Whole-cell patch clamp experiments were performed to examine the underlying currents to generate spontaneous activity in freshly isolated single longitudinal muscle cells of pregnant rat uterus (18-day gestation). Isolated single cells were spindle- or round-shaped (50-700 μm in length and 2-30 μm in diameter). The holding potential was -30 mV. Long-duration (3 sec) hyperpolarizing pulses were applied to -40 to -120 mV, in increments of 10 mV. Experiments were performed at room temperature (22℃). A　hyperpolarization-activated inward current (I_f) was produced. Current density at -120
mV was -1.03±0.31 pA/pF (n=5). The average capacitance was 64.3±2.3 pF (n=8). The threshold potential for activation of I_f was about -50 to -60 mV. The reversal potential was -18.6±2.1 mV (n=4). In the presence of Cs⁺ (3 mM), the I_f current
at -120 mV was decreased by 76.5±2.1% (P<0.01, n=5). These results indicate that the Cs⁺⁻sensitive hyperpolarization-activated inward current is present in the longitudinal muscle cells of pregnant rat uterus. This I_f current may contribute somewhat to the electrogenesis of the spontaneous activity

Nakashima, Toshikatsu

Satoh, Hiroyasu

English

Global Institutional repository of Nara Medical University

奈医誌. (J. N ara Med. Ass.) 44， 59~63 ， 1193 PACEMAKER CURRENT (HYPERPOLARIZATION -ACTIV ATED INWARD CURRENT) IN ISOLATED SINGLE PREGNANT RAT UTERINE MYOCYTES HIROYASU SATOH and TOSHIKATSU NAKASHIMA Dψartment 01 Pharmacology， Nara Medical UniversiかReceived恥1arch8， 1993 (59 ) Summary: Whole-cel1 patch c1amp experiments were performed to examine the underlying currents to generate spontaneous activity in freshly isolated single longitudinal musc1e cel1s of pregnant rat uterus C18-day gestation). Iso1ated single cel1s were spindle-or round-shaped (50-700μm in length and 2 -30μm in diameter). The holding potential was -30 m V. Long-duration (3 sec) hyperpolarizing pulses were applied to -40 to -120 mV， inincrements of 10 mV. Experiments were performed at room temperature (22 'C). A hyperpolarization-activated inward current CIf) was produced. Current density at -120 mV was -1.03土0.31pA/ pF (n = 5). The average capacitance was 64.3土2.3pF(n=8). The threshold potential for activation of 11 was about -50 to -60 m V. The reversal potential was -18.6土2.1m V (n = 4). ln the presence of Cs+ (3 mM)， the lf current at -120 m V was decreased by 76.5土2.1% (Pく0.01，nニ 5).These results indicate that the CsLsensitive hyperpolarization-activated inward current is present in the longitudinal musc1e cel1s of pregnant rat uterus. This lf current may contribute somewhat to the electrogenesis of the spontaneous activity. Index Terms hyperpolarization-activated inward current， pregnant rat uterus， single smooth musc1e cel1， whole-cell voltage-c1amp， electrophysiology INTRODUCTION Changes in the membrane potential of uterine smooth musc1e play an important role in regulation of the contraction and relaxation of the uterus. The functions are modulated by neurotransmitters and hormones pharmacomechanical1y and electrophysiological1yll2l. Rat uterus possesses spontaneous contraction and automaticity. ln cells possessing the automaticity， there might generally be an inward pacemaker current flowing during the pacemaker potential (or phase 4 depolarization). The hyperpolarization-activated inward curr百 ltCIf) is one of the inward currents contributing to depolarize the membrane to the threshold potential for action potential during diastole Recently， many informations about the ionic currents underlying the myometrial activity of isolated uterine single cells have been obtained by applying the patch-c1amp technique3)-8l. However， litle is known yet about the ionic currents underlying the spontaneous activity of uterine smooth musc1e. ln the present experiments， thus， we sought to examine the existence of the hyperpolar包 ation-activatedinward current (the pacemaker current)， lf， in 18-day (60 ) H. Satoh， etal gestation cells using whole-cell patch c1amp experiments. MATERIALS AND METHODS Single smooth musc1e cells were obtained from the longitudinallayer of pregnant rat uterus 08-day gestation)， according to the methods reported previously6)7)8). The tissues were incubated in nominally Ca2+ -free solution with 0.2% protease (Type X1V， Sigma Chemical， St.Louis， MO， USA) for 20 min after 60-min incubation with 0.2% collagenase (Worthington Biochemical). Spindle-and/or round-shaped cells (50-700μm in length， 2 -30μm in diame田ter) were isolated. Whole-cell voltage-c1amp recording was carried out with patch electrodes ( 3 -5 MO) using usually round-shaped cells and with an Axopatch patch-c1amp amplifier (Axon 1nstruments， Burlingame， CA， USA)9)-12). The patch electrodes were made from borosi1icate glass capillary tubing. The series resistance error was less than 3 -7 m V ; compensation was not used. All experiments were performed at room temperature (220C). The data were stored and analyzed on a 1BM-AT microcomputer， using the PCLAMP analysis program (Axon 1nstruments). Current traces were filtered， using a cutoff frequency of 1 KHz， for plotting. The values were expressed as mean :tSEM. The differences of the mean values were analyzed with Student's t-test for paired data. P value less than 0.05 was considered significant. The composition of the modified Tyrode solution was Cin mM) : N aCl 137， KCl 5.4， CaC12 1. 8， MgC12 1.0， N aH2P04 0.3， glucose 5.0， and HEPES 5. To avoid the interferences of other currents， 10μM TTX， 3 mM  CdC12， and 3 mM  BaC12 were added to external solution to block the fast N a+ current， the Ca2十 currentand the inward rectifier K十 current，respectively. The pipette solution contained Cin mM) : KOH 110， KCl 20， MgC12 2， EGT A 10， MgA TP 5， creatine phosphate 5， aspartic acid 100， and HEPES 5 (pH 7.2). The concentration of Ca2+ in pipette solution was adjusted to pCa 7， according to the calculations of Fabiato and Fabiato'3) and Tsien and Rink'4). RESULTS Long-duration (3 sec) hyperpolarizing pulses were applied to -40 to -120 m V from holding potential of -30 m V (Fig. 1A). Small 1f current was produced， as shown in Figs. 1B and C. The current was activated by hyperpolarizing pulses， and it had a very slow activation. Its amplitude was so small， and at -120 mV， the 11 current density was -1.03:t0.31 pA/pF (n= 5) (Fig. 2). The average capacitance was 64.3:t 2.3 pF (n = 8). The threshold potential was 60 to -70 mV. 1n the presence of Cs+ (3 mM)， the 11 current was inhibited to -0.25:t0.07 pA/pF at -120mV (Figs. 1D and 2). The percentage decrease was 76.5:t2.1%恒三5，P< 0.01) To examine the reversal potential for 11， test pulses were applied to -100 to + 10 m V， following conditioning potential of -120 m V， as shown in Fig. 3A. The current traces elicited by the test pulses are represented in Fig. 3B. Figure 4 shows the current-voltage relationship for the currents elicited by the test pulses. The reversal potential was -18.6:t2.1 mV (n=3). Pacemaker curr巴nt(hyperpolarization-activated inward current) in isolat巴dsingle pregnant rat uterin巴myocytes (61 ) A四30-.二空旦旦~B-よnlrol 一一升T胃ιし幽c-D-..，..制欄削削醐刷制~:Fig目1.Existence of hyperpolarization】activatedinward current (If) in single rat uterine ぜsmooth muscle cels during pregnancy (18-day). A: The voltag巴 clampprotocol Test pulses were applied from-40 m V to -120 mV， in 10 mV increments， from a holding potential of -30 m V. B: Presence of If in pregnant rat uterus. C: Activation of If at 120 m V from panel B. Dashed line indicates initial time-independ巴ntcurrent lev巴1. D: Blockade of If by Cs+ (3 mM) pA/pF O 0.5 1.0 帽 120 -80 回 40mV . . . 一・:瓦:6;8;⑨z・--e-・".. /【】/y./ O /白?t伽n圃 5剖} Fig. 2. Current-voltag巴 r巴lationshipfor If current density in the absence (open circles) and presenc巴 (fi1ledcircl巴s)of Cs+ (3 mM). Points plotted are the mean土SEM(n二 5);th巴SEMbars are les than the stickness of th巴symbols. External solution included 3 mM BaC12，1μM TTX， and 3 mM CdCl2・Experi伺ments were performed at room temperatur巴(22"C)ー Dashedline pr巴sentsthe zero curr巴ntlev巴l目4-Aminopyridine (3 mM) was added further to the external solution to block the transient outward current. DISCUSSION In the present experiments， the longitudinal muscle cell from pregnant rat uterus (18-day gestation) exhibited the If current (but smal) by hyperpolarizing pulses. The If was activated in a time-and voltage-dependent manner. The If required long hyperpolarizing steps for 3 sec or more to reach maximum. The results were as follows: (1) The If current density was approximately 1.0 pA/pF. The average capacitance was about 64 pF. (2) The threshold potential was around -60 to一70m V. (3) The reversal potential was about -20 m V. (4) Cs+ (3 mM) inhibited the I f • The characteristics for the If current in this study are consistent with those from cardiac muscle cells'5)16)17)， and also with those from the circular muscle cells of pregnant rat uterus'8). Therefore， these results indicate that the Cs十 一sensitivehyperpolariza回tion-activated inward current in the longitudinal muscle of pregnant rat uterus is similar to the I current in cardiac muscles as well as in the circular muscle cells of pregnant rat uterus. The membrane hyperpolarizes during gestation， whereas it depolarizes during parturition'9). Smooth muscle cells of isolated rat myometrial strips generate bursts of electrical activity， regardless of pregnant or non-pregnant status. Although the mechanisms generating this spontaneous activity remain unclear， the If current would be one of many factors involved. In cardiac muscles， If plays an important role in the pacemaker potential of Purkinje fibers'5) and a relatively minor role in sino-atrial node cells'6)20)2'). In pregnant rat uterus. the I current has ( 62 ) H. Satoh， etal目A -301ニ120費7B Fig. 3. Reversal potential for If current in rat uterine myocyte during 18-day pregnancy. A: Volt回age-c1amp protocol. Current r巴cordingsat test pulses ( -100 to十10mV) from condition同ing puls巴 (at-120m V) of 3 sec. The holding potential was -30m V. B: The current traces by the conditioning pulse and test pulses (-100 to十10mV). Th巴 dashedline indi. cat巴szero current level. Ext巴rnalsolution induced 3 mM BaCl" 1μM TTX， 3 mM CdCI2， and 3 mM 4-aminopyridine (4-AP). pA +20 ノO J O 叩 -80 -60 寸o叩 cJ.1p，0.' 0 mV ト n，o0''"' 〆圃 20ト _0_0，00-0-Fig. 4. Curr巴nt-voltagecurves showing to reversal potential. The curv巴 cross巴dbetween -20 to -10 m V. The data were plotted against voltage axis from th巴 recordingsin a single pregnant uterine smooth musc1e cel Cin Fig. 3B). been reported to be present in only circular smooth muscle cells， but not in longitudinal muscle cells18). If so， the spontaneous activity to be generated by the If current would be produced from only the circular muscle cels. However， the If current also actually existed in the longitudinal muscle cells， which has so small current density. Unfortunately， it is unlikely that the If current from the longitudinal muscle makes a major contribution to the spontaneous activity in pregnant rat uterus smooth muscles. Because the maximum diastolic potential of spontaneous action potential is not so negative (-50 to -60 m V)， and the If current required long (3 sec) and high negative voltages (-60 to -120 m V) of hyperpolarizing pulses. In addition， because the If current amplitude was so much smaller， as compared to that of cardiac muscles. Inoue and Sperelakis8) have reported that the fast Na+ current increased in the single longitudinal muscle cells of rat pregnant uterus C18-day gestation)， as term approaches. The ratio of Ca干/Na+current decreases from 0.57 to 0.31 as term approaches in pregnant rat myometrium22)， suggesting that the Na+ current as a major factor may be mainly involved in the spontaneous activity during gestation and near parturition. The N a+ current may play an important role for parturition 0. e. spread of excitation throughout the uterine smooth muscle and its contraction). ln conclusion， the pregnant rat uterus smooth muscle cells exhibit a lot of ionic currents， as term approaches. Presumably the existence of these ionic currents seems to be preparation for parturition. Therefore， the If current in the pregnant rat uterus， as one factor， also must play an important role during gestation and near parturition. REFERENCES 1) Mironneau， J.: Excitation-contraction coupling in voltage c1amped uterine musc1e. ]. Physiol. (Lond.) 233 : 127-141，1973. Pacemak巴rcurrent (hyperpolarization-activated inward current) in isolated single pregnant rat uterine myocytes (63 ) 2) Honore， E.， Martin， C.， Mironneau， C. and Mironneau， J. : An ATP-sensitive conductanc巴 incultured smooth muscle c巴lsfrom pregnant rat myometrium目 Am.]. Physiol. 257 : C297-C305， 1989. 3) Coleman， H. A. and Parkington， H. C. : Single chann巴1Cl-and K+ curr巴ntsfrom cells of uterus not treat巴dwith enzymes. Pflug巴rArch. 410 : 560-562， 1987. 4) Jmari， K.， Mironneau， C. and Mironneau， J. : Inactivation of calcium chann巴1current in rat uterine smooth muscle : evidence for calcium and voltage-mediated mechanism. ]. Physiol. CLond.) 380 : 111-126， 1986. 5) Kao， C. Y. and McCullough， J.R. : Ionic currents in the uterine smooth muscle. ]. Physiol. CLond目)246 : 1-36， 1975 6) Ohya， Y. and Sperelakis， N. : Fast Na+ and slow Ca2+ channels in single uterine muscle cells from pr巴gnantrat. Am.]. Physiol. 257 : C408-C412， 1989. 7) Ohya， Y. and Sperelakis， N. : Tocolytic agents act on calcium channel curr巴ntin singl巴 smoothmuscle cells of pregnant rat uterus. ]. Pharmacol. Exp. Th巴r.253・580-585，1990. 8) Inoue， Y. and Sperelakis， N. : Gestational chang巴 inNa+ and Ca2+ channel current densities in rat myometrial smooth muscl巴 cels.Am. ]. Physiol. 260 : C658-C663， 1991. 9) Satoh， H. : Inhibition of L-typ巴 Ca2+channel by stimulation of protein kinase C in isolated guinea-pig ventricular cardiomyocytes. Gen. Pharmacol. 23 : 1097-1102， 1992. 10) Satoh， H. and Sperelakis， N. : Id巴ntificationof the hyperpolarization-activated inward curr巴ntin young embryonic chick heart myocytes. ]. Devel. Physiol. 15 : 247-252， 1991. 11) Satoh， H. and Sperelakis， N. : Taurine inhibition of Na+current in embryonic chick v巴ntricularmyocytes. Eur ]. Pharmacol目 218: 83-89， 1992. 12) Satoh， H.呂ndSperelaki主ds，N. : Taur加巴巴ぱf白巴ctson Ca2肝十 curr巴nt匂si泊nyoung embryonic chick cardiomyocy計te白s.Eur目].Pharmacol. 231・443-499，1993. 13) Fabiato， A. and Fabiato， F. : Calculator programs for computing the composition of the solutions contain. ing multipl巴metalsand ligands used for experimemts in skinned muscl巴cels.]. Physiol. CLond.) 75 : 463 505， 1979. 14) Tsien， R. Y. and Rink， T. J. : N巴utralcarri巴rion-selective microelectrodes for measurements of intrac巴1.lular fr巴巴 calcium. Biochim. Biophys‘Acta 559 : 623-638， 1980 15) DiFrancesco， D. and Ojeda， C. : Properties of the pacemaker current i， inthe sinoatrial node of th巴rabbit : a comparison with the current ik2 

PACEMAKER CURRENT (HYPERPOLARIZATION-ACTIVATED INWARD CURRENT) IN ISOLATED SINGLE PREGNANT RAT UTERINE MYOCYTES

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PACEMAKER CURRENT (HYPERPOLARIZATION-ACTIVATED INWARD CURRENT) IN ISOLATED SINGLE PREGNANT RAT UTERINE MYOCYTES

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