Effects of extracellular pH on anodal break excitation in rabbit sino-atrial (SA) nodal cells were investigated using the two-microelectrode voltage-clamp technique. The method of anodal break excitation was developed by Weidmann²⁸⁾，and is regarded as a measurement of the activity of the fast Na⁺ channel. Increasing external pH from 7.4 to 8.5 enhanced the maximum rate of depolarization by anodal break excitation，as compared with the values of control (at pH 7.4). In contrast，a decline of pH from 7.4 to 5.5 inhibited the maximum rate of depolarization，accompanied with depression in the activity. Acidification shifted the inactivation curves (h∞) of the fast Na⁺ current in the depolarizing
direction，and alkalinization shifted it in the hyperpolarizing direction. These results
suggest that proton would modulate the membrane surface charge of the SA nodal cells，resulting in alteration of the gating kinetics of ionic channels

Satoh, Hiroyasu

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奈医誌. (]. Nara Med目 As.)45， 665~671 ， 1994 (665) MODULATIONS BY EXTERNAL PH ON  ANODAL BREAK EXCITATION IN RABBIT SINO-ATRIAL NODAL CELLS HIROYASU SATOH Dψartment 01 Pharmacology， Nara Medical University Received October 1， 1994 Abstract: Effects of extracellular pH on anodal break excitation in rabbit sino-atrial (SA) nodal cells were investigated using the two-microelectrode voltage-clamp technique. The method of anodal break excitation was developed by Weidmann28)， and is regarded as a measurement of the activity of the fast N a + channel. Increasing external pH from 7.4 to 8.5 enhanced the maximum rate of depolarization by anodal break excitation， as compared with the values of control (at pH 7.4). In contrast， a decline of pH from 7.4 to 5.5 inhibited the maximum rate of depolarization， accompanied with depression in the activity Acidification shifted the inactivation curves (hoo) of the fast N a + current in the depolarizing direction， and alkalinization shifted it in the hyperpolarizing direction. These results suggest that proton would modulate the membrane surface charge of the SA nodal cells， resulting in alteration of the gating kinetics of ionic channels. Index Terms extracellular pH， fast Na+ channel， anodal break excitation， voltage-clamp， sino-atrial nodal cells INTRODUCTION Acidification under the conditions of ischaemia and cardiac failure alters cardiac perfor. mance. Low pH decreased myocardial contraction18).26).27)， whereas high pH enhanced it8). The authors have also reported on the effects of protons on cardiac muscle cels. In puppy sino atrial (SA) node preparations perfused with Tyrode solution through the sinus node artery， the spontaneous activity was decreased by low pH solution， whereas it was increased by high pH solution20). In addition， we have demonstrated the effects of different pH solutions on the kinetics of the ionic channels in rabbit voltage-clamped SA and atrio-ventricular (A V) nodal cells; low pH inhibited the ionic currents， whereas high pH enhanced them21-23). External proton inhibits the fast N a + current (INa) in nerve山 1).29)，skeletal muscle4)， and cardiac muscles31). It is generally agreed that increasing the extracellular proton concentration would exert two actions30): (1) protons cause a shift in channel gating to more positive potentials， and (2) protons reduce the maximal conductance of the N a+ channels. However， the alteration of pH did not affect the kinetics of the ionic channels (the Ca2+ and delayed K+ currents， Ica and IK) of the pacemaker cells of the SA and A V nodes. The nodal cells also possess the fast Na+ channels， which may make a minor contribution to normal spontaneous beating. In the present experiments， we attempted to examine the modulation of the INa in Send correspondence to: Dr. Hiroyasu Satoh， Department of Pharmacology， Nara Medical University， Kashihara， Nara 634， ]apan (66) H. Satoh rabbit SA nodal cells in different pH solutions using anodal break excitation， since it is too fast to measure the precise values of the activation of INa • METHODS Preparations of sino-atrial node Rabbits of either sex， weighing 1.5-2.0 kg， were used. The preparations were made by the same method as described previously19-21). The rabbits were anesthetized with pentobarbital sodium (30 mg/kg， i. v.)， and exsanguinated. The chest was opened， the heart was quickly removed， and the right atria with the SA node region left intact was dissected in the bath solution. The preparation was made smaller by dissection to a final dimension of about 0.25 x 0 . 25mm. The preparations were usually smaller than the length constant (0.3-0.8 mm). The preparations were superfused with the bath solution oxygenated by 100 % O2 at 36 'c， and were left spontaneously beating. Procedure of anodal break excitation The two-microelectrode voltage-c1amp technique used has already been described19)，21)，2)， and it is similar to the method developed by N oma and Irisawa 16). Conventional glass microelectrodes fi1led with 3 M KCl were used， and their resistances were 20-30 Mn. According to the methods developed by Weidmann (1955) as shown in Fig. 1A， the test pulses of voltage c1amp (for 300 msec) were applied from -50 to -120 m V， with increment of 10 m V， by using a feedback amplifier (Dia Medical DP 100， Tokyo， Japan). The interval between test pulses was 1 min or more. Between applications of the voltage-c1amp， the preparations invariably showed spontaneous rhythmicity. The membrane potential and the maximum rate of depolar-ization (V max) were recorded with a pen-recorder (Nihon Kohden RJG-4124)， and were also displayed on an osci1loscope (SONY Tektono， Tokyo， J apan) and photographed with a camera (Nihon Kohden， RLG-6201) Solutions The bath solution had the following composition (mM) : N aCl137， KCl 2.7， CaC12 1.8， MgC12 0.5， HEPES [N-2-hydroxy ethylpiperazine-N'-2-ethansulfonic acid] (Wako Pure Chemical Industry， Ltd.， Osaka， J apan) 5.0， and glucose 5. O. The pH in each solution was adjusted with NaOH. Since the bath solution was completely exchanged with solutions of different pH within at least 3 min， the data were obtained 5-7 min after changing to a different solution. Statistical analysis Values are represented as arithmetical mean土 S.E. M. The significance of differences between mean values was assessed by Student's t-test for paired data， and were considered significant when P values were less than 0.05. RESULTS The experimental procedure is i1lustrated in Fig. 1 A. Voltage-c1amp pulses for 300 msec Extracellular pH on anodal br巴akexcitation (667) A 品 EjLJ」L C 40 10 A ，H U o ，" u ・，H6.5 ムー .:.! 10圃S目 調B pH 8.5 pH 7.4 pH 6.5 』ー-80 四時 [10~C 』，. 潤-10 申 ー占 -、ι '120 .10 -叩 .&0 田『] 申圃占 蜘耐醐 ，.個tilMM・-目白v，.1 • Fig. 1. Modulations of the anodal break excitation by changes in extracellular pH in rabbit spontaneously beating sino-atrial nodal cels. A: Protocol of th巴 anodalexcitation. The pr巴parationwas appli巴dfrom -50 to -120 m V by voltag巴 clampmethod. The values of the maximum rate of depolarization was measured. when the voltage-clamp was released. B: The maximum rate of d巴polarizationsat difer巴ntpot巴ntialsof voltage-clamp in three external pH solutions. C: Inactivation curves at differnt pH solutions. Th巴valueswere plotted from the panel B目 Symbolsare pH 8.5 (triangles). pH 7.4 (open circles) and pH 6.5 (squares)ーwere applied from -50 to -120 m V. At the end of test pulses. the feedback circuit was broken. The courses of the membrane potential and the maximum rate of rise of depolarization (V max) at the end of clamp period were measured. The stronger hyperpolarizing pulses produced larger amplitude of V max， but the values at more negative voltages than -120 m V reached a steady state. On switches of a superfusing solution having a normal pH (7.4) to one of pH 6.5 or 8.5， the spontaneous activity of SA nodal cells was depressed in acidic solution and was stimulated in alkaline solution. Changes in the action potential parameters are summarized in Table 1. Increasing external pH shortened the cycle length (CL)， and the action potential duration at 50 % repolarization (APD) (but not significantly). The action potential amplitude (APA) and the maximum rate of depolarization (V max) were increased. The maximum diastolic potential (MDP) was hyperpolarized by elevating pH. On the other hand， the V max at the end of release of feedback voltage-clamp was examined at the various hyperpolarizing steps (Fig. 1 B). When the more negative potentials were applied， larger V max was elicited. The V max at -120 m V was inhibited by 19.1土3.4% (n=6， (668) H. Satoh Table 1. Modulation by different extracellular pH solutions on the action potential parameters in the rabbit sino-atrial nodal cells pH 6.5 pH 7.4 pH 8.5 n 10 l4 1 APA (mV) 65士8** 81土9 90:t8* APD (ms巴c) 63土6 61士6 58士6MDP (mV) -52士4** 62士3 69土3*Vmax (V/s) 6土2** * 1土2 14士3** CL (msec) 294土4* 273土6 246士5*Values represent mean土 S.E.M.n: Number of experi. ments. APA: Action potential amplitude. APD: Action potential duration at 50 % repolarization. MDP: Maxi同mum diastolic potential. Vmax: Maximum rate of rise of depolarization. CL・Cyc1elength. *: P<0.05， * * : P< 0.01， * * * : P < 0.001， with respect to control values (at pH7.4). h∞ 1.0 545 818 HHH P1 80ロ「Rυ nu .120 .10 .80 .60 mV Membrane potential Fig. 2. N ormaliz巴dinactivation curves of the I¥a+ current in dif巴rentpH solutions in rabbit sino-atrial nodal cells目 Symbolsare pH 8.5 (triangl巴S)，pH 7.4 (open circles) and pH 6.5 (squar巴s)P<O.Ol) at pH 6.5， and was enhanced by 18.5:t2.6 (rt=6， P<O.Ol) at pH 8.5， as compared with the values at pH 7.4. Figure 1 C shows the inactivation curves in different pH solutions in which the values (obtained from Fig. 1 B) are plotted against the membrane potentials. In addition， the inactivation curves were normalized by taking the value at -120 m V as 1.0 (hoo) ， as shown in Fig. 2. The half-maximal potential was -86.1:t 2.5 m V (n = 6) at pH 8.5， -81.7 士2.1m V  (n=8) at pH 7.4， and -78.9土2.9mV(n=6) at pH 6.5 Extracellular pH on anodal br巴akexcitation (669) DISCUSSION The present experiments showed the following: (1) the pacemaker activity was stimulated by high pH， but it was depressed by low pH; (2) the V max induced by the anodal break excitation was enhanced in high pH solution， whereas it was inhibited in low pH solution; and (3) the inactivation kinetic (hoo) of the INa was shifted in the hyperpolarizing direction with an increase in the external pH. According to the ionic theory of electrical activity， a change in membrane potential depends on the displacement of charged partic1es across the membrane13). The upstroke of the cardiac action potential (from the resting potential at more negative potentials than -80 or -90 m V) is due to N a ions entering the cells7)，9). Since the driving force made up by membrane potential and Na+ concentration difference would be similar， the V max may thus be regatded as a measure for the inward Na+ current28). Therefore， the present experimental data are recognized as an indicator of the INa current， and could be fitted satisfactorily by the following equation14) : h=l/{l +exp(Vh - V)/5} where h is the fraction of the highest value observed for the V max， V is a c1amp potential (m V)， and V h is the potential at which h is half-maximal inactivation. Two mechanisms can be proposed to explain the effects of altering pH on the ionic currents of the cel membane: (1) art alteration in the surface potential (or surface charge) of the membrane， and (2) a change in conductance due to protonation of the channels. It is generally agreed that dec1ine of external pH causes a shift in channel gating to more positive potentials and reduces the maximal N a + conductance， similar to the results of this study. The shift in gating is commonly thought to result from titration of external negative surface charges2)，3)，12). These are in accord with previous reports on the frog node of Ranvierll)， frog skeletal musc1es5)， and Myxicola and crayfish giant axons24)，25). On the other hand， the second mechanism is also supported by some evidences. In rabbit SA and AV nodal cells (from my laboratory)， the decreases in gs and gk by low pH are voltage independent. These results suggest that the gating systems for these channels would be electrically isolated from the surface charge， and that the binding site should be located outside the transmembrane field. This is consistent with the proposal of Campbe1l4)，15) that the proton bining site in Na+ channels is located at or near the mouth of the channel on the surface of the membrane. These results suggest for litle or no contribution of a change in the surface potential to the changes in membrance currents. In the present experiments， the V 1/2 of V max was shifted by approximately 7 m V in the hyperpolarizing direction with an increase in the external pH from 6.5 to 8.5. A change in V 1/2 can be regarded as a good indication of a difference in surface potentiaPO)，17). Therefore， the shift may be considered to be due to the alteration of the surface charge， although the kinetics of Ica and h currents were not affected， as reported previously19-23). The SA and A V nodal cells as pacemaker cells also play a chemoreceptive role. There would be distinct binding sites for external H+ (which might cause the direct action). Protons compete with Ca2+ for the Ca2L binding sites and reduce the open times6). Further extensive experiments are required to (670) H. Satoh elucidate the mechanisms of pH -induced complex phenomena. REFERENCES 1) Begenisich， T.and Danko， M. : Hydrogen ion block of the sodium pore in squid giant axons. J. Gen Physio1. 82: 599-618， 1983 2) Brown， H. 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MODULATIONS BY EXTERNAL PH ON ANODAL BREAK EXCITATION IN RABBIT SINO-ATRIAL NODAL CELLS

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MODULATIONS BY EXTERNAL PH ON ANODAL BREAK EXCITATION IN RABBIT SINO-ATRIAL NODAL CELLS

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