351
Journal of Physiology (1991), 442, pp. 351-375 With 12 figures Printed in Great Britain
SODIUM CURRENTS IN SMOOTH MUSCLE CELLS FRESHLY ISOLATED FROM STOMACH FUNDUS OF THE RAT AND URETER OF THE GUINEA-PIG
BY KATSUHIKO MURAKI, YUJI IMAIZUMI* AND MINORU WATANABE From the Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467, Japan
(Received 26 November 1990) SUMMARY
1. Inward currents elicited by depolarization from holding potentials of -80 to -10 mV in single smooth muscle cells isolated from stomach fundus of the rat and ureter of the guinea-pig had two components. The initial fast component (Ifi) was activated and mostly inactivated within 1 2 and 10 ms, respectively, at 21 'C. The following sustained component (ISi) lasted over 50 and 500 ms in fundus and ureter cells, respectively. Iii was blocked by tetrodotoxin but not affected by 05 ,/M-ftconotoxin in both types of cells. 'Si was abolished by the substitution of extracellular Ca2+ with Mn2+. 2. The sensitivity of Ifis to TTX was markedly different in fundus and ureter cells. The half-inhibition was obtained at 870 and 11 nm, respectively. The amplitude of Ifi was highly dependent on extracellular Na+ concentration in a solution containing 2-2 mM-Mn2' and 0 mM-Ca21 in both cells. It is concluded that Ifis in these cells are TTX-sensitive and pu-conotoxin-insensitive Na+ currents. 3. Some of the kinetics of INa measured at 10 'C were markedly different in fundus and ureter cells. The current-voltage relationships for Ifi in fundus and ureter cells had peaks at about -10 and 0 mV, respectively. The voltage dependence of the steady-state inactivation of Ifi was also significantly different in these cell types. The half-inactivation voltages were about -74 and -45 mV, respectively. The recovery time course from inactivation in fundus cells was about 10 times slower than that in ureter at -80 mV, where it was 25 ms. 4. The contribution of Ifi to the rising phase of an action potential was examined using TTX under current clamp mode at 21 'C. A fast notch-like potential elicited by a subthreshold stimulus for action potential generation was blocked by TTX in both types of cells. Action potentials elicited by a stimulus around threshold were occasionally suppressed by TTX, whereas an action potential was never observed when extracellular Ca2+ was replaced with Mn2+. 5. In conclusion, the existence of at least two types of Na+ channel currents, which were distinguished by their TTX sensitivity and kinetics, was strongly suggested in smooth muscle cells from the rat fundus and the guinea-pig ureter. INa in these cells may have a physiological role to accelerate the generation of an action potential by * To whom all correspondence should be addressed. MS 8963
352
K. MURAKI, Y. IMAIZUMI AND M. WATANABE
triggering a rapid activation of ICa. while not being essential for activation of action potentials. INTRODUCTION
It is of considerable importance to obtain precise understanding about basic ionic mechanisms underlying the generation of the action potential in an excitable cell. With regard to smooth muscle cells, it is widely accepted that the major carrier ion of the inward current responsible for the rising phase of the action potential is Ca2+ (Tomita, 1970, 1981; Prosser, 1974). Since the tight seal voltage clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was introduced using single smooth muscle cells to analyse ionic current exactly, evidence has been accumulated in many types of smooth muscles that the major inward current upon depolarization is a current through voltage-dependent Ca2+ channels under physiological conditions (see reviews by Kitamura, Inoue, Inoue, Ohya, Terada, Okabe & Kuriyama, 1989; Watanabe, Imaizumi, Muraki & Takeda, 1989). Interestingly, a small ( < 20 pA) fast Na+ current highly sensitive to TTX has been found in cells from rabbit pulmonary artery in which electrical activity is not observed under physiological conditions (Okabe, Kitamura & Kuriyama, 1988). In primary cultured smooth muscle cells isolated from azygos vein of the neonatal rat, a large Na+ current has been observed (Sturek & Hermsmeyer, 1986). Recently, it has been reported that freshly isolated smooth muscle cells from rat myometrium (Ohya & Sperelakis, 1989) and portal vein (Okabe, Kajioka, Nakao, Kitamura & Kuriyama, 1990; Mironneau, Martin, Arnaudeau, Jmari, Rakotoarisoa, Sayet & Mironneau, 1990) also have a fast Na+ current which is highly sensitive to TTX. However, a clear line of evidence for a substantial contribution of a fast Na+ current to the rising phase of action potentials under physiological conditions has not been shown yet in any smooth muscle cells, while the possibility has been suggested in myometrium (Amedee, Renaud, Jmari, Lombet, Mironneau & Lazdunski, 1986). We here report Na+ currents which have amplitudes larger than or comparable to that of Ca2+ current under physiological conditions in freshly isolated smooth muscle cells from the rat stomach fundus or the guinea-pig ureter, respectively. The results strongly suggest the possibility that Na+ current contributes, in part, to the early rising phase of action potentials in these tissues under physiological conditions. Moreover, marked differences were observed in the characteristics of Na+ currents between these two types of smooth muscle cells. METHODS
Cell isolation Male rats (Wistar) weighing 180-200 g and guinea-pigs weighing 200-250 g were anaesthetized with ether or stunned and were killed by bleeding. Single smooth muscle cells were obtained from guinea-pig ureter as described previously using collagenase (Yakult, Tokyo) (Imaizumi, Muraki & Watanabe, 1989b; Imaizumi, Muraki, Takeda & Watanabe, 1989a). Those from the stomach fundus of the rat were also isolated in a similar manner, except for the simultaneous use of 0-1 % collagenase and 0-1 % elastase (Sigma, type III). Isolated cells were kept in HEPES-buffered solution containing 50 ,uM-Ca2+ at room temperature. Only relaxed cells having a spindly shape and length over about 90 ,um were used for electrical recordings within 3 h after isolation. The shape of fundus smooth muscle cells was similar to that of ureter cells. The total numbers of fundus and ureter cells used in the present study were approximately 200 and 150, respectively. A few drops of cell suspension were placed in the recording bath (0 5 ml) mounted on the stage of a phase-
SODIUM CURRENT IN SMOOTH MUSCLE
353
contrast microscope (Nikon, TMD) for electrical measurements. The cells in the bath were continuously perfused with solutions at a rate of 5 ml/min.
Electrical recordings. Whole-cell voltage clamp records were obtained by using a single suction micropipette method (Hamill et al. 1981). The amplifiers used were those reported previously (Clark & Giles, 1987) and List EPC-7. Pipette resistances ranged from 1 to 3 MQ, when filled with the pipette solution. The liquid junction potential of the pipette solution containing 148 mM-Cl- was less than 4 mV and not corrected for. The series resistance was partly compensated electronically and decreased to 2-5 MQ. To examine the space clamp of the cell during Na+ current recording, the membrane potential was monitored by applying another suction micropipette to the cell in some experiments (Seyama & Yamaoka, 1988). The distance between the tips of two pipettes was about 70,cum. Experiments were performed at room temperature (21 + 2 °C) except when mentioned otherwise. Most of the kinetics of INa were measured at 10 + 1 °C, as well as at room temperature. In the text, the temperatures were simply given as 21 or 10 °C, omitting the ranges.
Data storage and analysis Data storage and analysis were performed in the same way as described previously (Imaizumi et al. 1989a, b). In brief, electrical events were monitored on a storage oscilloscope (VC-6041, Hitachi, Tokyo, Japan) and stored on videotape after being digitized by a PCM-recording system (PCM 501ES, SONY; modified to obtain a frequency response from DC to 20 kHz). The data on the tape were replayed off-line through an A-D converter (Datatranslation; DT2801A) for analysis. Data acquisition (AQ, Robinson & Giles, 1986) and analysis software (DAS) for IBM-AT were supplied by Dr Wayne Giles (University of Calgary, Canada). Since the fastest sampling rate of the A-D converter was 25 kHz, the data directly digitized from the videotape did not have a good enough quality for analysis of fast INa kinetics. To improve the relationship between signal and data acquisition speed, some of the stored data on the videotape were occasionally re-stored on another cassette tape-recorder (TEAC, MR-10; frequency response of 20 kHz) and were replayed at slower tape speed (one-eighth) for data acquisition. In most experiments, both capacitive transient and leakage current were corrected for on the computer. Capacitive transient was removed by subtraction of current obtained by applying a pulse of the same voltage and opposite direction. The leak at positive potentials was obtained by extrapolating the linear relationship between voltage and current obtained in the range of -80 to -50 mV and was subtracted from the total current. Selected records were printed out using a laser printer (Hewlett-Packard Laser Jet Series II) or X-Y plotter (Loland DXY-1300). Curve fitting was performed using the algorithm of damping Gauss-Newton or modified Marquardt method on an IBM-AT. The pooled data were averaged and illustrated as a mean and the S.E.M. was given as a vertical bar. Statistical significance was examined using Student's t test.
Solutions and drugs The composition of normal medium was as follows (in mM): NaCl, 137; KCI, 5*9; MgCl2, 1P2; CaCl2, 2-2; glucose, 14-0; HEPES, 10-0. The pH was adjusted to 7-2 by NaOH. When the concentration of extracellular Na+ was decreased, Na+ in the normal solution was replaced with equimolar tetraethylammonium+ (TEA+), Tris+ or K+. In some cases, to block Ca2+ channel current, 2-2 mM-Ca2+ in the normal solution was replaced with equimolar Mn2+ (the Mn2+ solution). The solution in the recording pipette for current clamp contained (in mM): KCI, 140; ATP.2Na, 5; MgCl2, 4; HEPES, 10; EGTA, 1. The pH was adjusted to 7-2 with KOH. For voltage clamp, potassium in the pipette solution was replaced by equimolar caesium to block outward currents and EGTA in the pipette solution was increased to 5 mm to prevent contraction. The following toxins were used: tetrodotoxin (Sigma), ,u-conotoxin (Peptide Institute Inc.). RESULTS
Current-voltage relationship of inward current Figure IA shows inward currents upon depolarization from two different holding potentials (a, -80 mV; b, -60 mV) in a smooth muscle cell obtained from the rat stomach fundus. Depolarization to -20 mV from a holding potential of -80 mV 12
PHY 442
K. MURAKI, Y. ILMAIZUMI AND M. WATANABE
354
elicited a large fast inward current (Ifi) in the standard HEPES-buffered solution (Fig. I Aa). The inactivation of Ifi was almost accomplished within 20 ms at -20 mV. A following sustained inward current (ISi) was more clearly observed at potentials positive to -10 mV. When the holding potential was changed to -60 mV, Ifi was A
Aa
-80mV
B
b
-60mV ~~ f____@ ~~~~~t
-20 mV
-30
-0
10
. 30 mV
00.
-10 mV
0 mV
0pA 10 ms
[pA 10 ms
Fig. 1. Inward current was measured from a single fundus smooth muscle cell at 21 'C. A, inward current was elicited by depolarization from a holding potential of -80 (a) or -60 mV (b) to the potentials denoted on the left side. Initial 30 ms of a 50 ms recording is shown in A. Pulses were applied at 0 I Hz. B, peak amplitude of the inward current was plotted against potentials of test pulse as current-voltage (I-V) relationships. 0 and denote the peak amplitude of inward current elicited by depolarization from -80 and -60 mV, respectively. The continuous lines in B are fitted by eye.
markedly reduced and, therefore, 'si was more apparent (Fig. lAb). By changing the holding potential, the peak inward current was decreased by 56 % at -10 mV (Fig. lAb). Figure I B shows the current-voltage relationships (I-V curves) of peak inward current elicited by depolarization from -80 and -60 mV. The holding potential change had, however, little effect on the peak inward current in the positive voltage range between + 10 and + 30 mV in which inward current mainly consists of ISi (not shown). In normal solution, the average amplitude of peak inward current upon depolarization from -80 to -10 mV was 304 9 + 38 pA (n = 9). The inward currents elicited by depolarization to -20 and -10 mV were decreased to 26-0 + 2-0 (n = 4) and 46-8 + 4-1 % (n = 4), respectively, by the change in holding potential from -80 to -60 mV. Almost the same protocol was applied to smooth muscle cells isolated from guineapig ureter (Fig. 2). Unlike fundus cells, ISi appeared to be predominant in ureter cells, whereas Ifi was distinct from I.i as an initial notch at -10 and 0 mV when depolarized from -80 mV as shown in Fig. 2A. The I-V curves obtained by depolarization from holding potentials of -80 and -50 mV overlapped each other
SODIUM CURRENT IN SMOOTH MUSCLE 355 in the whole voltage range examined in ureter cells (Fig. 2B). A small hump at -10 mV in the I-V curve was observed when the holding potential was -80 mV and is considered to be due to the Ifi. From the fast kinetics of activation, inactivation and the voltage dependence of Ifi, it was most probable that Ifis in both types of cells were transient-type Ca2+ channel A
a
-80 mV
B
b
-30 -10 10
30 mV
-50 mV
-100
-20 mV
-300
pA
-10 mV
0 mV
250pA 10 ms Fig. 2. Inward current was measured from a single ureter smooth muscle cell at 21 'C. A, inward current was elicited by depolarization from a holding potential of -80 (a) or -50 mV (b) to the potentials denoted on the left side. B, peak amplitude was plotted against potentials of test pulse as I-V relationships. 0 and A denote the peak amplitude of inward current elicited by depolarization from -80 and -50 mV, respectively. The continuous lines in I-V relationships in B are fitted by eye.
currents (T-type Ca21 channel currents; Bean, 1985). This possibility was examined by substituting extracellular Ca2+ with Mn2 , which has a far lower permeability through voltage-dependent Ca2+ channels (Hess, Lansman & Tsien, 1986).
Effects of Mn2+ and TTX on inward current Ifis in both fundus and ureter cells were resistant to the replacement of Ca21 with Mn2+ (Fig. 3A and B, respectively). The replacement completely suppressed ISi in both types of cells, whereas substantial Ifis remained (Fig. 3Ab and Bb). The effects were completely reversible (Fig. 3Ac and Bc). With the replacement of Ca2+ with Mn2+, the peak inward currents induced by depolarization to -10 mV in the fundus and ureter cells were decreased to 73-9 + 7-0 % (n = 3) and 40-8% (n = 2) of the control, respectively. The Isis which were inhibited by Mn2+ in both cells are thought to be inward currents which go through voltage-dependent Ca2+ channels, but the Ifis are not Ca2+ channel currents. The sustained currents shown in Fig. 3Ad and Bd were obtained by subtracting b from a in Fig. 3 A and B, respectively, and correspond to Mn2+-sensitive currents. Another candidate for inward current which has fast 12-2
K. MURAKI, Y. IMAIZUMI AND M. WATANABE
356
kinetics of activation and inactivation is Na+ channel current. Therefore, in Fig. 4, effects of tetrodotoxin (TTX) on inward current in both types of cell were examined, since TTX is a specific Na+ channel blocker (Narahashi, Moore & Scott, 1964). Application of 10 /M- and 5 nM-TTX to fundus and ureter cells abolished or A
a
-10 mV
b
c
d
c
d
-80 mV
pA B
a
-10 mV
b
lOims
-70 mV
DA
10 ms Fig. 3. Effect of replacement of extracellular Ca2+ with equimolar Mn2+ (the Mn2+ solution) on inward current upon depolarization in fundus (A) and ureter (B) cells. a, inward current elicited by depolarization from -80 (A) or -70 mV (B) to -1O mV in the standard HEPES-buffered solution. b, extracellular solution was changed from the HEPES-buffered solution to the MnI2+ solution. Note that only the sustained inward current was blocked in the Mn2+ solution. c, the inward current recovered by changing the solution back to the standard HEPES-buffered solution containing Ca2+. d, the current obtained by subtracting b from a. Note that the current is a Mn2+-sensitive component (Isi)
markedly reduced the Ifis (Fig. 4Ab and Bb), respectively, but had no effect on Isis. Similar observations were obtained in four separate cells from each muscle. Like the action of Mn2+ on 'si (see Fig. 3Ac and Bc), the effects of TTX on Ifi were completely removed by wash-out (Fig. 4Ac and Bc). It is strongly suggested that Ifis in both types of cells are Na+ channel currents (INa). The currents obtained by subtraction (Fig. 4Ad and Bd) were quite transient and correspond to TTX-sensitive currents. It is notable that the currents in Fig. 3Ab and Bb are comparable to those in Fig. 4Ad and Bd, respectively, and those in Fig. 3Ad and Bd are also comparable to those in Fig. 4Ab and Bb, respectively. Space clamp during fast inward current Na+ currents in cardiac and neuronal cells have very fast kinetics of activation and large amplitude. In these cells, space clamp of the whole cell and accurate measurement of INa are very difficult under normal conditions at room temperature when Na+ currents are fully activated. To examine the capability of space clamp
SODIUM CURRENT IN SMOOTH MUSCLE 357 under the experimental conditions in the present study, membrane potential was monitored under the whole-cell voltage clamp in Mn+ solution at 20 °C using an additional recording pipette which was placed about 70 /sm from the first pipette for whole-cell clamp in a manner similar to that reported previously (Seyama & A
a
-10 mV
c
b
d
-80 mV
B a
-10 mV
-
~0 FpA
~
~~~~~-
b
10 ms
c
d
-70 mV
Journal of Physiology (1991), 442, pp. 351-375 With 12 figures Printed in Great Britain
SODIUM CURRENTS IN SMOOTH MUSCLE CELLS FRESHLY ISOLATED FROM STOMACH FUNDUS OF THE RAT AND URETER OF THE GUINEA-PIG
BY KATSUHIKO MURAKI, YUJI IMAIZUMI* AND MINORU WATANABE From the Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467, Japan
(Received 26 November 1990) SUMMARY
1. Inward currents elicited by depolarization from holding potentials of -80 to -10 mV in single smooth muscle cells isolated from stomach fundus of the rat and ureter of the guinea-pig had two components. The initial fast component (Ifi) was activated and mostly inactivated within 1 2 and 10 ms, respectively, at 21 'C. The following sustained component (ISi) lasted over 50 and 500 ms in fundus and ureter cells, respectively. Iii was blocked by tetrodotoxin but not affected by 05 ,/M-ftconotoxin in both types of cells. 'Si was abolished by the substitution of extracellular Ca2+ with Mn2+. 2. The sensitivity of Ifis to TTX was markedly different in fundus and ureter cells. The half-inhibition was obtained at 870 and 11 nm, respectively. The amplitude of Ifi was highly dependent on extracellular Na+ concentration in a solution containing 2-2 mM-Mn2' and 0 mM-Ca21 in both cells. It is concluded that Ifis in these cells are TTX-sensitive and pu-conotoxin-insensitive Na+ currents. 3. Some of the kinetics of INa measured at 10 'C were markedly different in fundus and ureter cells. The current-voltage relationships for Ifi in fundus and ureter cells had peaks at about -10 and 0 mV, respectively. The voltage dependence of the steady-state inactivation of Ifi was also significantly different in these cell types. The half-inactivation voltages were about -74 and -45 mV, respectively. The recovery time course from inactivation in fundus cells was about 10 times slower than that in ureter at -80 mV, where it was 25 ms. 4. The contribution of Ifi to the rising phase of an action potential was examined using TTX under current clamp mode at 21 'C. A fast notch-like potential elicited by a subthreshold stimulus for action potential generation was blocked by TTX in both types of cells. Action potentials elicited by a stimulus around threshold were occasionally suppressed by TTX, whereas an action potential was never observed when extracellular Ca2+ was replaced with Mn2+. 5. In conclusion, the existence of at least two types of Na+ channel currents, which were distinguished by their TTX sensitivity and kinetics, was strongly suggested in smooth muscle cells from the rat fundus and the guinea-pig ureter. INa in these cells may have a physiological role to accelerate the generation of an action potential by * To whom all correspondence should be addressed. MS 8963
352
K. MURAKI, Y. IMAIZUMI AND M. WATANABE
triggering a rapid activation of ICa. while not being essential for activation of action potentials. INTRODUCTION
It is of considerable importance to obtain precise understanding about basic ionic mechanisms underlying the generation of the action potential in an excitable cell. With regard to smooth muscle cells, it is widely accepted that the major carrier ion of the inward current responsible for the rising phase of the action potential is Ca2+ (Tomita, 1970, 1981; Prosser, 1974). Since the tight seal voltage clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was introduced using single smooth muscle cells to analyse ionic current exactly, evidence has been accumulated in many types of smooth muscles that the major inward current upon depolarization is a current through voltage-dependent Ca2+ channels under physiological conditions (see reviews by Kitamura, Inoue, Inoue, Ohya, Terada, Okabe & Kuriyama, 1989; Watanabe, Imaizumi, Muraki & Takeda, 1989). Interestingly, a small ( < 20 pA) fast Na+ current highly sensitive to TTX has been found in cells from rabbit pulmonary artery in which electrical activity is not observed under physiological conditions (Okabe, Kitamura & Kuriyama, 1988). In primary cultured smooth muscle cells isolated from azygos vein of the neonatal rat, a large Na+ current has been observed (Sturek & Hermsmeyer, 1986). Recently, it has been reported that freshly isolated smooth muscle cells from rat myometrium (Ohya & Sperelakis, 1989) and portal vein (Okabe, Kajioka, Nakao, Kitamura & Kuriyama, 1990; Mironneau, Martin, Arnaudeau, Jmari, Rakotoarisoa, Sayet & Mironneau, 1990) also have a fast Na+ current which is highly sensitive to TTX. However, a clear line of evidence for a substantial contribution of a fast Na+ current to the rising phase of action potentials under physiological conditions has not been shown yet in any smooth muscle cells, while the possibility has been suggested in myometrium (Amedee, Renaud, Jmari, Lombet, Mironneau & Lazdunski, 1986). We here report Na+ currents which have amplitudes larger than or comparable to that of Ca2+ current under physiological conditions in freshly isolated smooth muscle cells from the rat stomach fundus or the guinea-pig ureter, respectively. The results strongly suggest the possibility that Na+ current contributes, in part, to the early rising phase of action potentials in these tissues under physiological conditions. Moreover, marked differences were observed in the characteristics of Na+ currents between these two types of smooth muscle cells. METHODS
Cell isolation Male rats (Wistar) weighing 180-200 g and guinea-pigs weighing 200-250 g were anaesthetized with ether or stunned and were killed by bleeding. Single smooth muscle cells were obtained from guinea-pig ureter as described previously using collagenase (Yakult, Tokyo) (Imaizumi, Muraki & Watanabe, 1989b; Imaizumi, Muraki, Takeda & Watanabe, 1989a). Those from the stomach fundus of the rat were also isolated in a similar manner, except for the simultaneous use of 0-1 % collagenase and 0-1 % elastase (Sigma, type III). Isolated cells were kept in HEPES-buffered solution containing 50 ,uM-Ca2+ at room temperature. Only relaxed cells having a spindly shape and length over about 90 ,um were used for electrical recordings within 3 h after isolation. The shape of fundus smooth muscle cells was similar to that of ureter cells. The total numbers of fundus and ureter cells used in the present study were approximately 200 and 150, respectively. A few drops of cell suspension were placed in the recording bath (0 5 ml) mounted on the stage of a phase-
SODIUM CURRENT IN SMOOTH MUSCLE
353
contrast microscope (Nikon, TMD) for electrical measurements. The cells in the bath were continuously perfused with solutions at a rate of 5 ml/min.
Electrical recordings. Whole-cell voltage clamp records were obtained by using a single suction micropipette method (Hamill et al. 1981). The amplifiers used were those reported previously (Clark & Giles, 1987) and List EPC-7. Pipette resistances ranged from 1 to 3 MQ, when filled with the pipette solution. The liquid junction potential of the pipette solution containing 148 mM-Cl- was less than 4 mV and not corrected for. The series resistance was partly compensated electronically and decreased to 2-5 MQ. To examine the space clamp of the cell during Na+ current recording, the membrane potential was monitored by applying another suction micropipette to the cell in some experiments (Seyama & Yamaoka, 1988). The distance between the tips of two pipettes was about 70,cum. Experiments were performed at room temperature (21 + 2 °C) except when mentioned otherwise. Most of the kinetics of INa were measured at 10 + 1 °C, as well as at room temperature. In the text, the temperatures were simply given as 21 or 10 °C, omitting the ranges.
Data storage and analysis Data storage and analysis were performed in the same way as described previously (Imaizumi et al. 1989a, b). In brief, electrical events were monitored on a storage oscilloscope (VC-6041, Hitachi, Tokyo, Japan) and stored on videotape after being digitized by a PCM-recording system (PCM 501ES, SONY; modified to obtain a frequency response from DC to 20 kHz). The data on the tape were replayed off-line through an A-D converter (Datatranslation; DT2801A) for analysis. Data acquisition (AQ, Robinson & Giles, 1986) and analysis software (DAS) for IBM-AT were supplied by Dr Wayne Giles (University of Calgary, Canada). Since the fastest sampling rate of the A-D converter was 25 kHz, the data directly digitized from the videotape did not have a good enough quality for analysis of fast INa kinetics. To improve the relationship between signal and data acquisition speed, some of the stored data on the videotape were occasionally re-stored on another cassette tape-recorder (TEAC, MR-10; frequency response of 20 kHz) and were replayed at slower tape speed (one-eighth) for data acquisition. In most experiments, both capacitive transient and leakage current were corrected for on the computer. Capacitive transient was removed by subtraction of current obtained by applying a pulse of the same voltage and opposite direction. The leak at positive potentials was obtained by extrapolating the linear relationship between voltage and current obtained in the range of -80 to -50 mV and was subtracted from the total current. Selected records were printed out using a laser printer (Hewlett-Packard Laser Jet Series II) or X-Y plotter (Loland DXY-1300). Curve fitting was performed using the algorithm of damping Gauss-Newton or modified Marquardt method on an IBM-AT. The pooled data were averaged and illustrated as a mean and the S.E.M. was given as a vertical bar. Statistical significance was examined using Student's t test.
Solutions and drugs The composition of normal medium was as follows (in mM): NaCl, 137; KCI, 5*9; MgCl2, 1P2; CaCl2, 2-2; glucose, 14-0; HEPES, 10-0. The pH was adjusted to 7-2 by NaOH. When the concentration of extracellular Na+ was decreased, Na+ in the normal solution was replaced with equimolar tetraethylammonium+ (TEA+), Tris+ or K+. In some cases, to block Ca2+ channel current, 2-2 mM-Ca2+ in the normal solution was replaced with equimolar Mn2+ (the Mn2+ solution). The solution in the recording pipette for current clamp contained (in mM): KCI, 140; ATP.2Na, 5; MgCl2, 4; HEPES, 10; EGTA, 1. The pH was adjusted to 7-2 with KOH. For voltage clamp, potassium in the pipette solution was replaced by equimolar caesium to block outward currents and EGTA in the pipette solution was increased to 5 mm to prevent contraction. The following toxins were used: tetrodotoxin (Sigma), ,u-conotoxin (Peptide Institute Inc.). RESULTS
Current-voltage relationship of inward current Figure IA shows inward currents upon depolarization from two different holding potentials (a, -80 mV; b, -60 mV) in a smooth muscle cell obtained from the rat stomach fundus. Depolarization to -20 mV from a holding potential of -80 mV 12
PHY 442
K. MURAKI, Y. ILMAIZUMI AND M. WATANABE
354
elicited a large fast inward current (Ifi) in the standard HEPES-buffered solution (Fig. I Aa). The inactivation of Ifi was almost accomplished within 20 ms at -20 mV. A following sustained inward current (ISi) was more clearly observed at potentials positive to -10 mV. When the holding potential was changed to -60 mV, Ifi was A
Aa
-80mV
B
b
-60mV ~~ f____@ ~~~~~t
-20 mV
-30
-0
10
. 30 mV
00.
-10 mV
0 mV
0pA 10 ms
[pA 10 ms
Fig. 1. Inward current was measured from a single fundus smooth muscle cell at 21 'C. A, inward current was elicited by depolarization from a holding potential of -80 (a) or -60 mV (b) to the potentials denoted on the left side. Initial 30 ms of a 50 ms recording is shown in A. Pulses were applied at 0 I Hz. B, peak amplitude of the inward current was plotted against potentials of test pulse as current-voltage (I-V) relationships. 0 and denote the peak amplitude of inward current elicited by depolarization from -80 and -60 mV, respectively. The continuous lines in B are fitted by eye.
markedly reduced and, therefore, 'si was more apparent (Fig. lAb). By changing the holding potential, the peak inward current was decreased by 56 % at -10 mV (Fig. lAb). Figure I B shows the current-voltage relationships (I-V curves) of peak inward current elicited by depolarization from -80 and -60 mV. The holding potential change had, however, little effect on the peak inward current in the positive voltage range between + 10 and + 30 mV in which inward current mainly consists of ISi (not shown). In normal solution, the average amplitude of peak inward current upon depolarization from -80 to -10 mV was 304 9 + 38 pA (n = 9). The inward currents elicited by depolarization to -20 and -10 mV were decreased to 26-0 + 2-0 (n = 4) and 46-8 + 4-1 % (n = 4), respectively, by the change in holding potential from -80 to -60 mV. Almost the same protocol was applied to smooth muscle cells isolated from guineapig ureter (Fig. 2). Unlike fundus cells, ISi appeared to be predominant in ureter cells, whereas Ifi was distinct from I.i as an initial notch at -10 and 0 mV when depolarized from -80 mV as shown in Fig. 2A. The I-V curves obtained by depolarization from holding potentials of -80 and -50 mV overlapped each other
SODIUM CURRENT IN SMOOTH MUSCLE 355 in the whole voltage range examined in ureter cells (Fig. 2B). A small hump at -10 mV in the I-V curve was observed when the holding potential was -80 mV and is considered to be due to the Ifi. From the fast kinetics of activation, inactivation and the voltage dependence of Ifi, it was most probable that Ifis in both types of cells were transient-type Ca2+ channel A
a
-80 mV
B
b
-30 -10 10
30 mV
-50 mV
-100
-20 mV
-300
pA
-10 mV
0 mV
250pA 10 ms Fig. 2. Inward current was measured from a single ureter smooth muscle cell at 21 'C. A, inward current was elicited by depolarization from a holding potential of -80 (a) or -50 mV (b) to the potentials denoted on the left side. B, peak amplitude was plotted against potentials of test pulse as I-V relationships. 0 and A denote the peak amplitude of inward current elicited by depolarization from -80 and -50 mV, respectively. The continuous lines in I-V relationships in B are fitted by eye.
currents (T-type Ca21 channel currents; Bean, 1985). This possibility was examined by substituting extracellular Ca2+ with Mn2 , which has a far lower permeability through voltage-dependent Ca2+ channels (Hess, Lansman & Tsien, 1986).
Effects of Mn2+ and TTX on inward current Ifis in both fundus and ureter cells were resistant to the replacement of Ca21 with Mn2+ (Fig. 3A and B, respectively). The replacement completely suppressed ISi in both types of cells, whereas substantial Ifis remained (Fig. 3Ab and Bb). The effects were completely reversible (Fig. 3Ac and Bc). With the replacement of Ca2+ with Mn2+, the peak inward currents induced by depolarization to -10 mV in the fundus and ureter cells were decreased to 73-9 + 7-0 % (n = 3) and 40-8% (n = 2) of the control, respectively. The Isis which were inhibited by Mn2+ in both cells are thought to be inward currents which go through voltage-dependent Ca2+ channels, but the Ifis are not Ca2+ channel currents. The sustained currents shown in Fig. 3Ad and Bd were obtained by subtracting b from a in Fig. 3 A and B, respectively, and correspond to Mn2+-sensitive currents. Another candidate for inward current which has fast 12-2
K. MURAKI, Y. IMAIZUMI AND M. WATANABE
356
kinetics of activation and inactivation is Na+ channel current. Therefore, in Fig. 4, effects of tetrodotoxin (TTX) on inward current in both types of cell were examined, since TTX is a specific Na+ channel blocker (Narahashi, Moore & Scott, 1964). Application of 10 /M- and 5 nM-TTX to fundus and ureter cells abolished or A
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10 ms Fig. 3. Effect of replacement of extracellular Ca2+ with equimolar Mn2+ (the Mn2+ solution) on inward current upon depolarization in fundus (A) and ureter (B) cells. a, inward current elicited by depolarization from -80 (A) or -70 mV (B) to -1O mV in the standard HEPES-buffered solution. b, extracellular solution was changed from the HEPES-buffered solution to the MnI2+ solution. Note that only the sustained inward current was blocked in the Mn2+ solution. c, the inward current recovered by changing the solution back to the standard HEPES-buffered solution containing Ca2+. d, the current obtained by subtracting b from a. Note that the current is a Mn2+-sensitive component (Isi)
markedly reduced the Ifis (Fig. 4Ab and Bb), respectively, but had no effect on Isis. Similar observations were obtained in four separate cells from each muscle. Like the action of Mn2+ on 'si (see Fig. 3Ac and Bc), the effects of TTX on Ifi were completely removed by wash-out (Fig. 4Ac and Bc). It is strongly suggested that Ifis in both types of cells are Na+ channel currents (INa). The currents obtained by subtraction (Fig. 4Ad and Bd) were quite transient and correspond to TTX-sensitive currents. It is notable that the currents in Fig. 3Ab and Bb are comparable to those in Fig. 4Ad and Bd, respectively, and those in Fig. 3Ad and Bd are also comparable to those in Fig. 4Ab and Bb, respectively. Space clamp during fast inward current Na+ currents in cardiac and neuronal cells have very fast kinetics of activation and large amplitude. In these cells, space clamp of the whole cell and accurate measurement of INa are very difficult under normal conditions at room temperature when Na+ currents are fully activated. To examine the capability of space clamp
SODIUM CURRENT IN SMOOTH MUSCLE 357 under the experimental conditions in the present study, membrane potential was monitored under the whole-cell voltage clamp in Mn+ solution at 20 °C using an additional recording pipette which was placed about 70 /sm from the first pipette for whole-cell clamp in a manner similar to that reported previously (Seyama & A
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