-APB
and dantrolene (Dant.), respectively] did not significantly alter CBD responses in
neurons, a sample trace of which is also shown (C). In the presence of CGP
37157 (CGP), but not in the presence of the mPTP inhibitor cyclosporin A (CsA),
CBD responses were also blocked in normal and high-excitability HBS; CBD
responses under high-excitability conditions no longer differed from standard
HBS responses. Data are presented as % F/F + SEM; n.s., not statistically
significant; **p < 0.01, ***p < 0.001.
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Thus, our data strongly suggested that [Ca2+]i regulation via CBD is achieved via mitochondrial uptake and
release, which could potentially be achieved via either the mPTP or the mitochondrial Na+/Ca2+-exchanger
(NCX) (Griffiths, 1999 ). Experiments with the mPTP inhibitor CsA showed no difference to control CBD
responses, implying that the mPTP is not the principal mechanism of CBD's actions (Fig. 6). When the role
of the NCX in CBD-mediated responses was investigated using the specific antagonist CGP 37157 (CGP)
(Chiesi et al., 1988 ; Medvedeva et al., 2008 ), preapplied and coapplied (10 µM), CBD (1 µM) responses
were abolished [remaining response: neurons: 10 ± 10% F/F (n = 8), glia: 3 ± 7% F/F (n = 14), p
values <0.001] (Fig. 6). To confirm that NCX was also fundamental to [Ca2+]i reducing CBD responses, the
experiment was repeated in the presence of elevated [K+]e (as above). The reversal of neuronal CBD
responses normally seen under these conditions was no longer observed. Accordingly, the CBD response in
CGP no longer differed between high-K+ and standard HBS in both neurons and glia (p values >0.05) (Fig.
6). Therefore, we conclude that CBD is acting via the mitochondrial NCX to elevate or decrease cytosolic
Ca2+ levels, dependent on resting [Ca2+]i.
Protection by CBD against mitochondrial toxins
The apparent mitochondrial site of action of CBD led to the hypothesis that CBD may act as a
neuroprotectant against mitochondrially acting toxins, acting either directly on mitochondrial sites or
downstream thereof (Fig. 1). Initial tests used the mitochondria-reliant viability assay Alamar Blue in SHSY5Y cells, with protective actions of CBD confirmed in hippocampal cultures using a live–dead stain (Fig.
7).
Figure 7. Determination of cell death in hippocampal cultured neurons
(live–dead cell staining kit) by multichannel image capture in cells
treated with 20 µM oligomycin. A, Transmission image. B, Cells with
compromised cell membranes (rhodamine filter). C, Healthy cells (FITC
filter). D, Merged image. A dead sample neuron is circled in each
image. For further details, see Materials and Methods.
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Application of hydrogen peroxide (H2O2), produced in response to cell stress and metabolic impairment as
a byproduct of the dismutation of the superoxide (O ) free radical, to SH cells at 100 µM for 3 h reduced
cell viability by 50% (range: 40–60%). As a positive control for the mode of cell death, the peroxidespecific catalyzing enzyme catalase was coapplied. With and without 1 h preapplication, catalase (at both
500 and 1000 U/ml) fully protected against peroxide-induced cell death. Next, CBD (0.1 and 1 µM) was
assessed as a potential neuroprotectant and was initially coapplied with H2O2. The lower concentration of
CBD proved to be marginally, though significantly, protective (by 16 ± 5% (n = 18); p < 0.05), whereas
the higher concentration had no significant effect (Fig. 8A). This experiment was repeated with cells
preexposed to CBD (concentrations as above) for 1 h before H2O2 exposure. The neuroprotective effects
of 100 nM CBD were no longer evident, while 1 µM CBD worsened the fate of cells (p < 0.05, compared
with peroxide controls). Overall, this pattern argues against a simple antioxidant action of CBD.
Figure 8. Cannabinoids as potential neuroprotectants against mitochondrial
stressors in SH-SY5Y cells. Data are expressed as percentage protection (+SEM)
relative to within-experiment controls and shown for peroxide (0.1 mM) (A) and
oligomycin (20 µM) (B) toxicity. CBD conferred protection against oligomycin
toxicity in both SH-SY5Y cells and hippocampal cultures (HIPP.). Pre-Inc.,
Following 1 h preincubation; *p < 0.05, ***p < 0.001.
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Next, ATP production was targeted with oligomycin, an inhibitor of ATP synthase (Fig. 1) that blocks the
phosphorylation of ADP at this complex of the electron transport chain (Penefsky, 1985 ; Duchen, 2004 ).
Following overnight dose–response experiments, a concentration of 20 µM was selected for further
experimentation (average reduction in cell viability: 35%). A modest, though significant, protection was
conferred by coapplication of the toxin with 1 µM CBD [improved by 15 ± 4% (n = 23), p < 0.05] (Fig.
8B), but not with the lower CBD concentration (100 nM, data not shown). As a confirmation of mPTP
involvement in this toxicity assay, the inhibitor of mPTP formation, CsA (1 µM), was applied and proved to
be protective [increase in cell viability: 53 ± 10% (n = 24), p < 0.001 compared with oligomycin controls].
In comparison, catalase conferred no protection against oligomycin toxicity, coapplication of CsA and CBD
also proved not to be additive (data not shown). Notably, CsA alone (in the absence of any toxin) improved
cell viability (CsA control being 110 ± 3% of control value, p < 0.05), implying under resting conditions
there may be some activation of mPTP (data not shown). Neuroprotection of CBD was also confirmed in
hippocampal cultures, where CBD (with 1 h preincubation) again proved to be neuroprotective by 31 ± 3%
(n = 9, p < 0.01) (Fig. 8B).
Finally, as a continuation of our imaging data, CBD was again tested in combination with the uncoupler of
ATP synthesis, FCCP (see above and Fig. 1). To further confirm the mitochondrial site of action of FCCP,
SH-SY5Y cells were loaded with the MitoCapture fluorescent dye, also used as a marker for apoptosis. In
healthy cells, the reagent congregates in the mitochondria and is detected as a red fluorescence signal.
Conversely, in apoptotic cells, MitoCapture remains in the cell cytosol (due to the disrupted mitochondrial
membrane potential) and can be monitored as a green fluorescent signal. Following FCCP incubation (20
µM, overnight) green fluorescence was increased by 45 ± 8%, while red fluorescence was decreased by 51
± 6% (in both cases n = 30 and p < 0.001 compared with controls) (Fig. 9C). This indicates that FCCP is
acting to primarily depolarize mitochondria.
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Figure 9. Effects of CBD and other potential neuroprotectants against
toxicity induced by the mitochondrial uncoupler FCCP in SH-SY5Y cells. A,
The phytocannabinoid CBD is neuroprotective with and without
preincubation (Pre-inc.), although protection was enhanced when CBD
was present before FCCP was applied. Such neuroprotection was similarly
observed in hippocampal cultures (HIPP.) Maximal protection with CBD
was comparable to that observed with cyclosporin A (CsA). B,
Comparison of antioxidant effects of CBD and butylated hydroxytoluene
(BHT). Note the apparent synergism between these two compounds,
achieving full (100%) protection. C, Confirmation of FCCP's actions to
disrupt mitochondrial potential was obtained using MitoCapture,
demonstrating both a reduced number of healthy cells (red signal) and
increased number of cells with disrupted mitochondrial potential (green
signal) in the presence of FCCP. Data are presented relative to controls
(+SEM). *p < 0.05, ***p < 0.001; n.s., not statistically significant.
The Alamar Blue assay indicated that 20 µM FCCP (overnight) caused a mean reduction in cell viability of 70
± 2%. When CBD (100 nM and 1 µM) was coapplied with FCCP it was neuroprotective at both
concentrations [percentage protection: 10% and 15%, respectively, n = 12 for both (p < 0.01)] (Fig. 9A),
in line with the evidence from previous acute imaging experiments. This experiment was next repeated
with the cells exposed to CBD for 1 h before FCCP application. A markedly enhanced protection was
observed (100 nM CBD yielding 35 ± 3% protection and 1 µM conferring 53 ± 2% protection). This level of
protection was significantly greater than that seen without CBD preexposure in each case (p values
<0.05). CsA was also found to be protective in this model in a dose-dependent manner, reaching maximal
protection (43 ± 2%, n = 24, p < 0.001) at 20 µM (shown in Fig. 9A). As for oligomycin, when the FCCP
toxicity experiment was repeated in cultured hippocampal neurons (with 1 h CBD preincubation), CBD
proved to be protective by 27 ± 3% (n = 6, p < 0.01) (Fig. 9A).
Since an antioxidant capacity has been widely reported for CBD (Hampson et al., 1998 ; Chen and Buck,
2000 ), its neuroprotective properties were subsequently compared with the protective capabilities of the
free radical scavenger butylated hydroxytoluene (BHT). Interestingly, coapplication of FCCP with BHT (at 3
and 10 µM; n values = 11 and 12, respectively) conferred no significant protection (p values >0.05), yet
joint application of CBD (1 µM) applied with the higher concentration of BHT (10 µM) provided a complete
prevention of FCCP's toxic effects [100 ± 7% protection (n = 12), p < 0.001 compared with FCCP
controls], significantly more potent than CBD alone (p < 0.001) (Fig. 9B). The superadditive nature of this
protection strongly suggests independent but synergistic modes of action. Overall, our data suggest that
CBD directly acts on mitochondria, and this action offers protection against toxins that directly target
mitochondria.
Discussion
Enhanced excitability and epileptiform activity
We here report bidirectional regulation of [Ca2+]i and protection provided by CBD. This was evident acutely
as CBD reduced cytosolic Ca2+ levels in high-K+ solution, and also silenced and prevented epileptiform-like
activity induced by 4AP. The latter experiments were performed in the absence of TTX (as sustained
spontaneous firing and neurotransmitter release is fundamental for epileptiform activity), hence one
possibility is that anticonvulsant activity could be mediated by actions on transmitter release, a property
already identified for a number of cannabinoids with respect to glutamate (Szabo and Schlicker, 2005 ;
Shen et al., 1996 ) and GABA (Katona et al., 1999 ; Köfalvi et al., 2005 ). Such actions can potentially
alter excitability but would require CBD to act on the endocannabinoid system. While modulatory
interactions between CBD and endocannabinoids were demonstrated in our previous work (Ryan et al.,
2007 ), this did not involve agonism on known CB receptors, although an indirect action on these
receptors via inhibition of endocannabinoid reuptake and hydrolysis remains a possibility (Bisogno et al.,
2001 ; Ligresti et al., 2006 ). A number of other studies have identified CBD as an anti-epileptic agent
both in vitro and in vivo (for review, see Pertwee, 2004 ), and our data imply that this can be achieved by
a mitochondrial regulation of [Ca2+]i. We also propose that this action would offer beneficial protection in
disease states that involve hyperexcitability, as CBD's mode of action may allow it to functioning as a Ca2+
sensor and regulator.
The reversal of Ca2+ responses in hippocampal cultures in the presence of an already elevated [Ca2+]i (as a
result of increased K+ in the perfusion media) ruled out the ER receptors as the primary source of Ca2+ in
CBD responses, but instead echoed the theory of the mitochondrial Ca2+"set point" (Nicholls, 2005 ), i.e.,
the cytosolic concentration of Ca2+ at which mitochondrial uptake and efflux of Ca2+ are equal: interactions
between Ca2+ influx and efflux mechanisms in the mitochondria maintain extramitochondrial Ca2+
concentrations at a fixed value (Nicholls, 1978 ; Thayer and Miller, 1990 ). Therefore, the opposing CBD
responses may be achieved via reversal of one and the same Ca2+ transport mechanism (see also Poburko
et al., 2006 ). Additionally, the ER as the principal source of Ca2+ released by CBD was effectively
discounted by the combined application of dantrolene and 2-APB, which did not prevent CBD responses. 2APB blocks sites other than IP3 receptors, including TRP channels (for review, see Bootman et al., 2002 ),
a subset of which can act as Ca2+ release channels from the ER. This is an important consideration, as
phytocannabinoids can raise Ca2+ via these channels (De Petrocellis et al., 2008 ), yet a contribution to the
CBD response in our experiments is unlikely as suggested by our data obtained with 2-APB (Fig. 6) (see
also Tsuzuki et al., 2004 ). Instead, the trend of increased CBD responses in the presence of 2-APB is in
agreement with similarly enhancing actions observed with TRPV1 and CB1 antagonists (Ryan et al., 2007).
NCX as a target for CBD
The ER is a fundamental player in Ca2+ homeostasis with perturbations of this organelle's functioning
associated with excitotoxicity (e.g., in Alzheimer's disease) (for review, see Mattson and Chan, 2003 ).
Moreover, the close interaction between ER and mitochondrial Ca2+ signaling is an important factor in
apoptotic signaling (for review, see Szabadkai and Rizzuto, 2004 ), although a recent study has suggested
mitochondrial regulation of cytosolic Ca2+ independent of the ER, with the NCX as the rate-limiting factor
for temporal decoding (Young et al., 2008 ).
A direct role for mitochondria in CBD signaling was confirmed here by the use of dual-loaded hippocampal
neurons with Ca2+-sensitive probes for mitochondrial and cytosolic compartments, with changes in
mitochondrial Ca2+ levels preceding the rise of cytosolic Ca2+. Moreover, the depletion of Ca2+ from
mitochondria (using FCCP) resulted in the inability of CBD to yield any subsequent response. CBD's point
of action upon mitochondria was identified to be the NCX (and not the mPTP), as CBD responses were no
longer present when this exchanger was blocked by CGP, yet unaffected by CsA. The NCX functions
normally to remove Ca2+ from the mitochondria but can reverse when the ionic gradients are sufficiently
altered, especially in disease states (Jung et al., 1995 ; Griffiths, 1999 ; Poburko et al., 2006 ). A
fundamental role for the mitochondrial NCX in Ca2+ signaling associated with ischemia and excitotoxic
events, where the influx of Na+ into the cell causes release of Ca2+ from mitochondria, has previously been
identified in hippocampal tissue (Zhang and Lipton, 1999 ).
CGP may act not only upon mitochondrial NCX, but also as an inhibitor of VGCCs in dorsal root ganglion
neurons (Baron and Thayer, 1997 ). However, our previous data with VGCC blockers (Drysdale et al.,
2006 ) are not consistent with an effect of CBD on this target. Others have found CGP to inhibit the NCX
in the plasma membrane of cerebellar granule cells (Czyz and Kiedrowski, 2003 ), although with an IC50 of
13 µM, a concentration higher than that used here, and higher than CGP's IC50 (4 µM) for the mitochondrial
NCX in cultured rat DRGs (Baron and Thayer, 1997 ). Indeed, the concentration of CGP used here is in
keeping with recent work by others in cultured neurons (Medvedeva et al., 2008 ).
CBD as a neuroprotectant
Our study uncovers a new intracellular, and potentially direct, target for CBD, which has so far been
largely elusive despite the wide-ranging use of this phytocannabinoid in diverse preparations and
applications. Evidence for actions of CBD on mitochondria was strongly supported by cell death models
with mitochondrial toxins. The most potent CBD protection was seen against FCCP toxicity (in SH cells and
reproduced in cultured hippocampal neurons), a mitochondrial uncoupler, causing the collapse of the
mitochondrial membrane potential and the release of Ca2+ into the cytosol. A more modest protection by
CBD was observed against other oxidative stress related agents, hydrogen peroxide and oligomycin. FCCP
causes the accumulation of protons into mitochondria leading to uncoupling of the mitochondrial potential
( m), ultimately causing a loss of ATP production (for review, see Wallace and Starkov, 2000 ), and has
previously been demonstrated to cause apoptosis in PC12 cells (Dispersyn et al., 1999 ) and primary
neurons (Moon et al., 2005 ). The loss of
m (confirmed by MitoCapture) and resultant cell death is also
assumed to involve mPTP formation (Marques-Santos et al., 2006 ), in keeping with our finding that CsA
can protect against FCCP toxicity in a dose-dependent manner.
Exogenous application of hydrogen peroxide (as well as the cellular generation of this oxidative agent) has
been shown to induce apoptosis in association with MAP kinase activation (Guyton et al., 1996 ). Thus,
CBD's neuroprotective action against H2O2 toxicity in SH cells is in line with CBD's reported inhibition of
p38 MAP kinase, although proposed to be secondary to CBD's antioxidant capacity (El-Remessy et al.,
2006 ), which was not apparent here. Ligresti et al. (2006) also showed protection in breast cancer cells
against H2O2 toxicity at low (nM) but not higher (µM) concentrations of CBD. The latter effect was
suggested to involve the generation of ROS, also reported in glioma cells (Massi et al., 2006 ). Therefore,
the possibility has been raised that CBD might have potential as an anticancer treatment (for review, see
Mechoulam et al., 2007 ). A comparison between the antioxidant properties of CBD and BHT has been
performed previously (Hamelink et al., 2005 ), with approximately equivalent antioxidant capacities
reported for both compounds. This is surprising in the light of the data generated here where CBD, but not
BHT, alone was neuroprotective against FCCP toxicity. Interestingly, the combination of CBD and BHT
caused complete protection against FCCP. Previous work conducted in our lab has shown an interaction
between this phytocannabinoid and BHT, with BHT preexposure (saturating antioxidant pathways) to
primary rat hippocampal cultures facilitating [Ca2+]i responses to a subsequent CBD application (our
unpublished observations). This implies a synergy between CBD and antioxidant pathways, with the latter
facilitating CBD's effects, rather than mediating them. Since CBD showed only little protection in the
peroxide model, it seems that its anti-oxidant properties are not of major relevance for its protective action.
While similar protection was seen for CsA and CBD in the FCCP model, differences in efficacy between CsA
and CBD and the lack of additivity between CBD and CsA in the oligomycin model (Comelli et al., 2003 )
suggest that the mPTP is not a major target in CBD's action.
Overall, the apparent capacity for CBD to reduce [Ca2+]i when it is abnormally elevated via interactions
with mitochondria-dependent Ca2+ regulation may be a valuable property for many disease states
associated with Ca2+ dysregulation. Moreover, neurodegenerative diseases linked directly to mitochondrial
malfunction, such as Huntington's disease and Friedreich's ataxia, may benefit greatly from CBD-based
medicines.
Footnotes
Received Sept. 4, 2008; revised Jan. 13, 2009; accepted Jan. 13, 2009.
C. Lafourcade's present address: Departments of Physiology and Psychiatry, University of Maryland School
of Medicine, 655 West Baltimore Street, BRB 5-025, Baltimore, MD 21201.
We thank GW Pharmaceuticals for the provision of CBD.
Correspondence should be addressed to Bettina Platt at the above address. Email: b.platt@abdn.ac.uk
Copyright © 2009 Society for Neuroscience 0270-6474/09/292053-11$15.00/0
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This article has been cited by other articles:
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In Vitro and In Vivo
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[Abstract] [Full Text] [PDF]
http://www.jneurosci.org/cgi/content/full/29/7/2053?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fullte
xt=CBD&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&resourcetype=HWCIT
Anandamide increases swelling and reduces calcium sensitivity of mitochondria
Giuseppina Catanzaroa, b, Cinzia Rapinoa, Sergio Oddia, b and Mauro Maccarronea, b,
aDepartment
,
of Biomedical Sciences, University of Teramo, Piazza Aldo Moro 45, 64100 Teramo, Italy
bEuropean
Center for Brain Research (CERC)/IRCCS S. Lucia Foundation, Via del Fosso di Fiorano, 00143
Rome, Italy
Received 22 July 2009.
Available online 11 August 2009.
Abstract
The endocannabinoid anandamide alters mitochondria-dependent signal transduction, thus controlling key
cellular events like energy homeostasis and induction of apoptosis. Here, the ability of anandamide to
directly affect the integrity of mitochondria was investigated on isolated organelles. We found that
anandamide dose-dependently increases mitochondrial swelling, and reduces cytochrome c release
induced by calcium ions. The effects of anandamide were independent of its target receptors (e.g.,
cannabinoid or vanilloid receptors), and were paralleled by decreased membrane potential and increased
membrane fluidity. Overall, our data suggest that anandamide can impact mitochondrial physiology, by
reducing calcium sensitivity and perturbing membrane properties of these organelles.
Keywords: Anandamide; Apoptosis; Cannabinoid receptors; Cytochrome c release; Membrane fluidity;
Mitochondria; Mitochondrial swelling; Vanilloid receptors
Article Outline
Introduction
Materials and methods
Results and discussion
Acknowledgements
References
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-4X01P811&_user=10&_coverDate=10%2F16%2F2009&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=
c&_searchStrId=1207623522&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10
&md5=cd9a71ff23c691a46dd4c652d1776a6b
Mitochondrial permeability transition
Wikipedia.org
Mitochondrial permeability transition, or MPT, is an increase in the permeability of the
mitochondrial membranes to molecules of less than 1500 Daltons in molecular weight. MPT results from
opening of mitochondrial permeability transition pores, also known as the MPT pores or MPTP.
The MPT pore is a protein pore that is formed in the membranes of mitochondria under certain
pathological conditions such as traumatic brain injury and stroke. Induction of the permeability transition
pore can lead to mitochondrial swelling and cell death and plays an important role in some types of
apoptosis.
The MPTP was proposed by Haworth and Hunter in 1979 and has since been found to be involved in,
among other things, neurodegeneration, a process that results in damage and death of neurons.[1]
MPT is frequently studied in liver cells, which have especially large numbers of mitochondria.
Roles in pathology
MPT is one of the major causes of cell death in a variety of conditions. For example, it is key in cell death
in excitotoxicity, in which overactivation of glutamate receptors causes excessive calcium entry into the
cell.[2][3][4] MPT also appears to play a key role in damage caused by ischemia, as occurs in a heart attack
and stroke.[5] However, research has shown that the MPT pore remains closed during ischemia, but opens
once the tissues are reperfused with blood after the ischemic period,[6] playing a role in reperfusion injury.
MPT is also thought to underlie the cell death induced by Reye's syndrome, since chemicals that can cause
the syndrome, like salicylate and valproate, cause MPT.[7] MPT may also play a role in mitochondrial
autophagy.[7] Cells exposed to toxic amounts of Ca2+ ionophores also undergo MPT and death by
necrosis.[7]
MPTP Structure
The MPT pore is a nonselective, high conductance channel with multiple macromolecular components.[8][9]
It forms at sites where the inner and outer membranes of the mitochondria meet.[10] Though the exact
structure of the MPTP is still unknown, several proteins probably come together to form the pore, including
adenine nucleotide translocase (ANT), the mitochondrial inner membrane protein transporter (Tim), the
protein transporter at the outer membrane (Tom), the outer membrane voltage-dependent anion channel
(VDAC) and cyclophilin-D.[11] Cyclosporin A blocks the formation of the MPT pore by interacting with
cyclophilin from the mitochondrial matrix and preventing its joining the pore.[12] Mice lacking the gene
for cyclophilin-D develop normally, but their cells do not undergo Cyclosporin A-sensitive MPT, and they
are resistant to necrotic death from ischemia or overload of Ca2+ or free radicals.[13] However, the cells do
die in response to stimuli that kill cells through apoptosis, suggesting that MPT does not control cell death
by apoptosis.[13]
MPTP blockers
Agents that block MPT include the immune suppressant cyclosporin A (CsA); N-methyl-Val-4-cyclosporin A
(MeValCsA), a non-immunosuppressant derivative of CsA; another non-immunosuppressive agent,
NIM811, 2-aminoethoxydiphenyl borate (2-APB)[14], and bongkrekic acid.
Factors in MPT induction
Various factors enhance the likelihood of MPTP opening. In some mitochondria, such as those in the
central nervous system, high levels of Ca2+ within mitochondria can cause the MPT pore to open.[15][16]
This is possibly because Ca2+ binds to and activates Ca2+ binding sites on the matrix side of the MPTP.[9][17]
MPT induction is also due to the dissipation of the difference in voltage between the inside and outside of
mitochondrial membranes (known as permeability transition, or ).[3][18] The presence of free radicals,
another result of excessive intracellular calcium concentrations, can also cause the MPT pore to open. [11][19]
Other factors that increase the likelihood that the MPTP will be induced include the presence of certain
fatty acids,[20] and inorganic phosphate.[21] However, these factors cannot open the pore without Ca2+,
though at high enough concentrations, Ca2+ alone can induce MPT.[22]
Stress in the endoplasmic reticulum can be a factor in triggering MPT.[23]
Things that cause the pore to close or remain closed include acidic conditions,[24] high concentrations of
ADP,[19][25] high concentrations of ATP,[26] and high concentrations of NADH.[16] Divalent cations like Mg2+
also inhibit MPT, because they can compete with Ca2+ for the Ca2+ binding sites on the matrix side of the
MPTP.[9]
Effects of MPT
Multiple studies have found the MPT to be a key factor in the damage to neurons caused by
excitotoxicity.[3][4][17]
The induction of MPT, which increases mitochondrial membrane permeability, causes mitochondria to
become further depolarized, meaning that is abolished. When is lost, protons and some molecules are able
to flow across the outer mitochondrial membrane uninhibited.[3][4] Loss of interferes with the production
of adenosine triphosphate (ATP), the cell's main source of energy, because mitochondria must have an
electrochemical gradient to provide the driving force for ATP production.
In cell damage resulting from conditions such as neurodegenerative diseases and head injury, opening of
the mitochondrial permeability transition pore can greatly reduce ATP production, and can cause ATP
synthase to begin hydrolysing, rather than producing, ATP.[27] This produces an energy deficit in the cell,
just when it most needs ATP to fuel activity of ion pumps such as the Na+/Ca2+ exchanger, which must be
activated more than under normal conditions in order to rid the cell of excess calcium.
MPT also allows Ca2+ to leave the mitochondrion, which can place further stress on nearby mitochondria,
and which can activate harmful calcium-dependent proteases such as calpain.
Reactive oxygen species (ROS) are also produced as a result of opening the MPT pore. MPT can allow
antioxidant molecules such as glutathione to exit mitochondria, reducing the organelles' ability to
neutralize ROS. In addition, the electron transport chain (ETC) may produce more free radicals due to loss
of components of the electron transport chain (ETC), such as cytochrome c, through the MPTP.[28] Loss of
ETC components can lead to escape of electrons from the chain, which can then reduce molecules and
form free radicals.
MPT causes mitochondria to become permeable to molecules smaller than 1.5 kDa, which, once inside,
draw water in by increasing the organelle's osmolar load.[29] This event may lead mitochondria to swell and
may cause the outer membrane to rupture, releasing cytochrome c.[29] Cytochrome c can in turn cause the
cell to go through apoptosis ("commit suicide") by activating pro-apoptotic factors. Other researchers
contend that it is not mitochondrial membrane rupture that leads to cytochrome c release, but rather
another mechanism, such as translocation of the molecule through channels in the outer membrane, which
does not involve the MPTP.[30]
Much research has found that the fate of the cell after an insult depends on the extent of MPT. If MPT
occurs to only a slight extent, the cell may recover, whereas if it occurs more it may undergo apoptosis. If
it occurs to an even larger degree the cell is likely to undergo necrotic cell death.[5]
Possible evolutionary purpose of the MPTP
The existence of a pore that causes cell death has led to speculation about its possible evolutionary
benefit. Some have speculated that the MPT pore may minimize injury by causing badly injured cells to die
quickly and by preventing cells from oxidizing substances that could be used elsewhere.[31]
There is controversy about the question of whether the MPTP is able to exist in a harmless, "lowconductance" state. This low-conductance state would not induce MPT[17] and would allow certain
molecules and ions to cross the mitochondrial membranes. The low-conductance state may allow small
molecules like Ca2+ to leave mitochondria quickly, in order to aid in the cycling of Ca2+ in healthy
cells.[32][25] If this is the case, MPT may be a harmful side effect of abnormal activity of a usually beneficial
MPTP.
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External links
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Mitochondrial Permeability Transition (PT) from Celldeath.de. Accessed January 1, 2007.
Retrieved from "http://en.wikipedia.org/wiki/Mitochondrial_permeability_transition"
Categories: Cellular respiration | Neurotrauma | Mitochondria
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http://www.righthealth.com/topic/mitochondrial_swelling/overview/wiki_detailed?modp=Mitochondrial_permeabilit
y_transition
mitochondrial swelling
medical dictionary
Increase in volume of mitochondria due to an influx of fluid; it occurs in hypotonic solutions due to osmotic
pressure and in isotonic solutions as a result of altered permeability of the membranes of respiring
mitochondria.
(12 Dec 1998)
Article
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The EMBO Journal (2000) 19, 6401 - 6407
doi:10.1093/emboj/19.23.6401
Respiring mitochondria determine the pattern of activation
and inactivation of the store-operated Ca2+ current ICRAC
Juan A. Gilabert1 and Anant B. Parekh1
1. Laboratory of Molecular and Cellular Signalling, Department of Physiology, University of Oxford, Parks
Road, Oxford OX1 3PT, UK
Correspondence to:
Anant B. Parekh, E-mail: anant.parekh@physiol.ox.ac.uk
Received 31 August 2000; Accepted 3 October 2000; Revised 3 October 2000
Abstract
In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a
biphasic increase in intracellular free Ca2+ concentration: an initial transient release of Ca2+ from
intracellular stores is followed by a sustained phase of Ca2+ influx. This influx is generally store dependent.
Most attention has focused on the link between the endoplasmic reticulum and store-operated Ca2+
channels in the plasma membrane. Here, we describe that respiring mitochondria are also essential for the
activation of macroscopic store-operated Ca2+ currents under physiological conditions of weak intracellular
Ca2+ buffering. We further show that Ca2+-dependent slow inactivation of Ca2+ influx, a widespread but
poorly understood phenomenon, is regulated by mitochondrial buffering of cytosolic Ca2+. Thus, by
enabling macroscopic store-operated Ca2+ current to activate, and then by controlling its extent and
duration, mitochondria play a crucial role in all stages of store-operated Ca2+ influx. Store-operated Ca2+
entry reflects a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.
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Keywords:
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Ca2+ buffering,
mitochondria,
store-operated Ca2+ entry
Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD.
Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF,
Reed JC.
The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
The Ca2+-activated protein phosphatase calcineurin induces apoptosis, but the mechanism is unknown.
Calcineurin was found to dephosphorylate BAD, a pro-apoptotic member of the Bcl-2 family, thus
enhancing BAD heterodimerization with Bcl-xL and promoting apoptosis. The Ca2+-induced
dephosphorylation of BAD correlated with its dissociation from 14-3-3 in the cytosol and translocation to
mitochondria where Bcl-xL resides. In hippocampal neurons, L-glutamate, an inducer of Ca2+ influx and
calcineurin activation, triggered mitochondrial targeting of BAD and apoptosis, which were both
suppressible by coexpression of a dominant-inhibitory mutant of calcineurin or pharmacological inhibitors
of this phosphatase. Thus, a Ca2+-inducible mechanism for apoptosis induction operates by regulating
BAD phosphorylation and localization in cells.
PMID: 10195903 [PubMed - indexed for MEDLINE]
Publication Types, MeSH Terms, Substances, Grant Support
Publication Types:
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Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.
MeSH Terms:
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14-3-3 Proteins
Animals
Apoptosis*
Calcineurin/antagonists & inhibitors
Calcineurin/genetics
Calcineurin/metabolism*
Calcium/metabolism*
Calcium/pharmacology
Carrier Proteins/chemistry
Carrier Proteins/metabolism*
Cell Line
Cells, Cultured
Dimerization
Enzyme Inhibitors/pharmacology
Glutamic Acid/pharmacology
Hippocampus/cytology
Humans
Mitochondria/metabolism
Neurons/cytology
Neurons/metabolism
Phosphorylation
Protein-Serine-Threonine Kinases/metabolism
Proteins/metabolism
Proto-Oncogene Proteins c-bcl-2/metabolism
Rats
Recombinant Fusion Proteins/metabolism
Transfection
Tyrosine 3-Monooxygenase*
bcl-Associated Death Protein
bcl-X Protein
Substances:
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14-3-3 Proteins
BAD protein, human
BCL2L1 protein, human
Bad protein, rat
Bcl2l1 protein, rat
Carrier Proteins
Enzyme Inhibitors
Proteins
Proto-Oncogene Proteins c-bcl-2
Recombinant Fusion Proteins
bcl-Associated Death Protein
bcl-X Protein
Glutamic Acid
Calcium
Tyrosine 3-Monooxygenase
Protein-Serine-Threonine Kinases
Calcineurin
Grant Support:
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AG-1593/AG/NIA NIH HHS/United States
CA-69381/CA/NCI NIH HHS/United States
HD25938/HD/NICHD NIH HHS/United States
etc
LinkOut - more resources
http://www.ncbi.nlm.nih.gov/pubmed/10195903
Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are
altered with type 1 diabetes due to the direct effects of hyperglycemia
Yvonne M Searls
, Rajprasad Loganathan
, Irina V Smirnova
and Lisa Stehno-Bittel
Cardiovascular Diabetology 2010, 9:8doi:10.1186/1475-2840-9-8
Published:1 February 2010
Abstract (provisional)
Background
Diminished calcium (Ca2+) transients in response to physiological agonists have been reported in vascular
smooth muscle cells from diabetic animals. However, the mechanism responsible was unclear.
Methodology/Principal Findings: VSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB)
rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate
receptors (IP3R) and the SR Ca2+ pumps (SERCA 2 and 3). Generally, a decrease in IP3R levels and
dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic
animals. Redistribution of the specific IP3R subtypes was dependent on the rat model. SERCA 2 was
redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the
STZ diabetic rat. The free intracellular Ca2+ in freshly dispersed VSMCs from control and diabetic animals
was monitored using ratiometric Ca2+ sensitive fluorophores viewed by confocal microscopy. In control
VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in
diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free
intracellular Ca2+ in the VSMCs from control rats with a prolonged and significantly blunted response in
the diabetic VSMCs. A slow rise in free intracellular Ca2+ in response to thapsigargin, a specific blocker of
SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic
rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments
were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control
conditions. In general, they demonstrated the same changes in protein levels and distribution as well as
the blunted Ca2+ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.
Conclusions/Significance: This work demonstrates that the previously-reported reduced Ca2+ signaling in
VSMCs from diabetic animals is related to decreases and/or redistribution in the IP3R Ca2+ channels and
SERCA proteins. These changes can be duplicated in culture with high glucose levels.
http://www.cardiab.com/content/9/1/8
Ca2+ : An Ion of Biological Cybernetics
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Felix Bast(formarly, Vadakke Madam Sreejith)
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ENTRY DATA
Introduction
The word 'Cybernetics' was first suggested by father of
control engineering, Norbert Weiner in 1947 as the
science of control and communication. Applications of
this stream have been influencing the control and
communication aspects of Biological methods as
evidenced by birth of specialized journals like
'Cybernetica', 'Kybernetica', 'Journal of Biological
Cybernetics' etc. The word 'Bionics' has been used by a
number of scientists to refer to the field of the simulation
of biological systems and this too should be thought of, as
dealing with important bridge between Biology and
Cybernetics.
Ca2+ effectively performs control of various life processes
and communicates in molecular level between living
cells. The importance of calcium in cell biology was
recognized as early as 1882 when Sidney Ringer
demonstrated that minute amounts of the divalent ion
were necessary to maintain heart muscle contractility
[See box 1]. About a century later, physiologists first
described calcium as a second messenger in analogy with
cyclic adenosine 3',5'-monophosphate (cAMP) which was
shown by Sutherland and others during the 1960's to be an
important mediator of hormonal responses. The term
second messenger implies that calcium is released
intracellularly as an intermediate and transient signal
produced upon excitation of the cell by external stimuli.
Calcium ions play a major role in controlling the
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functioning of all cells of the body by acting as carriers of
intracellular messages. Cells receive their instructions
from the body through hormones and neurotransmitters
that bind to receptors on their surface. Many of these
messages are relayed by the release of calcium ions from
internal stores into the cell cytoplasm, or by opening
channels in the membrane of the cell, allowing external
calcium to enter the cytoplasm. By these means calcium
controls a wide range of cellular functions such as muscle
contraction, neurotransmitter and hormone release,
metabolism, cell division and differentiation.
Calcium ions are quite remarkable in being able to control
numerous processes within the same cell simultaneously
[Figure 1]. This ability of calcium to provide a number of
signals encoded by the same ion is a result of the way in
which calcium levels can be altered in the cell. Outside
the cell, free calcium ion concentration is around 1mM,
whereas the free calcium ion concentration in the
cytoplasm of cells is maintained at around 0.1mM, mainly
by ATP powered pumps that pump calcium either out of
the cell or into an internal store called the endoplasmic
reticulum (E.R.). Because the cytoplasm contains high
concentrations of proteins that chelate calcium ions,
calcium is able to diffuse only short distances within the
cell. This means that a tight control is maintained over
the movement of calcium ions in the cytoplasm.
Ca2+ Signals: A Lingua Franca of 'Bionics' at Molecular
and Cellular Level.
The control configuration of Ca2+ signaling is negative
unity feed back. We can call it as dynamical system, as it
possesses memory. Let us explain this concept.
Y(t1) is dependent on the output applied before t= t1, if
y(t)1 is its output at t= t1. It can be modeled by an
algebraic equation. The output y(t1) at t= t1 depends
only on the input ‘u’ applied until that instant, but not
later. Hence time varying calcium-signaling system is
really a ‘casual system’ and is non-anticipatory.
Ca2+ signaling possesses almost universal biophysical
mechanisms. In common with cAMP, the key to Ca2+
activity as a secondary messenger is a rise in the
intracellular cytosolic concentration of the ion as a result
of hormone/neurotransmitter binding to its receptor at
cell membrane [see Figure 1.]. They may enter through
Voltage Operated Ca2+ Channels (VOCs) in neurons,
Receptor Operated Ca2+ Channels (ROCs) in response to
neurotransmitters, or Store Operated Ca2+ Channels
(SOCs) which open when the internal stores are emptied
of Ca2+. Ca2+ is released by two mechanisms, viz., by the
activity of Ins 1,4,5 P3 (Inositol 1,4,5,triphosphate; IP3)
on E.R. membrane or by Ryanodine receptors on E.R.
membrane. Ins 1,4,5 P3 is produced from Ptd.Ins 4,5 P2
(Phosphatidyl Inositol 4,5 bisphosphate; PIP2) by the
enzyme PLC (Phospholipase C). PLC is activated by Gs
protein, which in turn is activated by binding with first
receptor. Rise in intracellular Ca2+ activates Calmodulin
dependent Protein Kinase 2, which in turn will
phosphorylate cellular proteins triggering a cell response.
There is active research going on in the fields of
biophysical mechanisms of Ins 1,4,5 P3 dependent Ca2+
regulatory proteins and Ryanodine receptors on the
organelle membrane leading to the release of Ca2+ from
intracellular stores to the cytosol.
Some of the Ca2+ E.R. is rapidly taken up by the
mitochondria and is then returned to E.R., although most
of the stored Ca2+ resides in the lumen of E.R.. But if the
mitochondria are overloaded with Ca2+, the result is the
abnormal mitochondrial metabolism, which may activate
programmed cell death, to be discussed in a later section.
The Space-Time-Modulation Trilogy
The Ca2+ control mechanism depends on 1) the space
where Ca2+ concentration is higher or lower, 2) the time
through which Ca2+ transients operate and finally 3) the
modulation of Ca2+ signals.
Space
Ca2+ can be derived from extracellular as well as
intracellular stores. Ca2+ from intracellular stores can be
released through channels in E.R. or sarcoplasmic
reticulam. When a Ca2+ channel opens, a highly
concentrated mass of Ca2+ forms around its mouth and
when the channel is closed, it is rapidly dissipated by
diffusion. This Ca2+ spark is responsible for signaling
repertoire. These signals may be either localized in the
vicinity of channels or may generate a global Ca2+ wave,
which is intracellular or intercellular. If individual Ca2+
channels of neighboring cells are connected, an
intercellular global Ca2+ wave is produced which is
responsible for smooth muscle induced Nitric Oxide
synthesis in endothelium, glial cell function, wound
healing etc. Intracellular Ca2+ wave is responsible for
fertilization, cardiac muscle contraction, gene
transcription, cell proliferation etc. Membrane
excitability, synaptic plasticity etc. are under the control
of localized Ca2+ wave.
Time
Usually as an adaptive measure, cells avoid very high
concentration of Ca2+ by using low amplitude signals or
delivering signals as brief transients. When information
has to be relayed over longer time periods, cells use Ca2+
oscillations. The periods of localized and global Ca2+
oscillations differ widely. For example, period of
localized Ca2+ oscillation in arterial smooth muscle is 0.10.5 seconds; it is 10-60 s for global waves in liver cells, 135 minutes for Ca2+ waves in human eggs after
fertilization and 10-20 hrs for Ca2+ transients that control
cell division.
Modulation
To vary the intensity and nature of physiological output,
cells use frequency modulation (FM). Smooth muscle
lining of artery can be relaxed by increasing frequency of
Ca2+ signals. Calmodulin dependent protein kinase 2 is an
important enzyme which catalyses the phosphorylation of
cellular enzymes by counting Ca2+ transients, where by
depends on it's frequency [See Figure 2].
The key question concerning the repetitive transients is
their significance, because the "shape" of the Ca2+ signal
is an important factor in the modulation of targets.
Oscillations are an elegant and efficient way to transmit
Ca2+ signals, while at the same time avoiding deleterious
sustained elevations of Ca2+. Some Ca2+ -sensitive
events, e.g., muscle contraction or synaptic transmission,
require the rapid delivery of a spatially confined signal of
high intensity. Other processes require that Ca2+ be
delivered in a form that is not strictly localized and is
persistent over a longer period. These processes demand
that the signal change from elementary to global to
permit the prolonged exposure of targets to it.
Amplitude Modulated (AM) Ca2+ signals are generally less
reliable than FM. But there is growing evidence that
varying the amplitude of Ca2+ signals can activate
different genes.
In Fertilization and Development
Intracellular Ca2+ concentration of 50nM with in
spermatozoa is an extremely important factor required
for fertilization. Using flow cytometry it is found out that
in response to agonist progesterone, the Ca2+
concentration in side a sperm, is increased to 195nM,
before declining approximately 1/2 of the maximum level
with in 2 minutes.
During the interaction of sperm and ovum, a Ca2+
oscillation is produced, which might persist for several
hours. It is this oscillation, which triggers stimulation of
enzymatic machinery leading to ordered mitosis of
zygote. One celled Zygote divides into two when a
spontaneous Ca2+ oscillation activates it. This orderly
program of mitosis is under the control of two distinct
oscillators, viz., Ca2+ levels and oscillating levels of cell
division proteins. But the main mechanism is thought to
be Ca2+ signals, as it persists when dissociated from cell
cycle oscillator. During the development of fertilized
ovum, the process by which the concentration of
intracellular diffusible Ins 1,4,5 P3 concentration sharply
increases with each cell division leading to periodic Ca2+
oscillation is presently unknown. But the study shows that
hormones and growth factors produced during cytokinesis
activate the enzyme PLC, which catalyses the production
of Ins 1,4,5 P3 and Di Acyl Glycerol (DAG) from Ptd Ins 4,5
P2 as described previously.
During the cell differentiation of embryo, Ca2+ signaling
contributes to body polarity formation. This is clearly
proved by an experiment conducted in zebra fish embryo.
When anti Ins 1,4,5 P3 antibodies were incorporated,
dorso-ventral specification is altered in progeny.
Ca2+ signaling mechanism is extensively utilized during
the differentiation of specific cell types to form germ
layers on the gastrulation stage of embryo development.
During the formation of muscle, spontaneous Ca2+
transients control assembly of the contractile fibers. Ca2+
is also involved in the development of heart and nervous
system. Ca2+ dependent "Nuclear Factor of Activated Tcells" (NF-AT) helps to form cardiac septum (Atrioventricular) and semilunar heart valves, during the
development of heart. Role of Ca2+ transients with
varying frequencies on differentiation of developing
amphibian neurons was already established. Migration of
neuronal cells is another phenomenon, which is under the
control of Ca2+ signals. Primary stages of neuronal
reticulation leading to complex neuronal circuits of
developing brain are also controlled by Ca2+ control
mechanism.
In Neurons
Ca2+ plays a crucial role in neuronal function as well as in
information processing of brain. The prime control system
of our body itself is under the control of Ca2+. Let us
discuss how this happens.
Primarily Ca2+ plays 4 important functions in neurons,
viz., signal reception, signal transmission, synaptic
plasticity and long term memory.
Neurons have an input zone known as the "dendritic tree",
containing several spines, which are responsible for
receiving signals from pre-synaptic neuron. When a signal
is received (i.e., localized de/pre-polarization due to
Na+/K+ ion exchange devise) Ca2+ will enter the spines
either from outside (Through VOCs or ROCs) or from
internal stores like E.R. (through IP3 operated channels).
Hence IP3 and Ca2+ are involved in signal reception. If
both IP3 and Ca2+ come together from separate neural
signals, it can integrate the information, which is thought
to be responsible for learning and memory.
There is growing evidence that local Ca2+ transients are
involved in release of neurotransmitter vesicles. Ca2+ can
also modulate neuronal sensibility, since it can activate
K+ channels leading the efflux of K+ and depressing the
nervous transmission.
Neuronal spines are referred to as microprocessors with in
the brain, even though it has only 0.1 µm3 volume. It is
generally believed that Ca2+ signals with in the spines are
responsible for "neuronal plasticity", a phenomenon
leading to short-term memory.
Ca2+ is also involved in the consolidation of short-term
memory by the genes of nucleus. For triggering the gene
transcription far away from the spines, Ca2+ produce
other signaling components that migrate in to the
nucleus. Thus a long-term memory is created in the
genomic level. This is how Ca2+ controls permanent
memories.
In Vision Perception
When the rhodopsin is exposed to light, it dissociates to
form all-trans retinal and opsin- the visual protein. This
conversion also triggers a conformational change to
calcium ion channel in the membrane of rod cell. The
rapid inflow of Ca2+ triggers a nerve impulse, allowing
light to be perceived by the brain.
In Lymphocyte Proliferation
When a specific antigen binds to its receptor site
(epitope) on the surface of T or B Lymphocytes, Ins 1,4,5
P3 is produced by unknown mechanisms, which in turn
activate the release of Ca2+ from internal stores. In order
to compensate decrease in the concentration of Ca2+,
these internal stores absorb Ca2+ from ECF through SOCs.
This influx of Ca2+ though SOCs is often in the form of
regular Ca2+ oscillations that activates factors such as
NF-AT, which enter the nucleus causing interactions with
operon leading to switch-on of genes. Recently
mechanism of immuno-suppression by drugs, such as
cyclosporin is discovered to be, by the inhibition of Ca2+
dependent activation of NF-AT. This discovery highlights
the relevance of Ca2+ signaling in lymphocyte
proliferation.
In Necrosis and Apoptosis
There are several proteases located with in the cell and
some of them are sensitive to Ca2+. It is widely accepted
that Ca2+ sensitive proteases are directly involved in
random cell death - Necrosis.
Ca2+ control mechanism is more relevant in the case of
programmed cell death (PCD or apoptosis). The
phenomenon of PCD is involved during normal
development of tissue patterns as well as in pathological
conditions such as AIDS, Cancer and Alzheimer's disease.
Normally most of the stored Ca2+ resides with in the E.R.
lumen while the mitochondrion carries very little. This
high concentration of Ca2+ inside of E.R. acts not only as
store but they are essential for the synthesis and
processing of proteins. Stress signals activates shuttling of
Ca2+ from E.R. to mitochondria, which in turn leads to
'switch on' of the genes associated with PCD.
Most of the malignant cells contain a mutated proteinBcl2, which is solely responsible for it's anti-apoptotic
activities. Bcl2 prevents cell death, leading to
proliferation of cancer cells by modifying the way in
which E.R. and mitochondria handle Ca2+ i.e. Bcl2
inhibits the shuttling of Ca2+ from E.R. to mitochondria
and thereby prevents Ca2+ depletion of E.R. or Ca2+
excess of mitochondria.
Conclusion
There are many other disciplines of life where Ca2+
mediated control/signal mechanisms particularly appeals.
Ca2+ signals control every attributes of life, perhaps with
very few exceptions. These ubiquitous and unique
properties make Ca2+, an ion of Biological Cybernetics.
Regulation of life processes with control of Ca2+ signaling
is expected to become as important as genetic
engineering with in next few years [Look box 2]. There
are multitudes of functions controlled by Ca2+, ranging
from initiation of cell division or cellular apoptosis
requiring several hours to the sub-second secretory and
contractile events. It can be concluded here that the
cybernetics of Ca2+ signaling exhibits significant
variations in pattern and mechanism of recognition.
Acknowledgements
I thank Council of Scientific and Industrial Research, Govt
of India for the support via Junior Research Fellowship
Scheme.
Suggested Reading
[1] Clapham, 'Calcium Signaling', Cell, Vol 80, pp.259-268,
1995.
[2] Ramakalyan,A and J R Vengateswaran, 'Systems and
Control Engineering - Basic Concepts of Systems',
Resonance, Vol 4, No 3, pp.45-52, 1999.
[3] ‘Signal Transduction Knowledge Environment’- an
animated web tool to locate signal transduction key
pathways, accessible at Science website
[4] Nand K Shah, Tarvinder K Taneja and Seyed E Hasnain,
'Mitochondria can Power Cells to Life and Death - Role of
Mitochondria in Apoptosis', Resonance, Vol 5, No. 4,
pp.74-84, 2000,
[5] Berridge, M.J, 'Differential activation of transcription
factors induced by Ca2+ response, amplification and
duration', Nature, Vol 386 No 6627, pp.855-858,1997.
[6] Berridge, M. J., 'Neuronal calcium signaling', Neuron
Vol 21, pp.13-26, 1998.
[7] D. H. Mac Lennan and N. M. Green, 'Pumping ions',
Nature, Vol 405, p.633, 2000.
[8] J.W. Putney, Jr., 'Calcium Signaling: Up, Down, Up,
Down…. What's the Point?', Science, Vol 279, pp.191 192, 1998.
[9] 'Molecular Cell Biology' (4th edn.), Harvey Lodish and
others, W.H.Freeman and Co., Part 4, Chapter 20, 2002.
Box1. "The London Tape Water Mystery"
It was 120 years ago, when Sidney Ringer was studying the
contraction of isolated rat hearts… an experiment which
by today's standards would be considered unacceptably
sloppy, marked the beginning of the Ca2+ signaling saga
[Ringer, S., J. Physiol. Vol. 4, pp. 29-43, 1883]. In earlier
experiments, Ringer had suspended them in a saline
medium for which he admitted to having used London tap
water, which is hard: The hearts contracted beautifully.
When he proceeded to replace the tap water with
distilled water, he made a startling finding. The beating
of the hearts became progressively weaker, and stopped
altogether after about 20 min. To maintain contraction,
he found it necessary to add Ca2+ salts to the suspension
medium. Thus, Ringer had serendipitously discovered that
Ca2+ hitherto exclusively considered as a structural
element, was active in a tissue that has nothing to do
with bone or teeth, and performed there a completely
novel function: It carried the signal that initiated heart
contraction. It was a landmark observation, which should
have immediately aroused wide interest. Unexpectedly,
however, for decades it attracted no particular attention.
Occasionally, farsighted pioneers argued forcefully for a
messenger role of Ca2+, offering compelling experimental
evidence. Among them, one could quote L. V. Heilbrunn
[Heilbrunn, L. V., Physiol. Zool. Vol. 13, pp. 88-94, 1940],
who contracted frog muscle fibers by applying Ca2+ salts
to their cut ends, but not to their surfaces. Heilbrunn
correctly concluded that Ca2+ had diffused from the cut
ends to the internal contractile elements to elicit their
contraction. One could also quote K. Bailey [Bailey, K.
Biochem. J. Vol. 36, pp. 121-139, 1942], who showed that
the ATPase activity of myosin was strongly activated by
Ca2+ (but not by Mg2+), and concluded that the liberation
of Ca2+ in the neighborhood of the myosin controlled
muscle contraction. Clearly, enough evidence was there,
but only a handful of people had the vision to see it and
to foresee its far-reaching implications. Perhaps O. Loewy
can offer no better example of clairvoyance than the quip
in 1959: "Ja Kalzium, das ist alles!"
Box 2. Drugs regulating Ca2+ signaling.
Generally, calcium related chemotherapeutic agents that
are in widespread use today target outer membrane
channels, which allow calcium to enter the cells from
outside. Drugs like verampil block influx of Ca2+ through
channels, and hence can be effectively administered for
the relaxation of muscles lining blood vessels in patients
with hypertension, or in angina patients as it enhances
blood flow in coronary arteries. As these drugs reduces
level of Ca2+ in cardiac muscle tissues, heart became less
excitable with normal action potentials and hence
patients with dysrhythmias (the irregular heart beating
which may lead to heart attack) can be treated with this
kind of drugs.
Other group of drugs block calcium pumps or Ca2+
channels - both situated inside the cells - which release
Ca2+ from the internal stores. The pump blocker
thapsigargin and channel blockers such as xestospongin
have proved to be invaluable tools in unlocking the
secrets of calcium signaling pathways. However, agents of
this type have not been used therapeutically (although
thapsigargin is an ancient folk remedy for arthritis). The
reason for the lack of pump and internal channel blockers
in the therapeutic arsenal may be related to another role
played by calcium in the life of cells.
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Peer Review: A2417654 - Ca2+ : An Ion of Biological Cybernetics
Ca: an element of great importance
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