Intracellular acidosis differentially regulates
KV channels in coronary and pulmonary vascular muscle
MARCIE G. BERGER,1 CHRISTOPHE VANDIER,2 PIERRE BONNET,2
WILLIAM F. JACKSON,3 AND NANCY J. RUSCH1
1Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226;
2Laboratoire de Physiologie des Cellules Cardiaques et Vasculaires, Centre National de la
Recherche Scientifique Université Francois-Rabelais 6542, Tours, France; and 3Department
of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008
Berger, Marcie G., Christophe Vandier, Pierre Bonnet, William F. Jackson, and Nancy J. Rusch. Intracellular acidosis differentially regulates KV channels in coronary
and pulmonary vascular smooth muscle. Am. J. Physiol. 275
(Heart Circ. Physiol. 44): H1351–H1359, 1998.—Decreases in
intracellular pH (pHi ) potently dilate coronary resistance
arteries but constrict small pulmonary arteries. To define the
ionic mechanisms of these responses, this study investigated
whether acute decreases in pHi differentially regulate K1
currents in single vascular smooth muscle (VSM) cells isolated from rat coronary and pulmonary resistance arteries. In
patch-clamp studies, whole cell K1 currents were elicited by
10-mV depolarizing steps between 260 and 0 mV in VSM
cells obtained from 50- to 150-µm-OD arterial branches, and
pHi was lowered by altering the NH4Cl gradient across the
cell membrane. Progressively lowering pHi from calculated
values of 7.0 to 6.7 and 6.4 increased the peak amplitude of
K1 current in coronary VSM cells by 15 6 5 and 23 6 3% but
reduced K1 current in pulmonary VSM cells by 18 6 3 and
21 6 3%, respectively. These changes were reversed by
returning cells to the control pHi of 7.0 and were eliminated
by dialyzing cells with pipette solution containing 50 mmol/l
HEPES to buffer NH4Cl-induced changes in pHi. Pharmacological block of ATP-sensitive K1 channels and Ca21-activated
K1 channels by 1 µmol/l glibenclamide and 100 nmol/l
iberiotoxin, respectively, did not prevent changes in K1
current levels induced by acidotic pHi. However, block of
voltage-gated K1 channels by 3 mmol/l 4-aminopyridine
abolished acidosis-induced changes in K1 current amplitudes
in both VSM cell types. Interestingly, a-dendrotoxin (100
nmol/l), which blocks only select subtypes of voltage-gated K1
channels, abolished the acidosis-induced decrease in K1
current in pulmonary VSM cells but did not affect the
acidosis-induced increase in K1 current observed in coronary
VSM cells. These findings suggest that opposing, tissuespecific effects of pHi on distinct subtypes of voltage-gated K1
channels in coronary and pulmonary VSM membranes may
differentially regulate vascular reactivity in these two circulations under conditions of acidotic stress.
coronary arteries; pulmonary arteries; potassium channels;
pH; ammonium chloride; vascular smooth muscle
ALTHOUGH VASCULAR SMOOTH MUSCLE (VSM) cells effectively buffer changes in intracellular pH (pHi ) under
physiological conditions (1), intracellular acidosis may
be an important signal linking metabolic demand to
blood flow during periods of metabolic challenge. Levels
of pHi measured in smooth muscle cells range between
7.0 and 7.2 under resting conditions (35). In pathophysiological states, tissue extracellular pH (pHo ) may fall
by 0.5–0.7 unit (2, 18), producing large corresponding
declines in VSM pHi (26). Interestingly, decreases in
arterial pH are associated with circulation-specific, and
sometimes opposing, vasoactive effects in vivo. For
example, arterial acidosis stimulates vasodilation of
the coronary microcirculation (16), augmenting coronary blood flow to regions of ischemic myocardium. In
contrast, acidosis constricts the pulmonary vasculature
(35), thereby diverting blood toward better-ventilated
alveoli to improve ventilation-to-perfusion matching.
Given the complexity of pH effects on the vasculature, it is not surprising that the cellular mechanisms
by which acidosis exerts its contrasting influence on
arterial muscle tone in the coronary and pulmonary
circulations are incompletely understood. For example,
changes in pH may regulate the contractile state of
VSM by altering the release and reuptake of intracellular Ca21, the activity of Ca21-permeable ion channels,
or the Ca21 sensitivity of contractile proteins (2). A
pH-sensitive release of vasoactive factors from the
endothelium may further modulate vascular tone (2,
36). However, the primary vasoactive response to acidosis may rely on the pH-sensing properties of the VSM
cells (16, 36). In fact, in vivo studies indicate that the
effects of acidosis on arterial muscle tone may occur
independently of muscarinic, b-adrenergic, or sympathetic nervous system innervation and may be
mediated by mechanisms inherent to the blood vessel
wall (20).
In this respect, several lines of evidence suggest that
VSM K1 channels may mediate pH-induced alterations
of coronary and pulmonary vascular tone. First, changes
in pH reportedly modulate the open-state probability of
several K1 channel families, including high-conductance Ca21-sensitive (BKCa ), ATP-sensitive (KATP ), and
voltage-activated (KV ) K1 channel types (3, 7, 16).
These same K1 channel families are reported to regulate the membrane potential (Em ) of coronary and
pulmonary VSM cells (4, 23, 37). Second, VSM cells of
rat cerebral arterioles hyperpolarize when bath pH is
reduced from 7.3 to 6.8, indicating that factors that
regulate resting membrane potential may be pH sensitive (9). Third, metabolic stimuli, other than pH, have
been shown to differentially influence the activity of K1
channels expressed in different VSM cell membranes.
For example, although hypoxia attenuates the openstate probability of KV channels in cultured rat pulmonary VSM cells, KV channels in mesenteric VSM cells
are unaffected by hypoxia (37). These findings raise the
possibility that a tissue-specific regulation of vascular
K1 channel types also may be involved in mediating the
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
H1351
H1352
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
opposing effect of acidosis on coronary and pulmonary
arterial smooth muscle tone. However, little is known
about the mechanisms by which acidosis may regulate
K1 channels in coronary or pulmonary VSM cells or
about the identity of the single channels that may
represent the conducting pathways. Furthermore, because the ionic effects of changes in pHo and pHi
generally are not examined independently (2), the
membrane location of a pH-sensing site on the K1
channel protein remains hypothetical.
Hence, this study examined the effect of lowering pHi
on whole cell K1 currents in patch-clamped rat coronary and pulmonary VSM cells. We used a method
based on the imposition of transmembrane NH4Cl
gradients to induce a selective decrease in the pHi of the
VSM cells, which permitted a focused, detailed analysis
of the effect of this single intracellular metabolic stimulus on K1 channel current (12). As outlined below, our
results provide initial evidence that a differential effect
of pHi on distinct KV channel subtypes in coronary and
pulmonary VSM cell membranes may provide one
explanation for the opposing effect of acidosis on coronary and pulmonary vascular muscle tone.
MATERIALS AND METHODS
Experimental animals. Sprague-Dawley rats were obtained from Sasco/Charles River Laboratories (Wilmington,
MA) at 8–12 wk of age. On the day of experiments, rats were
killed with an overdose of pentobarbital sodium (60 mg/kg ip),
and the heart and lungs were immediately removed and
placed in a dissecting dish filled with ice-cold physiological
saline solution (PSS) (11). The heart was examined at 320
magnification to locate the proximal origin of the left anterior
descending coronary artery. This artery was followed and
dissected as it approached the cardiac apex, and arterial
branches of 50- to 150-µm OD were dissected free and placed
in a vial of cold PSS. The lung was similarly examined to
locate the origin of the right and left main pulmonary
arteries. These arteries were followed and dissected as they
approached the lung apex, and fourth- and fifth-division
arterial branches of 50- to 150-µm OD were dissected free and
placed in cold PSS.
Cell isolation. Enzymatic isolation of VSM cells was performed as described recently in detail for rat microvessels
(17). Briefly, small segments of rat coronary or pulmonary
artery were placed for 10 min in a 1-ml aliquot of PSS
containing 100 µM Ca21 and 1 mg/ml BSA. Vascular segments were then transferred to a fresh 1-ml aliquot of the
same solution containing 1.5 mg/ml papain and 1 mg/ml
dithioerythritol (Sigma Chemical, St. Louis, MO), which was
warmed to 37°C for 7–10 min. Segments were then incubated
for 10–15 min in 1 ml of PSS containing (in mg/ml) 2
collagenase, 0.5 elastase, and 1 soybean trypsin inhibitor
(Sigma Chemical). Single smooth muscle cells were released
from the vessels by gentle trituration, and the resulting cell
suspensions were stored at 4°C for up to 6 h. Only long,
smooth, optically refractive cells were used for patch-clamp
measurements.
Patch-clamp recording. Whole cell K1 currents were recorded in single coronary and pulmonary VSM cells using
standard pulse protocols and a patch-clamp station previously described in detail (28, 29). The pipette solution contained (in mmol/l) 50 NH4Cl, 1 Na2ATP, 5 HEPES, 1 MgCl2,
1 EGTA, 100 glutamate, and 104 K1 (pH 7.0). By including 50
mmol/l NH4Cl in the pipette solution dialyzing the cells and
maintaining the NH4Cl concentration in the bath (extracellular) solution at 15, 7.9, or 4 mmol/l, we changed pHi between
calculated values of 7.0, 6.7, and 6.4, respectively, according
to the following equation (12)
1
pHi 5 pHo 2 log ([NH1
4 ]i /[NH4 ]o)
1
where [NH1
4 ]i and [NH4 ]o represent intra- and extracellular
concentration of NH1
.
As
such, the bath solution composition
4
for a calculated pHi of 7.0 was (in mmol/l) 15 NH4Cl, 100
HEPES, 1 MgCl2, 2 CaCl2, 1 EGTA, 45 NaCl, 10 glucose, and
2.4 K1 (pH 7.5). The calculated level of ionized Ca21 in the
bath solution was 1 mmol/l. By adjusting the NaCl concentration, a constant osmolarity of 290 mosmol/l was maintained
when the NH4Cl concentration in the bath solution was
lowered from 15 mmol/l to 7.9 or 4 mmol/l to change pHi. This
method for intracellular acidification, illustrated in Fig. 1, is
based on the imposition of transmembrane NH4Cl gradients,
taking advantage of the fact that NH4Cl only traverses the
cell membrane in its uncharged form (NH3 ). This process
leaves residual H1 in the cell interior. Hence, increasing the
gradient for NH3 efflux by reducing the NH4Cl concentration
in the bath solution results in predictable levels of intracellular acidification. Because bath pH (pHo ) is stabilized with 100
mmol/l HEPES to buffer changes in the extracellular H1
concentration, but the pipette solution contains a low concentration of HEPES (5 mmol/l), pHi can be selectively and
predictably modified in patch-clamped cells subjected to the
whole cell or perforated-patch configurations. This method
has been validated extensively by Grinstein et al. (12) using a
pH-sensitive fluoroprobe in mouse peritoneal macrophages.
To compare the effect of intracellular acidosis on outward
K1 currents between coronary and pulmonary VSM cells,
families of whole cell K1 currents were generated by progressive 10-mV depolarizing steps (400-ms duration, 5-s inter-
Fig. 1. Method used to modify intracellular pH (pHi ) of vascular
smooth muscle (VSM) cells clamped in whole cell mode. By including
a high concentration of buffer (100 mmol/l HEPES) in the bath
solution and a low concentration of buffer (5 mmol/l HEPES) in the
pipette solution dialyzing the cell, changes in transmembrane NH4Cl
gradient can alter pHi without affecting extracellular pH (pHo ) (12).
Decreasing extracellular NH4Cl concentration ([NH4Cl]o ) from 15 to
7.9 or 4 mmol/l at a constant intracellular NH4Cl concentration of 50
mmol/l enhances diffusion of NH3 out of cell. Residual intracellular
H1 ions change estimated pHi from 7.0 to 6.7 or 6.4. [HEPES]o and
[HEPES]i, extra- and intracellular HEPES concentration; [NH1
4 ]o
1
and [NH1
4 ]i, extra- and intracellular NH4 concentration.
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
vals) from a constant holding potential of 260 mV to a peak
command potential of 0 mV. To permit stable current amplitudes, readings were taken 2–5 min after conversion to the
whole cell recording mode and several minutes after superfusion with a new bath solution. The peak current elicited at a
single membrane potential was defined as the average of
1,000 sample points encompassing the maximal current
point. Currents were measured in cells sequentially exposed
to 15 mmol/l NH4Cl bath solution (calculated pHi 7.0) and
then to 7.9 mmol/l NH4Cl bath solution (calculated pHi 6.7) or
4 mmol/l NH4Cl bath solution (calculated pHi 6.4) (12). Cells
were returned to the 15 mmol/l NH4Cl bath solution to
examine reversibility of pHi-induced changes in current
amplitude. Trials in each bath solution were performed in
triplicate and averaged together to estimate peak current
amplitudes. Membrane capacitance was estimated in each
cell by integrating capacitive currents generated by 10-mV
hyperpolarizing pulses after electronic cancellation of the
pipette-patch capacitance, and peak K1 current amplitudes
were expressed in picoamperes per picofarad to normalize for
differences in cell membrane area between isolated vascular
myocytes (29). In some trials the bath solution contained 100
nmol/l iberiotoxin (IBTX) to provide pharmacological block of
BKCa channels, 1 µmol/l glibenclamide to inhibit KATP channels, 3 mmol/l 4-aminopyridine (4-AP) to antagonize KV
channels (23), 100 nmol/l a-dendrotoxin (a-DTX) to selectively inhibit KV1.1, KV1.2, and KV1.6 channels (6) or 1 µmol/l
nifedipine to block L-type Ca21 channels (24).
Drugs. All drugs were obtained from Sigma Chemical,
except IBTX, which was obtained from Research Biochemicals International (Natick, MA). Drugs were reconstituted as
concentrated stock solutions for direct dilution into the bath
solution. Glibenclamide was dissolved as a 10 mM stock in
0.1 M NaOH. Nifedipine was reconstituted as a 10 mM stock
solution in 70% ethanol. 4-AP was dissolved as 1 M aqueous
stock solution in distilled H2O, buffered to pH 7.4 with HCl.
IBTX and a-DTX were dissolved as 100 µM stock solutions in
distilled H2O. Addition of the drugs did not significantly affect
the pH of the bath solution and resulted in #0.01% dilution of
bath constituents.
Statistics. All averaged data are expressed as means 6 SE.
Statistical comparisons between groups were made with
one-way repeated-measures ANOVA with subsequent Duncan’s test. Significance was accepted at P , 0.05.
RESULTS
Intracellular acidosis differentially regulates outward current in coronary and pulmonary VSM cells.
Representative traces in Fig. 2, A and B, show that
incremental 10-mV depolarizing steps from 260 to
0 mV (at pHi 7) elicited families of outward current in
single coronary and pulmonary VSM cells, respectively.
Between the two cell types, outward currents observed
in coronary VSM cells at pHi 7 generally exhibited a
higher component of noisy current, although current
appearance, kinetics, and density varied significantly
even between cells from the same resistance artery.
Under these conditions, peak current density at 0 mV
averaged 2.20 6 0.15 pA/pF in coronary VSM cells
(range 0.32–7.44 pA/pF, n 5 61) and 6.48 6 0.8 pA/pF
in pulmonary VSM cells (range 1.54–18.08 pA/pF, n 5
65). Cell capacitance in the same cells averaged 17.4 6
0.6 and 18.7 6 2.3 pF, respectively. Although the
enzymatic isolation of cells is a potential cause of
variability in ion current levels, heterogeneity of chan-
H1353
Fig. 2. A and B: effect of changing pHi on outward current in
coronary and pulmonary VSM cells, respectively. Progressive 10-mV
depolarizing steps from 260 to 0 mV elicited outward currents in
both cell types. Representative traces demonstrate that reducing pHi
from a calculated level of 7.0 to 6.4 reversibly increased outward
current in coronary VSM cells but reversibly decreased outward
current in pulmonary VSM cells. Current-voltage (I-V) relationships
show effect of lowering pHi from 7.0 (pre) to 6.4 and returning to pHi 7
(post) on K1 current density in coronary and pulmonary cells (n 5 10
and 9, respectively). C and D: I-V relationships for coronary and
pulmonary VSM cells, respectively, serially exposed to pHi 7, 6.7, and
7 (n 5 7 and 8, respectively). * Current density at acidotic pHi was
significantly different from that measured at initial pHi 7 at same
membrane potential (Em ). † Current density at acidotic pHi was
significantly different from that measured on return to pHi 7 at same
Em. NH4Cl gradient was 50 mM in pipette and 15 mM in bath to
establish control pHi 7.0, 50 mM in pipette and 7.9 mM in bath to
lower pHi to 6.7, and 50 mM in pipette and 4 mM in bath to provide
pHi 6.4.
nel currents between cell populations also has been
documented in detail by other laboratories (4, 21).
Regardless of their initial level, outward currents in
coronary and pulmonary VSM cells responded predictably to NH1
4 -induced decreases in pHi. The original
traces in Fig. 2A show that reducing the NH4Cl concentration in the bath solution from 15 to 4 mmol/l to lower
the calculated pHi from 7 to 6.4 increased the amplitude of voltage-activated outward current in coronary
VSM cells. This effect was reversed on return to pHi 7.
In contrast, Fig. 2B shows that exposing pulmonary
VSM cells to a similar change in NH4Cl gradient to
induce intracellular acidosis reversibly attenuated outward current amplitudes. The corresponding currentvoltage (I-V) curves for coronary and pulmonary VSM
H1354
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
cells in Fig. 2, A and B, respectively, further demonstrate these relationships between outward current
amplitude, Em, and pHi. Outward current amplitudes
reversibly increased in coronary VSM cells during
intracellular acidification, and the current density elicited at 0 mV was 23 6 3% higher at pHi 6.4 than at pHi
7 (n 5 10). In pulmonary VSM cells (Fig. 2B) the
reduction in outward current accompanying intracellular acidosis reversibly depressed the I-V curve at more
positive potentials, and outward current density at 0
mV declined by 21 6 3% when the calculated pHi was
lowered from 7 to 6.4 (n 5 9). A smaller decrease in
predicted pHi from 7 to 6.7 also reversibly modulated
the level of voltage-elicited outward current in coronary
and pulmonary VSM cells, as shown by the I-V relationships in Fig. 2, C and D, respectively. Lowering pHi
from 7 to 6.7 reversibly increased current density at
0 mV by 15 6 5% in coronary VSM cells (n 5 7). In
pulmonary VSM cells a similar decline in pHi to 6.7
reversibly decreased outward current elicited at more
positive voltages and reduced current density at 0 mV
by 18 6 3% (n 5 8). In other experiments the K1
selectivity of outward currents obtained at calculated
pHi of 7.0 and 6.4 was verified by performing tail
current analysis at external K1 concentrations ([K1]o )
of 5, 10, and 30 mmol/l. With the use of a published
voltage protocol (28), the average reversal potential
obtained for each log [K1]o/[K1]i (where [K1]i is intracellular K1 concentration) was compared with the reversal potential predicted for a K1-selective channel by the
Nernst equation (15). Under these conditions, reversal
potentials in coronary and pulmonary VSM cells (n 5
5–10 cells) were not different from those predicted by
the Nernst equation for a purely K1-selective ion
Fig. 3. Effect of altering transmembrane
NH4Cl gradient, in presence of elevated
[HEPES]i, on whole cell K1 currents in
coronary (A) and pulmonary (B) VSM
cells. Currents were elicited by 10-mV
depolarizing steps from 260 to 0 mV. In
cells dialyzed with pipette solution containing 50 mmol/l HEPES to minimize changes
in pHi, alternating [NH4Cl]o between 15
and 4 mmol/l had no effect on K1 current
amplitudes. I-V relationships indicate that
lowering [NH4Cl]o from 15 to 4 mmol/l did
not affect K1 current densities measured
in coronary (n 5 6) or pulmonary (n 5 6)
VSM cells dialyzed with 50 mmol/l HEPES.
channel, implicating K1 as the primary charge carrier
for outward current observed at both levels of pHi.
Further experiments demonstrated that the L-type
Ca21 channel antagonist nifedipine (1 µmol/l) did not
prevent NH4Cl-induced changes in outward current
levels in either VSM cell type.
High intracellular HEPES prevents NH4 Cl-induced
changes in K1 current. Subsequent experiments verified that the NH4Cl-induced effects on outward current
in coronary and pulmonary VSM cells were associated
with pHi change and did not occur as a consequence of
the NH4Cl method distinct from pH manipulation. In
these experiments the same pulse protocol of 10-mV
steps was used to elicit K1 currents between 260 and
0 mV. However, the pipette solution dialyzing the cells
contained 50 mmol/l (rather than 5 mmol/l) HEPES, to
buffer the intracellular H1 generated when the transmembrane NH4Cl gradient was increased. The intracellular osmolarity was maintained at 290 mosmol/l by
substitution of HEPES for glutamate in the pipette
solution. Under these conditions, Fig. 3A shows that
when the NH4Cl concentration in the bath solution was
changed from 15 to 4 mmol/l to generate intracellular
H1, the level of K1 current was not altered in coronary
VSM cells dialyzed with 50 mmol/l HEPES, although it
was enhanced in similar cells dialyzed with only
5 mmol/l HEPES (Fig. 2A). Similarly, Fig. 3B shows
that outward current elicited in pulmonary VSM cells
dialyzed with 50 mmol/l HEPES was not affected by
reducing the NH4Cl concentration in the bath solution,
whereas a similar intervention significantly attenuated
outward current in pulmonary VSM cells dialyzed with
5 mmol/l HEPES (Fig. 2B). The average I-V relationships obtained from coronary and pulmonary VSM cells
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
dialyzed with 50 mmol/l HEPES (Fig. 3) confirm that
outward current did not change in either VSM cell type
in response to lowering [NH1
4 ]o from 15 to 4 mmol/l.
Hence, NH4Cl-induced changes in the level of pHi,
rather than nonspecific effects of the NH4Cl technique,
apparently triggered the contrasting changes in coronary and pulmonary VSM currents shown in Fig. 2.
Identification of the K1 channel type regulated by
intracellular acidosis. The identity of the conducting
pathways for K1 current activated at the control pHi of
7.0 was explored using specific pharmacological K1
channel blockers. A similar approach was used to
identify the K1 channel types underlying the pHiinduced changes in outward current levels. On the
basis of reports suggesting that several K1 channel
types are coexpressed in coronary and pulmonary VSM
membranes (3, 4, 23), we selected 1 µmol/l glibenclamide, 100 nmol/l IBTX, and 3 mmol/l 4-AP to antagonize KATP, BKCa, and KV channels, respectively (23). The
I-V relationships in Fig. 4, A and B, show that, under
our control conditions of pHi 7, addition of 1 µmol/l
glibenclamide to the bath solution to block KATP channels did not alter K1 current levels measured in
coronary or pulmonary VSM cells, respectively. The
sample traces in Fig. 4, C and D, and the corresponding
I-V curves plotted beneath, further demonstrate that
glibenclamide did not prevent the opposing regulation
of K1 current amplitude in coronary and pulmonary
VSM cells triggered by lowering pHi from calculated
levels of 7 to 6.4 (n 5 10 and 8, respectively).
Figure 5, A and B, shows a similar analysis examining the potential contribution of BKCa channels to
outward current in coronary and pulmonary VSM cells,
respectively. Selective block of BKCa channels by 100
nmol/l IBTX revealed a significant component of BKCa
current in both VSM cell types at the control pHi of 7.
IBTX reduced K1 current density elicited at 0 mV by
22 6 5 and 13 6 5% in coronary and pulmonary VSM
cells, respectively. However, the representative traces
and average I-V relationships in Fig. 5, C and D, show
that IBTX failed to block the acidosis-induced increase
in K1 current amplitude in coronary VSM cells, and
IBTX also failed to prevent the contrasting acidosisinduced attenuation of K1 current in pulmonary VSM
cells (n 5 7 and 6, respectively).
In contrast, the I-V relationships in Fig. 6, A and B,
show that a 4-AP-sensitive K1 current was the predominant component of outward current observed at pHi 7
in coronary and pulmonary VSM cells, respectively. At
the calculated control pHi of 7, 3 mmol/l 4-AP reduced
K1 current amplitudes elicited at 0 mV by 62 6 8 and
78 6 3% in coronary and pulmonary VSM cells (n 5 7
and 9, respectively). In a subset of cells, increasing the
concentration of 4-AP to 5 mmol/l did not cause additional block (n 5 5 each). Because intracellular acidosis
may enhance 4-AP potency (31), we next used the
maximal effective dose of 3 mmol/l 4-AP to examine the
contribution of KV channels to pHi-sensitive currents.
The sample traces in Fig. 6, C and D, from coronary and
pulmonary VSM cells, respectively, illustrate that 3
mmol/l 4-AP prevented the pHi-induced changes in K1
H1355
Fig. 4. A and B: glibenclamide (1 µmol/l) did not affect I-V relationships for macroscopic K1 current observed at a calculated pHi of 7 in
coronary and pulmonary VSM cells, respectively (n 5 6 and 5).
C: actual traces and I-V curve for averaged data show that glibenclamide did not block the reversible increase in K1 current density
associated with lowering pHi from 7 to 6.4 in coronary VSM cells (n 5
10). D: current traces and I-V relationship for averaged data show
that glibenclamide did not block the reversible decrease in K1 current
density associated with lowering pHi from 7 to 6.4 in pulmonary VSM
cells (n 5 8). * Current level at pHi 6.4 was significantly different from
that measured at initial pHi 7 at same Em. † Current level at pHi 6.4
was significantly different from that measured on return to pHi 7 at
same Em.
current amplitude observed in VSM cells when pHi was
reduced from 7 to 6.4. The corresponding I-V relationships beneath the actual tracings confirm that 4-AP
prevented changes in K1 current levels in the coronary
and pulmonary VSM cells in response to intracellular
acidosis (n 5 6 and 7, respectively).
a-DTX selectively prevents NH4 Cl-induced decreases
in pulmonary arterial K1 current. To further identify
which KV channel subtypes account for the 4-AP- and
pHi-sensitive K1 current component in coronary and
pulmonary VSM cells, we studied the effect of a-DTX on
the opposing regulation of K1 current by intracellular
acidosis. a-DTX is reported to preferentially block a
subset of KV channels, including KV1.1, KV1.2, and
KV1.6, with high affinity (6). The I-V relationships in
Fig. 7, A and B, show the sensitivities of coronary and
pulmonary VSM K1 currents, respectively, to the blocking action of this peptide toxin. At the baseline pHi of 7,
the addition of 100 nmol/l a-DTX reduced the level of
H1356
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
preferential blocker of distinct KV channel subtypes,
prevented acidosis-induced changes in K1 current only
in pulmonary VSM cells. To our knowledge, these data
provide the first detailed evidence to suggest that the
differential regulation of distinct KV channel subtypes
by intracellular acidosis may be a mechanism for the
opposing effect of reduced pHi on coronary and pulmonary arterial muscle cell excitability.
Evidence for pH regulation of K1 channels. A number
of recent reports suggest that several types of membrane K1 channels may be sensitive to pHi in nonvascular cell types (7, 19, 22, 25). Kume et al. (19) observed
that lowering pHi from 7.4 to 7.0 in rabbit tracheal
smooth muscle cells decreased the open-state probability of BKCa channels. Similarly, Copello et al. (7)
reported a decline in BKCa channel activity in Necturus
gall bladder epithelial cells with intracellular acidosis.
KATP channels also may be modulated by pHi. In
inside-out patches excised from rat pancreatic b-cells,
Misler et al. (22) showed that KATP channel activity
decreased in the presence of 50–100 µmol/l ATP as pHi
was lowered from 7.3 to 6.25. However, applying a
similar protocol for intracellular acidification to b-cells
from the mouse pancreas, Proks et al. (25) observed an
enhanced open-state probability of KATP channels. Taken
Fig. 5. A and B: I-V relationships showing effect of 100 nmol/l
iberiotoxin (IBTX) on averaged K1 current density measured at a
calculated pHi of 7 in coronary and pulmonary VSM cells, respectively (n 5 6 and 7). * IBTX significantly reduced K1 current density
at indicated Em. C: actual traces and I-V curve for averaged data
show that IBTX did not block the reversible increase in K1 current
density associated with lowering pHi from 7 to 6.4 in coronary VSM
cells (n 5 7). D: current traces and I-V relationship for averaged data
show that IBTX did not block the reversible decrease in K1 current
density associated with lowering pHi from 7 to 6.4 in pulmonary VSM
cells (n 5 6). * Current level at pHi 6.4 was significantly different from
that measured at initial pHi 7 at same Em. † Current level at pHi 6.4
was significantly different from that measured on return to pHi 7 at
same Em.
K1 current density activated at 0 mV by 6 6 9% in
coronary VSM cells (n 5 7) and by 23 6 3% in
pulmonary VSM cells (n 5 6). This inhibition was more
pronounced and only significant in the pulmonary VSM
cells. Importantly, the sample traces and corresponding
I-V curve in Fig. 7C illustrate that 100 nmol/l a-DTX
failed to prevent the enhancement of coronary VSM K1
current associated with lowering pHi from 7 to 6.4. In
contrast, a-DTX blocked the acidosis-induced decrease
of K1 current in pulmonary VSM cells exposed to the
same conditions (Fig. 7D).
DISCUSSION
The new findings of this study demonstrate that
1) lowering pHi from a calculated value of 7.0 to 6.7 or
6.4 produces a graded activation of whole cell K1
current in coronary VSM cells but a graded depression
of macroscopic K1 current in pulmonary VSM cells,
2) the pHi-induced changes in K1 current amplitude in
coronary and pulmonary VSM cells are blocked by
4-AP, an inhibitor of KV channels, and 3) a-DTX, a
Fig. 6. A and B: I-V relationships showing effect of 3 mmol/l
4-aminopyridine (4-AP) on averaged K1 current density measured at
a calculated pHi of 7 in coronary and pulmonary VSM cells, respectively (n 5 7 and 9). * 4-AP significantly lowered current level at
indicated Em. C: actual traces and I-V relationship for averaged data
show that 4-AP prevented the increase in K1 current density
associated with lowering pHi from 7 to 6.4 in coronary VSM cells (n 5
6). D: current traces and I-V relationship for averaged data show that
4-AP blocked the decrease in K1 current density associated with
lowering pHi from 7 to 6.4 in pulmonary VSM cells (n 5 7).
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
Fig. 7. A and B: I-V relationships showing effect of 100 nmol/l
a-dendrotoxin (a-DTX) on averaged K1 current density measured at
a calculated pHi of 7 in coronary and pulmonary VSM cells, respectively (n 5 7 and 6). * a-DTX significantly reduced K1 current density
at indicated Em. C: current traces and I-V relationship for averaged
data demonstrate that a-DTX did not block the increase in K1
current density associated with lowering pHi from 7 to 6.4 in coronary
VSM cells (n 5 7). D: actual traces and I-V curve for averaged data
demonstrate that a-DTX prevented the decrease in K1 current
density associated with lowering pHi from 7 to 6.4 in pulmonary VSM
cells (n 5 8). * Current level at pHi 6.4 was significantly different from
that measured at initial pHi 7 at same Em. † Current level at pHi 6.4
was significantly different from that measured on return to pHi 7 at
same Em.
together, these findings suggest the plausibility of a
direct effect of intracellular acidosis on K1 channel
gating in membranes from several types of nonvascular
cells and also imply that several K1 channel types may
show pHi sensitivity as a property.
However, only one recent study has directly examined the effect of acidosis on K1 channel function in
VSM cells. Ahn and Hume (3) observed that superfusing canine pulmonary VSM cells with 20 mmol/l sodium butyrate to lower pHi enhanced a 4-AP-sensitive
KV current by ,20% over control levels. Although this
finding seemingly contradicts our observation that KV
current is diminished by lowering pHi in rat pulmonary
VSM cells, there are some methodological differences
between the two studies that should be considered.
First, Ahn and Hume used VSM cells from second- to
fourth-generation canine pulmonary arteries in their
investigation, whereas we used VSM cells from smaller
fourth- to fifth-order rat pulmonary arteries. The de-
H1357
scription of morphological and functional diversity
within the pulmonary circulation by Archer and colleagues (4) suggests that K1 channel regulation may
differ between arterial divisions, and the response of
K1 channels to metabolic stimuli in smaller arteries
may be different from that in larger vessels. Second,
species-specific differences in pHi gating of pulmonary
VSM K1 channels also may exist, as described for the
opposing effect of intracellular acidosis on KATP channels in pancreatic b-cells of the rat and mouse (22, 25).
Third, our study used a standard whole cell recording
method employing NH4Cl gradients to alter pHi,
whereas Ahn and Hume used the perforated-patch
technique and sodium butyrate to decrease pHi in
pulmonary VSM cells. Because pHi levels were not
directly measured in either study and different patchclamp configurations were used, the relative pHi
changes between the two studies may not be comparable.
Mechanisms for differential regulation of KV channels
by intracellular acidosis. Our new findings provide
initial evidence that pHi may act at an intracellular site
to differentially regulate KV channels in coronary and
pulmonary VSM cells. In the present study, pharmacological block of BKCa channels by IBTX and of KATP
channels by glibenclamide failed to prevent the increase in coronary or the decrease in pulmonary VSM
K1 current accompanying intracellular acidosis. After
selectively blocking KV channels in these VSM cell
membranes with 3 mmol/l 4-AP (3, 23, 31), however, we
no longer observed changes in outward current amplitude related to lowering levels of pHi in either cell type.
In contrast, blocking KV1.1, KV1.2, and KV1.6 channels
with a-DTX (6) prevented only the pulmonary VSM K1
current response to intracellular acidosis. These observations, indicating that an a-DTX-sensitive channel
provides the pHi-sensitive K1 current in pulmonary,
but not in coronary, VSM cells, suggest that the opposing effects of pHi on distinct 4-AP-sensitive K1 channels
may constitute a novel mechanism to differentially
regulate VSM excitability in the coronary and pulmonary circulations under conditions of acidotic stress.
Our results, however, should not be interpreted to
suggest that other K1 channel types, such as BKCa or
KATP channels, are strictly insensitive to regulation by
pHi. Because our pipette solution contained 1 mmol/l
EGTA to buffer intracellular Ca21, K1 current through
BKCa channels represented only a relatively small
component of the whole cell K1 current measured in
our coronary and pulmonary VSM cells. Hence, small
pHi-induced changes in BKCa current amplitudes may
not have been detected in this study. Similarly, the
inclusion of physiological levels of ATP (1 mmol/l) in the
pipette solution also likely minimized the contribution
of KATP channels to whole cell current, as evidenced by
the absence of a glibenclamide-sensitive K1 conductance in coronary and pulmonary VSM cells. Indeed,
Ishizaka and Kuo (16) recently reported that 5 µmol/l
glibenclamide attenuated the vasodilator response of
isolated, bovine coronary arterioles to extracellular
acidosis (pHo 7.0–7.3), suggesting a potential role for
H1358
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
KATP channels in mediating the coronary vasodilator
response to this type of acidotic challenge. In contrast,
given their low level of baseline activity in pulmonary
VSM cells (14), it appears unlikely that closure of
vascular KATP channels could signal acidosis-induced
vasoconstriction in pulmonary resistance arteries.
Clearly, a detailed single-channel approach will be
required to resolve whether changes in pHi and pHo
levels directly influence the unitary properties of distinct K1 channel types expressed in these two vascular
beds.
The findings of the present study argue that protonation sites on the cytoplasmic side of the KV channel
protein may be involved in pH sensing. In this regard,
the protonation of intracellular residues on the poreforming a-subunit may elicit conformational changes
affecting channel activity (5). Alternatively, H1 may
bind to the membrane-associated b-subunits, thereby
altering their interaction with a-subunits to influence
KV channel gating (5, 32). Importantly, our data indicate that KV channels showing different pharmacological profiles mediate pHi-sensitive K1 current in coronary and pulmonary VSM cells. Thus the differential
expression of KV channel subtypes in coronary and
pulmonary VSM membranes may explain the divergent gating response of KV channels to changes in pHi
in these vasculatures. Notably, the localization of transcript for the KV1.2 channel subtype to aorta and
pulmonary artery, but not to renal artery or portal vein,
has provided initial evidence for circulation-specific
expression of KV channel genes (13, 27, 34). Because of
these complexities, uncovering the precise mechanisms
by which pHi divergently regulates K1 channels in
coronary and pulmonary VSM cells may require detailed examination of proton interactions with channel
subtypes and subunits in heterologous expression systems.
Methodological considerations. Several different techniques have been used to induce intracellular acidosis
in VSM cell or tissue preparations in earlier studies (1,
2, 35). Traditionally, levels of pHi have been lowered by
equilibrating the extracellular solution with gases containing elevated levels of CO2 (1, 2, 8, 10, 33, 35).
However, this method does not permit predicted levels
of pHi to be achieved, and interpretation of findings is
complicated by the fact that elevated CO2 levels per se
may directly regulate VSM excitability (1, 20). For
these reasons, changing the external concentration of
the permeable weak conjugate base NH3 in the presence of a large reservoir of the intracellular H1 equivalent NH1
4 has been used to produce calculated reductions in the level of pHi in VSM preparations. However,
although this technique has been carefully documented
(12), the quantitative relationship between NH4Cl transmembrane gradients and pHi values in this study was
not measured directly and should be regarded as
representing calculated, rather than absolute, pHi levels.
Regardless of this limitation, several control experiments in this study suggested that NH4Cl-induced
changes in pHi were indeed linked to changes in K1
current levels in coronary and pulmonary VSM cells.
First, the reversal potentials of voltage-elicited outward currents in this study showed high K1 selectivity
throughout the pHi range studied. Second, the L-type
Ca21 channel blocker nifedipine (24) did not prevent
NH4Cl-induced changes in whole cell outward current.
Therefore, acidosis-induced inhibition of inward Ca21
current, which could appear as a potentiation of outward current under our recording conditions, did not
contribute to NH4Cl-induced changes in current amplitudes. Additionally, including a high HEPES concentration (50 mmol/l) in the pipette solution to buffer intracellular H1 prevented the differential changes in K1
current levels induced by NH4Cl gradients in coronary
and pulmonary VSM cells. Thus a differential regulation of 4-AP-sensitive K1 channels by intracellular
acidosis may best explain the opposing changes in
outward current levels in coronary and pulmonary
VSM cells associated with increasing the transmembrane NH4Cl gradient.
Physiological relevance. Although investigators have
observed the opposing effects of acidosis on coronary
and pulmonary vascular tone for many years (8, 10, 16,
30, 33, 35), controversy remains as to whether acidosis
modulates coronary and pulmonary vascular tone at
intracellular or extracellular sites. Recent reports indicating that pHi levels in VSM cells may be particularly
sensitive to environmental acidosis, with 70–80% of
pHo change transmitted to the cytoplasm (26), suggest
that pHi represents a potential stimulus for modulating
vascular reactivity. Using the fluorescent indicator
carboxy-seminaphthorhodafluor to measure pHi in rat
coronary VSM cells, Ramsey et al. (26) measured a
decline in pHi from 6.97 to 6.56 as pHo was lowered
from 7.4 to 6.9. Although further studies in vascular
systems are required to clarify the physiological relevance of pHi-induced changes in KV current to the
contrasting regulation of coronary and pulmonary arterial tone, our patch-clamp findings are consistent with
a possible role for KV channels. Notably, an amplification of outward KV current in coronary VSM cells in
response to intracellular acidification would favor VSM
cell hyperpolarization and coronary vasodilation. Conversely, a decline of KV current in pulmonary VSM
membranes by intracellular acidosis would mediate
depolarization and constriction of small pulmonary
arteries. Furthermore, expression of distinct KV channels in coronary and pulmonary resistance arteries
may provide differential molecular targets for vasodilator therapies specific for these two circulations. A
detailed characterization of these KV channels and the
subsequent development of K1 channel-opening drugs
with site-specific actions could lead to significant therapeutic advances for pathologies in which blood flow to
the coronary and pulmonary vasculatures is diminished.
M. Berger was supported by National Heart, Lung, and Blood
Institute (NHLBI) Grants T32 HL-07729 and F33 HL-09798. N. J.
Rusch is supported by NHLBI Grants P01 HL-29587 and R01
HL-59238; W. F. Jackson is supported by NHLBI Grants R01
HL-09290 and R01 HL-32469. P. Bonnet was supported by the
ARTERIAL KV CHANNEL REGULATION BY ACIDOSIS
Ministere de l’Enseignement Superieur et de la Recherche and
Fondation pour la Recherche Medicale.
Address for reprint requests: N. J. Rusch, Dept. of Physiology,
Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.
Received 18 September 1997; accepted in final form 9 June 1998.
REFERENCES
1. Aalkjaer, C. Regulation of intracellular pH and its role in
vascular smooth muscle function. J. Hypertens. 8: 197–206,
1990.
2. Aalkjaer, C., and L. Poston. Effects of pH on vascular tension:
which are the important mechanisms? J. Vasc. Res. 33: 347–359,
1996.
3. Ahn, D. S., and J. R. Hume. pH regulation of voltagedependent K1 channels in canine pulmonary arterial smooth
muscle cells. Pflügers Arch. 433: 758–765, 1997.
4. Archer, S. L., J. M. C. Huang, H. L. Reeve, V. Hampl, S.
Tolarova, E. Michelakis, and E. K. Weir. Differential distribution of elecrophysiologically distinct myocytes in conduit and
resistance arteries determines their response to nitric oxide and
hypoxia. Circ. Res. 78: 431–442, 1996.
5. Breitwieser, G. E. Mechanisms of K1 channel regulation. J.
Membr. Biol. 152: 1–11, 1996.
6. Chandy, K. G., and G. A. Gutman. Voltage-gated potassium
channel genes. In: Handbook of Receptors and Channels, Ligandand Voltage-Gated Ion Channels, edited by A. North. Boca Raton,
FL: CRC, 1995, p. 1–71.
7. Copello, J., Y. Segal, and L. Reuss. Cytosolic pH regulates
maxi K1 channels in Necturus gall-bladder epithelial cells. J.
Physiol. (Lond.) 434: 577–590, 1991.
8. Daugherty, R. M., J. B. Scott, J. M. Dabney, and F. J. Haddy.
Local effects of O2 and CO2 on limb, renal, and coronary vascular
resistances. Am. J. Physiol. 213: 1102–1110, 1967.
9. Dietrich, H. H., and R. G. Dacey. Effects of extravascular
acidification and extravascular alkalinization on constriction
and depolarization in rat cerebral arterioles in vitro. J. Neurosurg. 81: 437–442, 1994.
10. Feinberg, H., A. Gerola, and L. N. Katz. Effect of changes in
blood CO2 level on coronary flow and myocardial O2 consumption.
Am. J. Physiol. 199: 349–354, 1960.
11. Gauthier-Rein, K. M., D. M. Bizub, J. H. Lombard, and N. J.
Rusch. Hypoxia-induced hyperpolarization is not associated
with vasodilation of bovine coronary resistance arteries. Am. J.
Physiol. 272 (Heart Circ. Physiol. 41): H1462–H1469, 1997.
12. Grinstein, S., R. Romanek, and O. D. Rotstein. Method for
manipulation of cytosolic pH in cells clamped in the whole cell or
perforated-patch configurations. Am. J. Physiol. 267 (Cell Physiol.
36): C1152–C1159, 1994.
13. Hart, P. J., K. E. Overturf, S. N. Russell, A. Carl, J. R. Hume,
K. M. Sanders, and B. Horowitz. Cloning and expression of a
KV1.2 class delayed rectifier K1 channel from canine colonic
smooth muscle. Proc. Natl. Acad. Sci. USA 90: 9659–9663, 1993.
14. Hasunuma, K., D. M. Rodman, and I. F. McMurtry. Effects of
K1 channel blockers on vascular tone in the perfused rat lung.
Am. Rev. Respir. Dis. 144: 884–887, 1991.
15. Hille, B. (Editor). Ionic Channels of Excitable Membranes (2nd
ed.). Sunderland, MA: Sinauer, 1992, p. 11–15 and 351–352.
16. Ishizaka, H., and L. Kuo. Acidosis-induced coronary arteriolar
dilation is mediated by ATP-sensitive potassium channels in
vascular smooth muscle. Circ. Res. 78: 50–57, 1996.
17. Jackson, W. F., J. M. Huebner, and N. J. Rusch. Enzymatic
isolation and characterization of single vascular smooth muscle
cells from cremasteric arterioles. Microcirculation 4: 35–50,
1996.
18. Kitakaze, M., S. Takashima, H. Funaya, T. Minamino, K.
Node, Y. Shinozaki, H. Mori, and M. Hori. Temporary acidosis
during reperfusion limits myocardial infarct size in dogs. Am. J.
Physiol. 272 (Heart Circ. Physiol. 41): H2071–H2078, 1997.
H1359
19. Kume, H., K. Takagi, T. Satake, H. Tokuno, and T. Tomita.
Effects of intracellular pH on calcium-activated potassium channels in rabbit tracheal smooth muscle. J. Physiol. (Lond.) 424:
445–457, 1990.
20. Ledingham, I. M., T. I. McBride, J. R. Parratt, and J. P.
Vance. The effect of hypercapnia on myocardial blood flow and
metabolism. J. Physiol. (Lond.) 210: 87–105, 1970.
21. Majesky, M. W., C. M. Giachelli, M. A. Reidy, and S. M.
Schwartz. Rat carotid neointimal smooth muscle cells reexpress
a developmentally regulated mRNA phenotype during repair of
arterial injury. Circ. Res. 71: 759–768, 1992.
22. Misler, S., K. Gillis, and J. Tabcharani. Modulation of gating
of a metabolically regulated, ATP-dependent K1 channel by
intracellular pH in b-cells of the pancreatic islet. J. Membr. Biol.
109: 135–143, 1989.
23. Nelson, M. T., and J. M. Quayle. Physiological roles and
properties of potassium channels in arterial smooth muscle.
Am. J. Physiol. 268 (Cell Physiol. 37): C799–C822, 1995.
24. Pfaffendorf, M., M.-J. Mathy, and P. A. van Zwieten. In vitro
effects of nifedipine, nisoldipine, and lacidipine on rat isolated
coronary small arteries. J. Cardiovasc. Pharmacol. 21: 496–502,
1993.
25. Proks, P., M. Takano, and F. M. Ashcroft. Effects of intracellular pH on ATP-sensitive K1 channels in mouse pancreatic b-cells.
J. Physiol. (Lond.) 475: 33–44, 1994.
26. Ramsey, J., C. Austin, and S. Wray. Differential effects of
external pH alteration on intracellular pH in rat coronary and
cardiac myocytes. Pflügers Arch. 428: 674–676, 1994.
27. Roberds, S. L., and M. M. Tamkun. Cloning and tissue-specific
expression of five voltage-gated potassium channel cDNAs expressed in rat heart. Proc. Natl. Acad. Sci. USA 88: 1798–1802,
1991.
28. Rusch, N. J., R. G. DeLucena, T. A. Wooldridge, S. K.
England, and A. W. Cowley, Jr. A Ca21-dependent K1 current
is enhanced in arterial membranes of hypertensive rats. Hypertension 19: 301–307, 1992.
29. Rusch, N. J., and A. M. Runnells. Remission of high blood
pressure reverses arterial potassium channel alterations. Hypertension 23: 941–945, 1994.
30. Shapiro, B. J., D. H. Simmons, and L. M. Linde. Pulmonary
hemodynamics during acute acid-base changes in the intact dog.
Am. J. Physiol. 210: 1026–1032, 1966.
31. Stephens, G. J., J. C. Garratt, B. Robertson, and D. G.
Owen. On the mechanism of 4-aminopyridine action on the
cloned mouse brain potassium channel mKV1.1. J. Physiol.
(Lond.) 477: 187–196, 1994.
32. Uebele, V. N., S. K. England, A. Chaudhary, M. M. Tamkun,
and D. J. Snyders. Functional differences in Kv1.5 currents
expressed in mammalian cell lines are due to the presence of
endogenous Kvb2.1 subunits. J. Biol. Chem. 271: 2406–2412,
1996.
33. Viles, P. H., and J. T. Shepherd. Relationship between pH, PO2,
and PCO2 on the pulmonary vascular bed of the cat. Am. J.
Physiol. 215: 1170–1176, 1968.
34. Wang, J., M. Juhaszova, L. J. Rubin, and X.-J. Yuan.
Hypoxia inhibits gene expression of voltage-gated K1 channel
a-subunits in pulmonary artery smooth muscle cells. J. Clin.
Invest. 100: 2347–2353, 1997.
35. Wray, S. Smooth muscle intracellular pH: measurement, regulation, and function. Am. J. Physiol. 254 (Cell Physiol. 23): C213–
C225, 1988.
36. Yamaguchi, K., T. Takasugi, H. Fujita, M. Mori, Y. Oyamada, K. Suzuki, A. Miyata, T. Aoki, and Y. Suzuki. Endothelial modulation of pH-dependent pressor response in isolated
perfused rabbit lungs. Am. J. Physiol. 270 (Heart Circ. Physiol.
39): H252–H258, 1996.
37. Yuan, X.-J., W. F. Goldman, M. L. Tod, L. J. Rubin, and M. P.
Blaustein. Hypoxia reduces potassium currents in cultured rat
pulmonary but not mesenteric arterial myocytes. Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8): L116–L123, 1993.