1124 Interrelation Between Pinacidil and Intracellular ATP Concentrations on Activation of the ATP-Sensitive K' Current in Guinea Pig Ventricular Myocytes Keiko Nakayama, Zheng Fan, Fumiaki Marumo, and Masayasu Hiraoka Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 The patch-clamp technique was used to study the relation between pinacidil and intracellular ATP concentration ([ATP]i) on the activation of the outward K' current in guinea pig ventricular myocytes. Pinacidil shortened the action potential duration, exhibiting stronger effect at 2 mM [ATP]i than at 5 mM [ATP]i. Pinacidil at 5 ,M or higher concentrations activated the time-independent outward current at potentials positive to -80 mV, and the pinacidil-activated current was suppressed by increasing [ATP]i from 2 to 5 mM. The dose-response curve of pinacidil at different [ATP]i showed a shift to the right and a depression of the maximum response at increased [ATP]j. The pinacidil-induced shortening of the action potential duration and outward current were inhibited by application of 0.3-1.0 JpM glibenclamide. In single-channel current recordings, pinacidil activated the intracellular ATP-sensitive K' channel current without changing the unitary amplitude, and increased open probability of the channel, an effect dependent on [ATP]i. The pinacidil-activated single-channel current was blocked by glibenclamide. These results prove the notion that pinacidil activates the ATPsensitive K' channel current, which explains the action potential shortening in cardiac cells after application of pinacidil. (Circulation Research 1990;67:1124-1133) R ecently, considerable attention has been paid to a group of agents -which includes cromakalim (BRL 34915), nicorandil, and pinacidil -because of their unique abilities to produce a potent vasodilatory action and to promote action on the potassium channel of vascular smooth muscle cells. Because of the latter action, these agents are called potassium channel openers.' All of these agents also affect cardiac membranes to shorten the action potential duration (APD) and, sometimes, to hyperpolarize the resting membrane potential.2-5 Therefore, they are assumed to modulate the potassium channel activity of cardiac membranes as well. An actual target of potassium channel openers has not been known until recently, when two of them, cromakalim and nicorandil, were shown with the single-channel recording technique6,7 to activate the ATP-sensitive K' channel current From the Department of Cardiovascular Diseases Medical Research Institute, (Z.F., M.H.), and the Second Department of Medicine (K.N., F.M.), Tokyo Medical and Dental University, Tokyo, Japan. Address for correspondence: Masayasu Hiraoka, MD, PhD, Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo-113, Japan. Received August 23, 1989; accepted July 2, 1990. (IK.ATP) of ventricular myocytes. As pinacidil has an effect similar to the others on the APD' and the background potassium current,8 its target is assumed to be the same K' channel. Indeed, Arena and Kass9 showed that pinacidil activated the time- and voltageindependent outward K' current, which was blocked by glibenclamide, a potent blocker of IKATP.10-13 From these observations, they suggested that the target for pinacidil in the heart is IK.ATP. A preliminary study by Escande et al14 with the single-channel recording technique indicated that pinacidil activated this channel, but the detailed mechanism of its action on IKATP remains unclear. The ATP-sensitive K' channel is characterized by a strong inhibition of activity when the intracellular ATP concentration ([ATP]1) is increased above the millimolar level.15 Therefore, modulation of this channel by any agent depends on the level of [ATP]i. Our previous study7 demonstrated that nicorandil produced a prominent enhancement of IK.ATP at low [ATP]j, whereas its effect was antagonized by increasing [ATP]i. The present study was done to characterize the pinacidil-activated K' current in intact cells under the condition of different [ATP]i. Direct evidence of the pinacidil action on K.ATP was further explored by means of the single-channel recording technique. Nakayama et al Pinacidil Activates ATP-Sensitive K' Current Methods Preparation Single ventricular myocytes were isolated from both ventricles of adult guinea pigs that weighed 250-350 g. Our technique to isolate single myocytes has been described in a previous report.16 Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 Solutions The composition of Tyrode's solution was (mM) NaCI 144, NaH2PO4 0.33, KCl 4.0, CaCl2 1.8, MgCl2 0.53, glucose 5.5, and HEPES 5.0, with pH adjusted to 7.3-7.4 by addition of NaOH. The high K+ low Cl- solution had the following composition (mM): glutamic acid 70, taurine 15, KCl 30, KH2P04 10, MgCl2 0.5, glucose 11, EGTA 0.5, and HEPES 10, with pH adjusted to 7.4 by addition of KOH. Pinacidil, a gift of Shionogi Pharmaceutical Co. (Osaka, Japan), was dissolved as a stock solution in 1% 0.1N HCl and diluted appropriately before study. Glibenclamide, a gift of Hoechst Japan (Tokyo), was dissolved as 0.2 mM stock solution in 2% dimethyl sulfoxide (DMSO) and diluted into the test solution to obtain the final concentration indicated in the text. The final concentration of DMSO contained in the test solution was less than 0.01%. Whole-Cell Current Recordings The patch-clamp technique of the whole-cell configuration17 was used to record membrane potentials and currents by using a patch-clamp amplifier (model 8900, Dagan Corp., Minneapolis). Details of the recording technique and the data-acquisition systems are described in our previous reports.7'16 When the ramp voltage-clamp method was used in some experiments, an intelligent arbitrary function synthesizer (model 1731, NF Instruments, Yokohama, Japan) was used to supply the command pulse. The composition of the pipette solution was (mM) KCI 120, K2ATP (Sigma Chemical Co., St. Louis) 5.0, HEPES 5.0, and K4BAPTA (Dojin Co., Tokyo) 5.0, with pH adjusted to 7.2 with KOH. In pipette solutions having different ATP contents, the concentration of K2ATP was changed, and at the same time, the concentration of KCI was varied so as to maintain the final K' concentration constant at 150 mM. The internal perfusion technique as originally described by Soejima and Noma18 was used for changing [ATP]i. Series resistance compensation was adjusted to minimize the duration of the capacitive surge on the current trace. At the end of each experiment, liquid junction potential was checked, and if more than +2 mV, the value of the membrane potential was corrected accordingly. The temperature of the perfusion chamber was maintained at 33-35° C. The solution in the chamber flowed continuously by gravity with a speed of 1.2-1.5 ml/min. The chamber volume of 0.8 ml was completely exchanged 1 minute after a solution change. Control data were taken 5 minutes after changing to the appropriate solution. Drug effects 1125 were tested 5-10 minutes after the drug-containing solution was changed. Single-Channel Current Recordings Single-channel current recordings were made at room temperature in the inside-out patch configuration'7 with the same patch-clamp amplifier used in the whole-cell experiments. Currents and voltage signals were recorded with a videocassette recorder (HR-S 5500, Victor Co., Tokyo) through a pulse code modulation converting system (RP- 882, NF Instruments). Recorded signals were filtered off-line through an eight-pole Bessel low-pass filter (48 dB/ octave, FV- 665, NF Instruments) at 500 Hz and digitized at 5 kHz using an analog-to-digital convertor (CED 1401, Cambridge Electronic Design Ltd., Cambridge, UK) to store into the disk of an IBM-AT personal computer for later analysis. When the single-channel recording with the inside-out patch configuration was done, the bath solution (intracellular solution) contained (mM) KCl 140, glucose 5.5, EGTA 2, and HEPES 5, with pH adjusted to 7.3 with KOH. The drug was dissolved in the bath solution at the concentration indicated in the text. The composition of the pipette solution (extracellular medium) was (mM) KCI 140, CaCl2 1.8, MgC12 0.53, glucose 5.5, and HEPES 5, with pH adjusted to 7.3 by addition of KOH. The drug effects usually were examined 2-3 minutes after the test solutions were changed, except for calculation of open probability at different [ATP]i as described below. Calculation of Open Probability (ATP-Sensitive K+ Channel Current) Open probability (PO) was calculated as NT where ti is the duration of an nith number of open channels, N is the available channel in the patch, and T is the total recording time (30 seconds in this study). The numbers of available channels were estimated as the maximum open channels at free [ATP]i. To avoid possible influence from the rundown of the channel, total exposure times to different [ATP]i with or without the presence of pinacidil were limited to be less than 4 minutes in any patch. The availability of the channels was reevaluated after washout of pinacidil and ATP. Only those patches showing PO> 0.98 at the second exposure to ATP-free solution were selected for the calculation (assuming Po=1.0 at the first exposure to 0 mM [ATP]i). Measurements of Cell Surface Area Cell surface areas were estimated from the cell capacitance assuming a specific membrane capacitance of 1 ,uF/cm2. Cell capacitance was calculated from current responses induced by 1-mV depolarizing pulses from a holding potential of 90 mV.19 1126 Circulation Research Vol 67, No 5, November 1990 A 5-ATP o~~~ -\ 1 - 0 ,. x mV A ** . (% °-100 B 2-ATP O :control O :P. A :P1OuM *PM1 OM 0 5M 1Os* 5S^l 1 D 50 11 \O wO 100* 100 5.mA I 0 - rD~ ~ ~C A Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 Lk 55Sa L100 200ms 2-ATP 1O CaM 5-ATP *p<O.05 **p<0.01 FIGURE 1. Effects ofpinacidil on action potential duration (APD) ofguinea pig ventricular myocytes in two different intracellular ATP concentrations ([A TP],). Panel A: Effect of 5 and 10 p.Mpinacidil (P) on action potentials recordedffrom a cell dialyzed with an intemal solution containing 5 mMATP (5-A TP). Panel B: Effects of 5 and 10 pMpinacidil on action potentials recorded from a cell dialyzed with an intemal solution containing 2 mMATP (2-ATP). Panel C: Summarized data of the effects ofpinacidil on APD. Values of APD are expressed by relative APD at 90% repolarization (APD90) compared with APD90 in the control as 100%. Multiple comparisons among data in the control, 5, and 10 pMpinacidil were done using the Student-Newman-Keuls method. Unpaired t test was used to judge statistical significance between the data of 2 and 5 mM [ATP]i at the same pinacidil concentration. Action potentials were elicited at a stimulation frequency of 0.067 Hz. Statistical Analysis Data are expressed as mean± SD. Comparisons between two groups of data were evaluated by paired or unpaired t test. Comparisons between more than two groups were evaluated by Student-NewmanKeuls method for critical difference. A value of p<0.05 was considered significant. Results Effect of Pinacidil on Membrane Potentials Effects of pinacidil on membrane potentials of ventricular myocytes were examined at two different [ATP]1, a normal (5 mM) and a low (2 mM) level, while the preparations were stimulated at a constant frequency of 0.067 Hz. At 5 mM [ATP]i, 10 gM pinacidil decreased action potential amplitude and shortened APD both at 20% (APD20) and 90% repolarization (APDgO), whereas 5 ,M pinacidil did not produce any changes in these parameters. At 2 mM [ATP]i, however, 5 ,M pinacidil decreased action potential amplitude and shortened both APD20 and APDg, as did 10 ,uM pinacidil. Furthermore, the degree of the APD shortening by the same concentration of pinacidil was significantly larger at the low than at the normal [ATP]i (Figure 1, Table 1). The resting potential was not changed by pinacidil at either [ATP]i. Effect of Pinacidil on Steady-State Current Figure 2A illustrates membrane current responses to depolarizing or hyperpolarizing voltage steps from the holding potential of -30 mV before and during the application of 10 ,uM pinacidil using a pipette solution containing 2 mM ATP. Pinacidil increased outward current at a potential positive to around -80 mV, and this increase was time-independent. At potentials negative to -90 mM, pinacidil decreased inward current during pulses (see "Discussion"). The steady-state current-voltage relation, in which current was measured at the end of the 2-second test pulses, showed increased outward current with a loss of negative slope region at voltages between -60 and -20 mV (Figure 2B). There was no change in the reversal potential. Similar results as shown in Figure 2 were confirmed in four cells, although the degree of increased current by pinacidil differed among different preparations. Effect of Intracellular ATP Concentration on the Pinacidil-Activated Outward Current The effects of pinacidil on membrane potentials indicated that pinacidil had a stronger effect on APD at the low [ATP]i than at the normal [ATPji. Therefore, we examined the pinacidil-activated outward Nakayama et al Pinacidil Activates ATP-Sensitive K' Current 1127 TABLE 1. Effects of Pinacidil on Membrane Potentials of Guinea Pig Ventricular Myocytes at Two Different Intracellular ATP Concentrations [ATP]i APD%o (msec) 2 mM Control 10 -87.6±4.1 134.7±4.4 507.2±218.1 713.2±299.9 Pinacidil 5 ,uM 9 -85.6±5.5 128.7±7.0* 300.6±168.6* 434.5±212.5* Pinacidil 10 ,uM 4 -86.5±3.6 123.4±11.2* 163.1±138.0* 245.8±187.1* 5 mM Control 5 -89.8±0.7 136.3±2.9 491.8±185.8 689.1±249.9 Pinacidil 5 ,uM 5 -88.0±0.9 135.4±2.0 521.4±209.4 719.6±207.9 Pinacidil 10 tM 5 -87.6±1.0 133.5±1.7* 405.2±202.5* 571.8±253.2* Values are mean±SD. [ATP]i, intracellular ATP concentration; RMP, resting membrane potential; APA, action potential amplitude; APD20, action potential duration at 20% repolarization; APD%, action potential duration at 90% repolarization. *p<0.05 vs. control. n RMP (mV) Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 current at different [ATP]i. First, antagonistic action of increasing [ATP]i on pinacidil-activated outward current was tested using the internal perfusion technique18 (Figure 3). Under the condition of 2 mM [ATP]i, 10 ,uM pinacidil markedly increased the time-independent outward current at potentials positive to -50 mV, and the current-voltage curve lost its negative slope region. When [ATP]i was increased to 5 mM in the presence of pinacidil, the increased outward current was nearly halved, and the currentvoltage curve regained a slight negative slope. In six myocytes, the outward current at 0 mV increased significantly from 0.13±0.07 nA in the control to 1.06+±0.63 nA after the pinacidil application at 2 mM ATP in the pipette solution (p<O.OS). An increase in [ATP]i to 5 mM in the presence of pinacidil decreased the current to 0.61+0.40 nA. The dose-response curve of pinacidil-activated current was examined at different [ATP]i. A ramp voltage-clamp method was used to produce a slow depolarization (200 mV/30 sec) from -120 to 80 mV. Figure 4 presents typical results obtained with different [ATP]i. The pinacidil dose-dependently activated the outward current at a voltage positive to -80 mV in each [ATP]i, and the degree of the increase also was dependent on [ATP]i. The pinacidil APA (mV) APD20 (msec) effect was the most prominent at 2 mM [ATP]i among the three conditions. A quantitative estimation of the type of experiments shown in Figure 4 was achieved by expressing the dose-response curve as the current density. The current density induced by pinacidil was measured as the current level at 0 mV, because it was nearly zero during the control (Figure 5). At 2 mM [ATP]i, the current density showed a sigmoidal dependence on the pinacidil concentration, with a nearly maximal response at 100 gM (159.4+±14.8 ,NA/cm2; n=5). We could not judge whether the response at 100 ,uM pinacidil was really saturated or not, because all the cells died after application of the drug at higher concentrations. At 5 mM [ATP]i, a sigmoidal dependence of the current density on the pinacidil concentration shifted to the right, and at the same time, the maximal response at 100 ,uM pinacidil was markedly suppressed to 16.8±4.5 tuA/cm2 (n=4). The response was saturated when the concentration of pinacidil was higher than 100 ,uM. At 10 mM [ATP]i, the maximal current induced by pinacidil was reduced further (15.8+4.0 gANcm'; n=6) and shifted to develop at the higher concentration (300 ,uM). The current density decreased to 10.3+2.5 gA/cm' at 500 ttM. The decrease of the current density response to 500 ,iM pinacidil in comparison to that at B A VT mv 0 11[ nA +60 -O 0 o 01~~~ ----° -60 -100HP=-30 * o VT 29 O:control *:P. 10pM *--4 :control :P.1OyM FIGURE 2. Effect ofpinacidil (P) on membrane current. Panel A: Superimposed current traces before (o) and during (0) the application of 10 pM pinacidil at four different test voltages (VT). Voltage protocol is shown at the bottom. Panel B: Effect of 10 pM pinacidil on current-voltage relation. Current values were measured as the level at the end of 2-second depolarizing or hyperpolarizing test pulses. HP, holding potential. Circulation Research Vol 67, No 5, November 1990 1128 A B nA S 5.01 0-O :control,2-ATP *---:2-ATP + P -----A:5-ATP + P l.OnA o z~ 'l X0~~~~-U o :control,2-ATP *:2-ATP + P * :5-ATP + P -e.- - t 0of 1 -60 -4o -20 -30mV -1.0f is Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 300 ,uM was observed in four of six cells. By these sigmoidal curves, the following Kd values were calculated: 77.0 and 69.0 ,M at 2 and 5 mM [ATP]i, respectively, and 158 gM at 10 mM [ATP]i. Block of the Pinacidil-Induced Changes in Membrane Potential and Current by Glibenclamide We examined the effects of glibenclamide on the changes in action potentials and outward current induced by pinacidil because Arena and Kass9 described the suppression of the pinacidil-activated current by glibenclamide. Figure 6A illustrates the effects of glibenclamide on the pinacidil-induced action potential change at 2 mM [ATP]i. Application of 0.3 and 1.0 ,M glibenclamide prolonged APD, which was shortened by the pretreatment of 100 ,M pinacidil. APD90 was shortened by 100 ,M pinacidil from 481+77 (n =5) to 177 ±70 msec. The shortened APDgO by pinacidil was prolonged by 0.3 ,uM glibenclamide to 292±167 msec (p<0.01 versus APD%0 in 100 ,M pinacidil) and further by 1.0 ,M to 416±135 msec (p<0.01) (Figure 6B). Therefore, 1.0 4M glibenclamide completely antagonized the shortening of APD induced by 100 ,uM pinacidil. The pinacidil-activated outward current also was almost 2-ATP | 40 600 80 80 mVV completely suppressed by 0.3 jM glibenclamide (Figure 7). This result was confirmed in three myocytes. Effect of Pinacidil on the Single-Channel Current The effect of pinacidil on the single-channel current was studied using the inside-out patch configurations. Figure 8 shows a typical example of the records at a membrane potential of +40 mV (Figure 8A), as well as amplitude histograms (Figure 8B). When the intracellular solution did not contain ATP, prominent openings of the single channels were observed (Figure 8A, recording a); the openings were largely inhibited by 2 mM [ATPJi (recording b). Application of 30 j.M pinacidil in the presence of 2 mM [ATP]i activated the current again (recording c). The amplitude of the pinacidil-activated channels at 2 mM [ATP]i (2.53 pA) was not different from that in the ATP-free solution (2.48 pA, calculated by the difference between the third and fourth open level in recording a), confirming our recent report.20 Addition of 0.01% DMSO did not influence the pinacidilactivated channel activity (recording d), whereas 0.3 ,uM glibenclamide dissolved in 0.01% DMSO completely inhibited channel activity (recording e). Values of PO before and after the addition of DMSO in 1O-ATP 5-ATP nAA |, 20 FIGURE 3. Effect of different intracellular ATP concentrations ([ATP]1) on the pinacidil-activated outward current. Panel A: Current traces recorded in response to depolarization to 0 mV for 2 seconds from -30 mVat different stages of treatment. o, Control with 2 mM [ATP]i (2-A TP); *, application of 10 M pinacidil (P) in 2 mM [ATP],, A, application of 10 /Mpinacidil in 5 mM [ATTPFi (5-A TP). Panel B: Effect of 10 pMM pinacidil on the currentvoltage relation at different ATP concentrations in the pipette solution. Data were taken from the same experiment shown in panel A. Current values were measured as the level at the end of 2-second test voltages from -30 mV. 20 AL -100 -5. 80mV - 1 20mV O control *:P.50pM A 30sec P.loopM FIGURE 4. Effects of pinacidil on the background current induced by 30-second depolarizing ramp from -120 to +80 mV at different intracellular ATP concentrations ([ATP]i). [ATP]i is indicated at the top of each graph as 2-ATP, 2 mM [ATP]i; 5-ATP, 5 mM [ATP]i; and 10-A TP, 10 mM [ATP]li. o, Control current before the application ofpinacidil (P); *, current in the presence of 50 p!pinacidil; A, current in the presence of 100 puM pinacidil. Nakayama et al Pinacidil Activates ATP-Sensitive K' Current 1129 A 60 CO cC4Q 40 0) -100 O:Controi *A:P+G0.3pM A:P+GI.OMM :20 0) o a a 1 10 100 a 200ms B 500 Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 Pinacidiil FIGURE 5. Dose-response cune of the pinacidil-activated current at different intracellularATP concentrations ([A TP]J. Ordinate indicates current density, and abscissa is the pinacidil concentration. Current density was calculated by the background current at 0 mV induced by 30-second depolarizing ramp pulses from -120 to +80 mV Each value represents offour to six measurements from different preparations; bars indicate±SD. Maximum current density activated by pinacidil was depressed with increasing [ATP]I. 2 mM [ATP]i (2-ATP); *, 5 mM [ATPfi (5-ATP); o, 10 mM [ATP]I (10-A TP). a mean [mS] 500o " 400- 300-1 lovv 200- 100- e, the presence of 2 mM [ATP]i and 30 ,uM pinacidil were 12.6+5.1% and 9.7+3.2%, respectively (n=3). Further application of 0.3 ,uM glibenclamide reduced PO to less than 0.1% in five preparations. At 2 mM [ATP]i, activation of the single-channel current sensitive to the ATP-free solution also was observed in eight of 10 patches in 30 ,uM pinacidil, and two of five patches in 10 ,M pinacidil. With either pinacidil concentration, the amplitude of the single-channel current was not changed from that with the ATP-free solution. PO of IK.ATP in different [ATP]i was estimated with and without the presence of 30 .M pinacidil. Figure 9A shows typical recordings of the single-channel current; Figure 9B summarizes data from four to six patches. At each [ATP]i (0.1-2.0 mM), pinacidil significantly increased PO. Furthermore, PO of the channel in the presence of pinacidil was dependent on [ATP]i, because an increase in the latter decreased the former. Discussion The present study demonstrated that a vasodilator, pinacidil, shortened APD and increased the timeindependent outward current in ventricular myocytes. The effects of pinacidil were dependent on [ATP]i, being accentuated by lowering [ATP]i and attenuated by increasing it or by application of glibenclamide. Single-channel current recordings confirmed an activation of the [ATP]i-sensitive current by pinacidil without changing the unitary amplitude of the current and the dependence of the activation on [ATP]i. These results indicate that O- Control P P + P + G G 0.3PM 1.0MM FIGURE 6. Effects of glibenclamide on the pinacidil-induced action potential shortening. Panel A: Superimposed action potentials. o, Action potential in control with 2 mM intracellularATP concentration ([A TPJd, *, application of 10 pMpinacidil (P); A, application of 10 tMpinacidil and 0.3 pMglibenclamide (G); A, application of 10 ,Mpinacidil and 1.0 iM glibenclamide. Panel B: Effects of pinacidil and glibenclamide on action potential duration at 90% repolarization (APD90). Data show mean ±SD collected from five preparations. pinacidil increases PO of the ATP-sensitive K' channel, which explains the shortening of APD of heart cells after drug treatment. Pinacidil has been shown to shorten the APD of heart muscles and to modify the shape of the T wave on surface electrocardiograms. The effects are attributed to an increase in K' permeability of cardiac membranes.5,21 lijima and Taira8 actually showed an increase in the outward current using isolated ventricular myocytes, and they interpreted this action as an activation of the background K' current. Arena and Kass9 recently explored the target of the membrane current system activated by pinacidil and confirmed that pinacidil actually activated the K' current in guinea pig ventricular myocytes, but they interpreted the pinacidil-activated current as IK.ATP on the basis of its time- and voltage-independent properties, as well as its pharmacological characteristics. 1130 Circulation Research Vol 67, No 5, November 1990 -100 +BOmV -12OmV,V30sec, 0: control *: P.30,0M A: P.30,M+G.0.3pM Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 FIGURE 7. Effect of glibenclamide (G) on the pinacidil (P)-induced background current. Current was induced by 30-second depolarizing ramp from -120 to +80 mV Note that 0.3 ,M glibenclamide (A) completely suppressed the pinacidil-activated background current (e). A a. 0-ATP .... b o1-1 ...^..- C-. -1001 bill 01 W-1 11 w-T -1-7 - . b. 2-ATP 1 C-_ c. 2-ATP + P 30uM A-0- ~ a A -- m --- I c-~~~~~~~~~~~~~~-e. 2-ATP + P 3OpM + ool%MO + G 03PM --- ONR o 1- l JL__--- B 0 x CO) z W *RO#Wb~ -1- 1 ---&- $_,;_j d. 2-ATP + P 30MM + o.ol%DMSO C~ S- Our study confirmed that pinacidil shortened APD of ventricular myocytes and activated the time-independent outward current. These effects were antagonized by raising [ATP]i. All of the above actions by pinacidil were easily blocked by a low concentration of glibenclamide, which has been claimed as a specific inhibitor of IK.ATP.10-13 Furthermore, the singlechannel recordings in this and the previous study20 proved the actual activation of IK.ATP by pinacidil. Therefore, our data presented the confirming evidence that the target of the pinacidil action in heart is indeed K.ATP. The mode of channel activation by pinacidil appears to increase the PO Of IKATP, because marked openings were seen without changing the unitary amplitude. An increase in [ATP)i decreased the channel opening without affecting the unitary current amplitude, and the decreased PO of the channel activity was restored by application of pinacidil (Figures 8 and 9). The results may indicate that ATP and pinacidil share the same binding site or process for channel modulation. Kinetic analysis of channel activity showed an antagonistic action by ATP and WU Ic %W~ h SpA FIGURE 8. Effects of pinacidil (P) and glibenclamide (G) on the ATP-sensitive single-channel current. Panel A: Current recordings obtained from a single patch. Membrane potential is held at +40 mV in the inside-out patch configuration. C, Closed leveL Recording a: Presence of 0 mMATP (0-ATP) in the intracellular solution. There are marked openings ofthe channel activity up to four levels. Recording b: Presence of 2 mMATP (2-ATP) in the intracellular solution. Most of the channel activity seen in recording a is now inhibited. Recording c: Application of 2 mMATP and 30 pMpinacidil in the intracellular solution. Marked openings of the channel again are seen. Recording d:Application of2 mMATP and 30 MMpinacidil and 0.01% dimethyl sulfoide (DMSO) in the intracellular solution. There is no apparent change in the channel activity compared with that in recording c. Recording e: Further application of 0.3 pMglibenclamide in the internal solution. Channel activity is completely suppressed. Panel B: Amplitude histograms recorded from the same patch in panel A. Nakayama et al Pinacidil Activates ATP-Sensitive K' Current 1131 A ro.1-AI 0-ATP T 'I __1 7 _ 1 VPin.1 3O-IM ~~~~~~~~~~~~~~~~~~~~~~~~~~l cCTwU~~~~~ ~~ O.1-ATP L 1 . .-- .5-ATP - C-~~~~~~~~~~~~~~~~~~~~7 ePf?hnacil3O>M& |0-ATP 0.5-ATP Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 5 pA C- ..... B 1.Oro I* PO 0.5 FIGURE 9. The open probability (Po) of the A TP-sensitive channel at different A TP concentrations in the presence and absence ofpinacidil (P). Panel A: Typical examples ofthe continuous current record. Holding potential is +40 mV Panel B: Summary data of Po obtained from four to six patches held at +40 mV The paired t test between the data at the same intracellular ATP concentration ([ATPJ) and Student-Newman-Keuls method among different [ATP]i are used to judge statistical significance. * O:control *:P.301*M 0 1-A-,. *p<0.05 0.1 0.5 A T P 2.0 mM pinacidil, where the channel was partially closed by ATP.20 However, the dose-response curve of the pinacidil action did not reveal a competitive inhibition by ATP, and the maximal current activated by pinacidil was suppressed by the higher [ATP]i in addition to a shift of the curve to the right (Figure 5). Partially, this shift may be due to experimental imperfection because of too few points on the curve near the maximal response, especially at 2 mM [ATP]i. Another possibility is that pinacidil may have at least two sites or processes to interact with the openings of IK.ATP. High concentrations of pinacidil may block the channel opening, in addition to its activation, or accelerate the "rundown" phenomenon of this channel activity.22 Further study is necessary to delineate an exact mechanism, as well as sites of action for pinacidil to activate lK.ATPThe effects of pinacidil on the current-voltage curve exhibit two characteristic changes worth mentioning in terms of the drug-Induced alteration in membrane potential, other than a marked increase of outward current. First, the increased outward current positive to around -60 mV caused a loss of the negative slope region in the current-voltage curve. Second, pinacidil produced little change in the current at the resting potential level around -80 mV and decreased inward current at potentials negative to -90 mV. These two factors could explain a marked shortening of APD without alternation of the resting potential (see Table 1). These effects were similar to those produced by cromakalim and nicorandil, which also were shown to activate IK.ATP.67 The result that pinacidil enhanced outward but not inward current may be related to the intrinsic properties of IK.ATP, because it has a sigmoidal currentvoltage curve, showing outward-going rectification at negative potentials and inward-going rectification at positive potentials.23 Consequently, activating IK.ATP will produce very little inward current negative to its reversal. This factor, however, cannot explain a decrease of the inward current negative to K' equilibrium potential (EK) after pinacidil, and therefore, the decrease of the current may be explained by several other possibilities. First, the activation of the current 1132 Circulation Research Vol 67, No 5, November 1990 by pinacidil may be voltage-dependent. Second, pinacidil may produce inhibition of other currents, such as the inward rectifier K' current (IK1). Third, pinacidil may display inhibition on IKATP in addition to activating action. A voltage-dependent activation is not likely because single-channel recording revealed that pinacidil activated K.ATP at potentials both negative and positive to EK.20 As to inhibitory action on IK.ATP, our preliminary study indicated that pinacidil Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 produced a suppression of channel activity at positive voltages to EK but no such action at negative voltages. Therefore, inhibitory action seems not to be the right explanation. Although we have not examined the drug effects on outside-out patches, the most likely explanation is that pinacidil inhibits 1K1 from the outer surface of the membrane, because internal application of the drug did not affect the channel activity of 1K120 The results of the present study point out a common mechanism of action on cardiac membranes shared by the three agents cromakalim, nicorandil, and pinacidil. These agents are claimed as K' channel openers,' which possess the ability to increase K' permeability in vascular smooth muscle cells and to produce a vasodilatory action. The exact mechanism of the three agents to induce relaxation of vascular smooth muscles is not known, but it is implicated that increased K' permeability has some crucial role for vasodilation.24 Recently, the existence of IKATP as well as its activation were reported.25 Pinacidil also was suggested to activate IK.ATP of vascular smooth muscles because of inhibition of the vasorelaxant effect of IK.ATP by glibenclamide.25 Although the properties of IK.ATP between cardiac and smooth muscle cells were not exactly the same, the present results obtained from cardiac myocytes may help to solve the mechanism of a vasodilatory action by K' channel openers. Study in this area continues,2627 and it is an interesting area for future studies. The physiological and clinical significance of the present finding is not known. It would be interesting to know why pinacidil, cromakalim, and nicorandil consistently are found to activate K.ATP even though their chemical structures differ. Information regarding the structure-function relation of these compounds may be helpful in clarifying the regulatory mechanism of IK.ATP itself and may lead to an understanding of the basic mechanism of the drug action on the heart. Pinacidil would be expected to be more effective in the ischemic heart than in the normal heart, because the ischemic heart would have less [ATP]i than the normal. Whether or not the action of pinacidil could bring about beneficial effects for the function of the ischemic heart is to be evaluated further. Acknowledgment The authors thank Ms. N. Fujita for her secretarial assistance. References 1. Cook NS: The pharmacology of potassium channels and their therapeutic potential. Trends Pharmacol Sci 1988;9:21-28 2. Taira N: Similarity and dissimilarity in the mode and mechanism of action between nicorandil and classical nitrates: An overview. J Cardiovasc Pharmacol 1987;10(suppl 8):S1-S9 3. Scholtysik G: Evidence for inhibition by ICS 205-930 and stimulation by BRL 34915 of K' conductance in cardiac muscle. Naunyn Schmiedebergs Arch Pharmacol 1987;335: 692-696 4. Osterrieder W: Modification of K' conductance of heart cell membrane by BRL 34915. Naunyn Schmiedebergs Arch Pharamcol 1988;337:93-97 5. Smallwood JK, Steinberg MI: Cardiac electrophysiological effects of pinacidil and related pyridylcyanoguancidine: Relationship to antihypertensive activity. J Cardiovasc Pharmacol 1988;12:102-109 6. Escande D, Thuringer D, Leguerns S, Cavero I: The potassium channel opener cromakalim (BRL 34915) activates ATPdependent K' channels in isolated cardiac myocytes. Biochem Biophys Res Commun 1988;154:620-625 7. Hiraoka M, Fan Z: Activation of the ATP-sensitive K' channel by nicorandil (2-nicotinamidoethyl nitrate) in isolated ventricular myocytes. J Pharmacol Exp Ther 1989;250:278-285 8. lijima T, Taira N: Pinacidil increases the background potassium current in single ventricular cells. Eur J Pharmacol 1987;141:139-141 9. Arena JP, Kass RS: Enhancement of potassium-sensitive current in heart cells by pinacidil: Evidence for modulation of the ATP-sensitive potassium channel. Circ Res 1989;65: 436-445 10. Trube G, Rorsman P, Ohno-Shosaku T: Opposite effects of tolbutamide and diazoxide on the ATP-dependent K' channel in mouse pancreatic B-cells. Pflugers Arch 1986;407:493-499 11. Sturgess NC, Ashcroft MLJ, Cook DL, Hales CN: The sulphonylurea receptor may be an ATP-sensitive potassium channel. Lancet 1985;ii:474-475 12. Schmid-Antomarchi H, De Weille JR, Fosset M, Lazdunski M: The receptor for antidiabetic sulfonylureas controls the activity of the ATP-sensitive K' channel in insulin-secreting cells. J Biol Chem 1987;262:15840-15844 13. Fosset M, De Weille JR, Green RD, Schmid Antomarchi H, Lazdunski M: Antidiabetic sulfonylureas control action potential properties in heart cells via high affinity receptors that are linked to ATP-dependent K' channels. J Biol Chem 1988;263: 7933-7936 14. Escande D, Thuringer D, Mestre M, Cavero I: Potassium channel openers activate the ATP-modulated K' channels in guinea-pig cardiac myocytes (abstract). Circulation 1988; 78(suppl II):II-26 15. Noma A: ATP-regulated K' channels in cardiac muscle. Nature 1983;305:147-148 16. Hirano Y, Hiraoka M: Barium-induced automatic activity in isolated ventricular myocytes from guinea-pig hearts. J Physiol (Lond) 1988;395:455-472 17. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high resolution current recording from cell and cell-free membrane patches. Pflugers Arch 1981;391:82-100 18. Soejima M, Noma A: Mode of regulation of the Ach-sensitive K-channel by the muscarinic receptor in rabbit atrial cells. Pflugers Arch 1984;400:424-431 19. Lindar M, Neher E: Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Arch 1988; 411:137-146 20. Fan Z, Nakayama K, Hiraoka M: Pinacidil activates the ATP-sensitive K' channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes. Pflugers Arch 1990;415:387-394 21. Goldberg MR: Clinical pharmacology of pinacidil, a prototype for drugs that affect potassium channels. J Cardiovasc Pharmacol 1988;12(suppl II):S41-S47 Nakayama 22. Ashcroft FM: Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Neurosci 1988;11:97-118 23. Noma A, Shibasaki T: Membrane current through adenosinetriphosphate-regulated potassium channels in guinea-pig ventricular cells. J Physiol (Lond) 1985;363:463-480 24. Cook NS, Quast U, Hof RF, Baumlin Y, Pally C: Similarities in the mechanism of action of two new vasodilator drugs: Pinacidil and BRL 34915. J Cardiovasc Pharmacol 1988;11: 90-99 25. Standen NB, Quayle JM, Davies NW, Brayden JE, Huang Y, Nelson MT: Hyperpolarizing vasodilators activate ATP- et al Pinacidil Activates ATP-Sensitive K' Current 1133 sensitive K' channels in arterial smooth muscle. Science 1989;178:177-180 26. Escande D, Thuringer D, Guern SL, Courteix J, Laville M, Cavero I: Potassium channel openers act through an activation of ATP-sensitive K' channels in guinea-pig cardiac myocytes. Pflugers Arch 1989;414:669-675 27. Arena JP, Kass RS: Activation of ATP-sensitive K channels in heart cells by pinacidil: Dependence on ATP. Am J Physiol 1989;257:H2092-H2096 KEY WORDS * ATP-sensitive K' current * intracellular ATP K' channel opener * pinacidil Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 Interrelation between pinacidil and intracellular ATP concentrations on activation of the ATP-sensitive K+ current in guinea pig ventricular myocytes. K Nakayama, Z Fan, F Marumo and M Hiraoka Downloaded from http://circres.ahajournals.org/ by guest on October 1, 2016 Circ Res. 1990;67:1124-1133 doi: 10.1161/01.RES.67.5.1124 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1990 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/67/5/1124 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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