Journal of Physiology (1992), 454, pp. 549-571 With 11 figures Printed in Great Britain

549

POTENTIAL-DEPENDENT INWARD CURRENTS IN SINGLE ISOLATED SMOOTH MUSCLE CELLS OF THE RAT ILEUM

BY S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA From the Department of Nerve-Muscle Physiology, A.A. Bogomoletz Institute of Physiology, Academy of Ukrainian Sciences, Kiev, Ukraine

(Received 24 June 1991) SUMMARY

1. Calcium (ICa) and sodium (INa) currents were studied in single smooth muscle cells freshly isolated from both the newborn (1-3 days old) and adult rat ileum, using the patch-clamp technique (whole-cell configuration). 2. Under conditions when INa was blocked, two components of 'Ca' low-voltage activated or ICalow and high-voltage activated or ICahigh' were observed in the newborn rat ileal cells. ICa high and ICalow have differing voltage ranges of activation and steady-state inactivation and time courses of recovery from inactivation. Potential dependence of ICalow was much steeper and shifted toward negative membrane potential than that for ICahigh (slope factors and the potential of halfmaximal inactivation were 13-6 and -60X6 and 8X8 and -49 mV for ICalow and ICahigh' correspondingly). 3. Nifedipine at the high concentration of 30 gim exerted no effect on ICa low and only slightly suppressed ICa high' decreasing its peak to 0'81 + 004 (n= 7) at the holding potential of -80 mV and to 0-66 + 0.05 (n = 3) at -50 mV. ICahigh was suppressed significantly by Cd2+ ions, while Ica,low was more sensitive to Ni2+ ions. 4. Results presented here suggest that the properties of high-voltage-activated (HVA) Ca2+ channels in the rat small intestine are quite different to those described for L-type Ca2+ channels found in other smooth muscles. It is proposed that HVA Ca2+ channels are similar to N-type Ca2+ channels. 5. Comparison of Ca2+ currents in newborn and adult rat ileal cells showed that the contribution of Ica,low to the net Ca2+ current was negligible in adults, whereas the properties of HVA Ca2+ channels were similar in the neonatal and adult animals. 6. INa, studied in nominally Ca2+-free physiological salt solution, activated in the voltage range between -50 and -40 mV and reached its peak at -10 mV. INa was blocked in a dose-dependent manner by TTX with an apparent dissociation constant of 4-5 nm. 7. INa decay was monoexponential in the voltage range studied and its time constant decreased monotonically with membrane depolarization from 4-7 + 0-2 ms (n = 6) at -30 mV to 0'51 +0-03 ms (n = 7) at 20 mV. Also, a process of slow inactivation of INa, which was completed in about 150 ms, was found. 8. Steady-state inactivation of INa was described by the Boltzmann equation with a half-inactivation potential of - 60-3 mV and a slope factor of 9-8 mV. Recovery of Ms 9500

550

S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA

INa from inactivation

was

monoexponential and slowed with depolarization,

from

1841 + 1P4 ms (n = 5) at the holding potential of -80 mV to 46f5 + 3.0 ms (n = 5) at -60 mV. 9. In the presence of 2-5 mM-Ca2+ the dependence of INa inactivation on the membrane potential was shifted by about 13 mV toward positive voltages, i.e. more than 50 % of Na+ channels are able to be activated in the voltage range between -60 and -50 mV which is close to the resting potential in many intestinal smooth muscle at the physiological Ca2` concentration. INTRODUCTION

It is generally accepted that the generation of action potentials and activation of contraction in most visceral smooth muscle cells (SMCs) is due to activation of potential-dependent calcium channels. Whole-cell and patch-clamp studies of single visceral SMCs have shown that the SMC membrane possesses only long-lasting or high-voltage-activated (HVA) Ca2` channel (Ganitkevich, Smirnov & Shuba, 1985; Mitra & Morad, 1985; Klickner & Isenberg, 1985; Ganitkevich, Shuba & Smirnov, 1986; Ohya, Terada, Kitamura & Kuriyama, 1986; Jmari, Mironneau & Mironneau, 1986; Yamamoto, Hu & Kao, 1989; Aaronson & Russell, 1991; but see Yoshino, Someya, Nishio, Yazawa, Usuki & Yabu, 1989). However, in contrast to visceral SMC, at least two populations, HVA and transient or low-voltage-activated (LVA) Ca2+ channels, were found in many vascular SMCs (Bean, Sturek, Puga & Hermsmeyer, 1986; Friedman, Suarez-Kurtz, Kaczorowski, Katz & Reuben, 1986; Hirst, Silverberg & van Helden, 1986; Loirand, Pacaud, Mironneau & Mironneau, 1986; Sturek & Hermsmeyer, 1986; Worley, Deitmer & Nelson, 1986; Benham, Hess & Tsien, 1987; Aaronson, Bolton, Lang & MacKenzie, 1988; McCarthy & Cohen, 1989; Ganitkevich & Isenberg, 1990; but see Caffrey, Josephson & Brown, 1986; Matsuda, Volk & Shibata, 1990). Recently, Gonoi & Hasegawa (1988) have shown that the contribution of at least two populations of Ca2+ channels, transient (LVA) and sustained (HVA), to the net membrane current in mouse skeletal muscle depends on the age of animals. The transient calcium current disappeared progressively in the postnatal period, whereas the contribution of a sustained calcium current was increased. These findings are also confirmed by other work performed on embryonic, neonatal and cultured (which are also usually obtained from neonatal animals) cells obtained from different tissues and where properties of LVA, as well as HVA, Ca2+ channels have been well described (e.g. Bean, 1985; Bossu, Feltz & Thomann, 1985; Fedulova, Kostyuk & Veselovsky, 1985; Carbone & Lux, 1987; Fox, Novycky & Tsien, 1987a, b). Taking into account these observations, and since visceral SMCs studied before were obtained as a rule from adult animals, it might be expected that for SMCs isolated from the intestine of the younger animals LVA. Ca2+ currents should be more pronounced. To check this assumption we performed a study of a Ca2+ currents (ICa) in single SMCs isolated from the small intestine of both newborn and adult rats. Also, the properties of a tetrodotoxin-sensitive potential-dependent sodium inward current (INa), which was found in these cells in contrast to other intestinal SMCs, have been described.

Ca2+ AND Na+ CHANNELS IN SMOOTH MUSCLE

551

METHODS

Experiments were performed on single isolated SMCs obtained from the longitudinal layer of the small intestine of both the newborn and adult Wistar rat. Solutions The composition of physiological salt solution (PSS) used for the isolation procedure was (mM): 130 NaCl, 5 KCl, 5 MgCl2, 2-5 CaCl2, 6 glucose, 10 HEPES. pH was adjusted to 7-2-7'3 with NaOH. Ca2l-free PSS was similar to PSS except that CaCl2 was omitted. The main Cs+-containing external solution for electrophysiological recordings of Ca2+ channel currents had the following composition (mM): 130 TEA-Cl, 5 CsCl, 2-5 CaCl2, 5 MgCl2, 6 glucose, 10 HEPERS. pH was adjusted with TEAOH to 7-2-7-3. TEA-Cl was used instead of NaCl in this kind of experiment to block sodium fluxes through potential-dependent Na+ channels. External solutions containing elevated Ca2" or Ba2+ concentration were prepared on the basis of the main solution by substitution of 2-5 mm-CaCl2 with an appropriate amount of CaCl2 or BaCl2. The composition of the external solution used for recording of INa was basically similar to the main CsW-containing solution except for the presence of 130 mM-NaCl instead of TEA-Cl. Also, Ca2+ ions were omitted to block ICa. Pipette solution contained (mM): 130 CsCl, 2-25 MgCl2, 3 Na2ATP, 3 4-aminopyridine, 5 HEPES, 5 EGTA. pH was adjusted to 7-2-7-3 using TEA-OH. cAMP (0-1 mM) was added to the pipette solution to prevent a run-down of Ca2+ channel currents. 4-Aminopyridine was present to block any current through potential-dependent fast-inactivating potassium channel (Smirnov, Zholos & Shuba, 1992). Isolation of SMCs Newborn rats (1-3 day-old, 5-9 g weight) were decapitated and a segment of small intestine 6-8 cm long was removed, cut into pieces about 5 mm long, and placed in Ca2+-free PSS. The contents of the intestine were removed using a syringe filled with the same solution. The longitudinal layer of SMC was carefully removed from the pieces under stereomicroscope and then placed in PSS for about 1 h at room temperature. After this time pieces were replaced into Ca2+-free PSS for 15-20 min and then transferred to 1-5 ml fresh Ca2+-free PSS which contained 1 mg collagenase (Sigma, type I), about 0-5 mg elastase (Reanal) and 3 mg bovine serum albumin (Sigma). After 13-17 min incubation at 36 °C pieces were transferred to fresh Ca2+-free PSS and gently agitated with a 2 ml Pasteur pipette until the solution had a cloudy appearance. Drops of cell suspension were placed into Petri dishes and after 2-3 min (to allow SMCs to adhere to the glass bottom) PSS was added. Cells were stored in the Petri dishes at room temperature for 4-6 h for duration of the experiments. Relaxed SMCs were 50-100 ,um long and 3-5 ,um in diameter. They contracted in response to application of 130 mM-KCl, 1 /tM-acetylcholine and 1 mM-ATP. Some cells were contracted and had a rounded form (15-30 um in diameter). It should be noted that some of these rounded cells were able to reversibly contract in response to drug application. The procedure for preparation of a single SMC from the ileum of the adult animal (90-200 g weight, 2-5-3 months old) was generally the same. However, concentrations of collagenase and elastase were increased, to 2 and 1 mg correspondingly, as was the incubation time (50-60 min). Single SMCs obtained from the adult rat ileum were about 2-3 times larger than those from the newborn rat and were also able to contract in response to the above mentioned agents.

Experimental procedure The technique of current recording did not differ from that previously described (Ganitkevich et al. 1986). A 500 MQ feed-back resistor was used in the current-voltage converter. All current recordings were made at 2 5 kHz. Pipettes filled with the pipette solution had a resistance of 2-4 MIQ. Pipette tips were coated with Sylgard 184 to minimize the capacitance artifact. Cells with a duration of capacitance current smaller than 0 5 ms were usually used. Series resistance compensation was introduced to give the fastest transient before oscillation. Leak conductance was smaller then 0-1-0-5 nS and the leak current was not usually compensated, unless otherwise indicated. Different test solutions were applied by pressure onto the cell through a four-barrel pipette, which was placed at a distance about 300 ,um from the cell. One barrel, connected to a peristaltic pump, was used for removal of the solution from the dish.

552

S. V. SMIRNOV, A. V. ZHOLOS AND M. F SHUBA

Experiments were performed at room temperature. Values are given as means and standard error of the mean (S.E.M.) with the number of cells in parentheses. Curve fitting was performed using a least-squares method. The significance of an experimental value was determined using a Student's two-tail t test unless otherwise indicated. Drugs Drugs were obtained from the following suppliers: N-2-hydroxyethylpiperazine-N' -2-ethanesulphonic acid (HEPES, Sigma); ethyleneglycol-bis-(fl-aminoethylether)-NVN',.-tetraacetic acid (EGTA, Sigma); tetraethylammonium chloride (TEA-Cl, Sigma); tetraethylammonium hydroxide (TEA-OH, Sigma); tetrodotoxin (TTX. Sankyo, Japan or Serva). Nifedipine (Sigma) was prepared as a 3 mm stock solution in ethanol immediately before use and diluted by the external solution to the final concentration. RESULTS

In conditions where K+ currents were blocked, two major components of inward current have been observed in the newborn rat ileal SMC: rapidly and slowly inactivating ones. These are due to fluxes of Na+ and Ca21 ions through separate potential-dependent Na+ and Ca2+ channels (Smirnov & Shuba. 1989). In these conditions both ICa and INa were observed in 89 % of the 128 cells studied. In most cells studied the ICa peak amplitude was about 2-3 times smaller (0 55 + 009, n = 20 at the holding potential of -50 mV and 0-38+004, n = 31 at -80 mV) in comparison to that of INa* In the following experiments different external solutions have been used to study a properties of ionic currents through Ca2+ and Na+ channels (see Methods).

Calcium currents at the normal and elevated Ca2+ In the presence of 2 5 mM-Ca 2+ ions in the Cs+-containing external solution with 130 mM-TEA' a family Of ICa could be elicited by 400 ms depolarizing pulses from the holding potential of -50 mV. ICa appeared between -30 and -20 mV, peaked at 0 mV and then its amplitude was reduced (Fig. IA). Increasing the holding potential to -80 mV resulted in an increase of ICa amplitude both at the beginning and at the end of the command step in the whole voltage range studied without any shift in the peak of the I-V relation of 'Ca (Fig. IB). The decay of 'Ca elicited from -80 mV was substantially accelerated compared to that from -50 mV and ICa was nearly completely inactivated during depolarizing pulses in the membrane potential range between -40 and -30 mV, whereas at more positive potentials a sustained component of Ica which inactivated more slowly was observed (Fig. IA). The peak amplitude of Ica varied between about 30 and 300 pA in different cells with an average value of 133 + 14 pA (n = 33) measured at 0 mV from the holding potential of -80 mV. A ratio of ICa peak elicited from -50 mV to that from -80 mV was equal to 0 56 + 0 04 (n = 23). The relatively small ICa amplitude in the presence of 2 5 mM-Ca2+ made a study of Ica difficult, especially in the negative voltage range where its characteristics seem to be different from those at more positive voltages. Therefore, the properties of Ica were studied in the external solution containing 10 mM-Ca2+ ions in all the following experiments. Increasing the external Ca2+ concentration to 10 mm led to a rise of 'Ca amplitude by a factor of 2-6 and 2 2 in two cells studied. Also, an acceleration of ICa decay was observed. In these conditions Ica elicited from the holding potential of -50 mV was not seen in the membrane potential range between -40 and -20 mV in contrast to

-~ ~ ~ -175

Ca2+ AND Na+ CHANNELS IN SMOOTH MUSCLE A

553

B

-40 -30

25

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_50

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V. (mv)

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/~~~~~~~~~~~~~~~~~~~~

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10

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~ -

(mV)

0

50

40

50 pA

-20

40

Membrane potential -40 . -100-80

-400

j 200 pA 50

/c. (pA) -

-600

100 ms

Fig. 1. Ca2, currents at 2-5 (A and B) and 10 (C and D) mM-external Ca2+ ions. A and C show a family of superimposed ICa elicited by depolarizing pulses from the holding potentials of -50 (upper trace) and -80 mV (lower trace) in each pair. Pulse duration was 400 (A) and 300 (C) ms. Membrane potential (in mV) indicated near each pair. B and D, current (ICa) versus membrane potential (V.) for ICa peak (circles) and current at the end of voltage step (triangles) at -50 and -80 mV (open and filled symbols, respectively. A and B, and C and D were obtained from two different cells.

84 ,0

S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA

554

ICa

elicited by depolarization from -80 mV to the same potentials (Fig. 10). A slower component of ICa appeared between -20 and -10 mV from both holding potentials and its amplitude increased when the cell membrane was hyperpolarized. ICa peak was shifted toward positive membrane potentials by more than 10 mV in A

B -50

0

50 J

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-20

-10

02

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20

Vm (mV)

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-150 -200

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1b

m

m

f

00 ms

-250

-300 /Ba (pA) Fig. 2. Ba2l currents in the presence of 10 mM-Ba2l. A, superimposed IBa elicited by 400 ms step depolarizations to potentials indicated (in mV) from -50 and -80 mV (upper and lower records, respectively). Calibration scale is 50 pA; note the different scale for records at -30, -20, -10 and 0 mV and 10, 20 and 30 mV. B, I-V relationship for ,Ba shown in A. Circles and triangles indicate the peak and current at the end of step depolarization, respectively, elicited from -50 mV (open symbols) and -80 mV (filled symbols). Leak current was compensated electronically.

comparison with that at 2-5 mM-Ca2+ (compare Fig. lB and D) and was usually observed between + 10 and + 20 mV. A ratio of ICa peak (measured at + 10 mV) at the holding potential -50 mV to that at -80 mV was equal to 0-65 + 0-03 (n = 12), which is similar to that found at 2-5 mM-external Ca 2+ ions. It should be stressed that, in spite of the presence of 5 mM-EGTA in the pipette solution, in some cells inward tail currents, as well as an acceleration of ICa decay, were observed during stepping the cell membrane to 0 or 10 mV. Our experiments with indirect measurements of conductance changes during ICa development (using 50 ms voltage steps of 10 mV in amplitude superimposed upon 1 or 3 s depolarization to 10 or 20 mV) have shown only a weak correlation between changes in ICa amplitude at the end of a depolarizing step and an amplitude of current activated by the additional voltage step. In some cases this conductance was even increased while Ica amplitude was decreased (not shown). This experiment may indicate that in the voltage range positive of 0 to +10 mV Ica decay might be distorted by some conductance(s) activated at positive voltages (presumably a Ca2+-activated one). Therefore, a quantitative kinetic analysis Of ICa inactivation has not been performed in the present study. As can be seen from Fig. 1, therefore, two components of Ica were observed in

Ca2+ AND Na+ CHANNELS IN SMOOTH MUSCLE

555

newborn rat ileal cells: one, activated at the voltage range close to -40 mV, is characterized by a fast decay during depolarization and complete inactivation at the holding potential of -50 mV; the other component of ICa had a slower time course of inactivation, was activated between -20 and -10 mV in the presence of 10 mmCa2l and dominated at positive potentials. Its amplitude also depended on the holding potential. We will refer to the fast component of Ca2+, activated at negative membrane voltages, as low-voltage activated or 'Ca low and the sustained component, which activated at more positive potentials, as high-voltage activated or ICa, high

Effect of Ba2+ ions Equimolar replacement of 10 mM-external Ca2+ by Ba2+ did not significantly change the Ca2+ channel current in the negative voltage range between -30 and -10 mV. IBa measured at the peak of I-V relationship increased by a factor of 1P8 + 0-13 (n = 8) in comparison with ICa- We found two types of effect of Ba2+ on Ca2+ channel current kinetics in the newborn rat ileum SMC. In half of the cases (5/10 cells studied) IBa amplitude was increased and its time course was considerably slowed down in comparison with ICa In the other five cells IBa decay was slowed only insignificantly, if at all, in spite of an increase in its peak amplitude (not shown). However, no marked differences in the dependence of IBa peak amplitude on the holding potential in comparison with Ica have been found (Fig. 2). A ratio of IBa peaks elicited by step depolarization to 0 mV from -50 mV to that from -80 mV was 0-48 + 0.05 (n = 13), which is similar to that found at 2-5 and 10 mM-external Ca2+ ions.

Effect of organic and inorganic Ca2+ channel antagonists. In contrast to other reports performed on SMCs, where low concentration of dihydropyridine antagonists effectively blocked ICa high' 1 /tM-nifedipine had no effect on both ICalow and ICa high in newborn rat ileal cells. At a very high concentration of 30 JtM, nifedipine reversibly decreased ICa peak amplitude elicited during a voltage step to + 10 mV from the holding potential of -80 mV to 0-81 + 0X04 of its control value (n = 7) (Fig. 3A). Amplitude Of Ica iow was not changed in the presence of this concentration of nifedipine (not shown). Changing the holding potential to -50 mV resulted in a further decrease Of ICa (ratio of ICa peak amplitude measured at + 10 mV with and without nifedipine was equal to 0-66 + 0-05 for three cells studied). The significance of the blocking effect of ICa high by nifedipine at two different holding potentials using a one-tailed t test is 0 05 < P < 041 and indicates some potential dependence of the nifedipine block on ICa Inorganic Ca2+ channel blockers Cd2+ and Ni2+ exert a different effect on ICalow and 'Cahigh Cd2+ ions (10 /tM) decreased 'Ca.high to 0-5 + 002 (n = 4) of its initial value, whereas the amplitude of ICaiow did not significantly change. Further increase in Cd2+ concentration to 100 /tM resulted in an essential block of both ICa (Fig. 3B). It should be noted that a fast inactivated component of ICa which remained in the presence of both 10 and 100 /tM-Cd2+ ions, completely disappeared when the holding potential was shifted to -50 mV (not shown). In contrast to Cd2+ ions, Ni2+ at a concentration of 0 5 mm almost completely suppressed the 'Calow and reduced the 'Ca high to 0-6 + 0 05 of its initial value (n = 5) (Fig. 3C).

S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA

556

Steady-state inactivation To study steady-state inactivation of both components of ICa, currents were elicited every 30 s by a step depolarization to -20 mV where ICa low was prominent, and to + 10 mV where ICa high was dominant, from different holding potentials (Fig. A 30 ,uM-nifedipine

Control

v

Wash I--

1000pA 50 ms

C

B

-30-10

120 pA

1lo

10

10

k-",

A&-

-

--

20 pA

i

10

100 pA

1100 pA 100 ms 100 ms

Fig. 3. Effect of organic and inorganic blockers on ICa* A, effect of nifedipine. I,. was elicited by a 200 ms pulse to 10 mV in the absence (control), in the presence of 30 ftMnifedipine and after 15 min wash-out of the drug from the external solution (wash). B, superimposed ICa recorded before (lower trace), in the presence of 10 /IM (middle trace) and 0 1 mM-(upper trace) Cd2+ ions. C, superimposed ICa records before (lower trace) and in the presence (upper trace) of 0 5 mM-Ni2+. Membrane potential indicated (in mV) near each current trace in B and C. Holding potential was -80 mV. A, B and C were obtained from three different cells.

4A and B). As can be seen from the figure, ICa,low was nearly completely inactivated at -40 mV, whereas ICa,high was inactivated only by about a half of this membrane potential. Actually, both ICa low and ICahigh start to inactivate in a similar voltage range. However, the inactivation curve for ICalow was steeper in comparison to that for ICa high (Fig. 4C). Both dependencies were well fitted to the Boltzmann equation

'/'max=

1

1 +exp (V-V0.5)/lc

(1)

Ca2+ AND Na+ CHANNELS IN SMOOTH MUSCLE A

B

-100

-80

557

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r

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k

|20 pA

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Va (mi

Fig. 4. Steady-state inactivation of Ca2+ currents. A and B, ICa elicited by 200 ms depolarizing steps to -20 mV (A) and 10 mV (B) from various holding potentials as indicated (in mV) near each record. A and B from two different cells. C, dependence of relative ICa peak amplitude measured at -20 mV (0) and 10 mV (a) on the membrane potential (Vm). I/Imax was defined as the ratio of ICa peak at the given membrane potential to maximally activated in the same cell. Each point and vertical bar represent mean and S.E.M. for six or eight cells studied at -20 and 10 mV, respectively, with a protocol shown in the inset. The continuous lines were drawn according to eqn (1) as described in the text. Here and in the following figures S.E.M.S smaller than the point size are not 'Ca

shown.

where V0.5, the potential of half-maximal inactivation, was 60-6 and -49 mV, and k, the slope factor, was 8-8 and 13-6 mV for ICa and lCahigh' respectively (Fig. 4C). It is noteworthy that we compared only the peak amplitude of Ica at each test potential where a contamination by other conductances especially at positive potentials (see above) should be negligible. -

low

S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA

558

Recovery of calcium currents from inactivation Recovery from inactivation of both ICalow and 'Cahigh was measured using a twopulse protocol. A 500 ms depolarizing pulse either to -20 or -10 mV eliciting ICa was followed, after a variable interpulse interval, by a test pulse to the same A I

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Fig. 5. Recovery of Ca2+ currents from inactivation. A and B represent current traces recorded at -20 mV and 10 mV, respectively, from two different cells using the experimental protocol shown in the inset of C. Prepulses of 500 ms and 100 ms test pulses following the 50, 200, 500 and 1000 ms interpulse intervals (A) and 50, 500, 1000 ms and 2 and 20 s ones (B) are shown. C, dependence of the normalized ICa peak amplitude measured at -20 mV (0) and 10 mV (a). 12/I1 is a ratio of Ica peak elicited by a 100 ms test pulse (P2) to that measured at the 500 ms prepulse (P,). Points and vertical bars are mean and s.E.M. for four (except points at 2 and 5 s where n = 2) and three (except point at 200 ms where n = 2) cells studied at -20 and 10 mV, correspondingly. Continuous lines were drawn by eye. Holding potential -80 mV.

membrane potential (Fig. 5A and B). It has been found that recovery of ICa low following a 500 ms depolarization was fast, and for the cell shown in Fig. 5A, ICa was completely recovered within 1 s (the time for a complete recovery of Icalow was between 2 and 5 s for three other cells studied).

Ca2+ AND Na+ CHANNELS IN SMOOTH MUSCLE 559 Restoration Of ICa high was significantly slower in comparison to that for ICalow and a period of at least 20 s was needed for ICa elicited by a step pulse to 10 mV to recover fully (Fig. 5B). Usually complete recovery of ICahigh was observed between 20 and 30 s (results from two other cells studied with a similar protocol). Dependence of normalized ICa peak amplitude for both Icalow and Ic highon the interpulse interval was complex and not monoexpontential (Fig. 5C).

ICa in a single SMC of the adult rat ileum When ileal SMC isolated from the adult rat was clamped in the presence of 10 mMCa2+, a typical family of ICa was recorded from two holding potentials, -50 and -80 mV (superimposed traces in Fig. 6A). A clear difference in ICa recorded in the adult rat was the absence of a visible fast ICa low in the negative voltage range (compare with Ca2+ currents in newborn rat cell in Fig. 1). However, it should be stressed that the dependence of 'Ca high on the holding potential was very similar to that found in newborn ileum muscle cell (Fig. 6B). An average ratio of peak ICa elicited from -50 mV to that from -80 mV was equal to 0-58 + 0-06 (n = 11) (compare to 0-65 for the newborn rat cells). Also, the dependence of Ica inactivation on the membrane potential using the experimental protocol described above for newborn rat ileal cells, was found to be virtually indistinguishable from that for newborn rat: the half-inactivation potential was -48-3 mV, and the slope factor was equal to 12-7 mV (Fig. 6C). It should be noted that in these cells the availability of the peak current at -20 mV was not studied in detail. However, an estimation of the ratio of Ica peak at the holding potentials of -50 and -80 mV, taking into account a leak current, was quite similar in the voltage range between -20 and + 20 mV (Fig. 6B), suggesting that in adult animals a component of Ica low seems to be small at these potentials in contrast to newborn rat ileal cells. An important property of Ca2+ channels found in leal SMC of the newborn rat was the low sensitivity of Ica to nifedipine. Therefore, the effect of 30 ,tM-nifedipine was tested on Ica in the adult rat leal cell membrane. It was found that Ica measured at 10 mV in adult rat cells possessed a significantly (P < 0 05 at both holding potentials) higher sensitivity to block by this concentration of nifedipine compared to newborn rats. Nifedipine reduced the current to 0-63 + 0-06 (n = 5) at the holding potential -80 mV and to 0 37 + 0-08 (n = 5) at -50 mV. It should be pointed out that in spite of the higher sensitivity of ICa in adult rat leal cells, this concentration of the drug failed to completely block Ca2+ channels even at the positive holding potential. It should be noted that in a few of the sixteen cells studied a fast inactivating component of Ica could be observed in the negative voltage range in ileal cells from adult rats; it was, however, too small to be studied in detail.

Potential-dependent Na+ current In Ca2+-free Cs+-containing external solution with 130 mM-Na+ INa appeared in the membrane potential range between -50 and -40 mV (holding potential -80 mV), reached a maximum at -10 mV and with further depolarization its amplitude decreased (Fig. 7A). It should be noted that there are difficulties in determining a true reversal potential for INa because at membrane potentials more positive then 20 mV

560

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V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA

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-40 -20 0 (mV) Fig. 6. Ca2+ currents in a single ileal SMC isolated from the adult rat. A, a family of superimposed ICa elicited by 200 ms depolarizing pulses to the potential indicated (in mV) near each current record from the holding potentials of -50 and -80 mV (upper and lower trace in each pair, respectively). B, normalized I-V relationships for ICa peak from -50 mV (0) and -80 mV (@). Points and vertical bars show mean and s.E.M. for five cells studied. Currents were normalized to the peak of ICa measured during a step from -80 to 10 mV in each cell studied. C, steady-state inactivation of ICa in SMC of the adult rat ileum. The experimental protocol was the same as described in the legend to Fig. 4C. Test depolarization to 10 mV. Each point and vertical bar represents mean and S.E.M. for five cells. Continuous line was drawn according to eqn (1) with VO.5 = 48 3 mV and k = 12-7. -100

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Ca2+ AND Nat CHANNELS IN SMOOTH MUSCLE A

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-30

0

Vm (mV)

_

I 60 L

-1*0

eU

_

4 ms

C

D

* -30 o -20 A -10

3 1

< 0*3

o

0

+

10

86w

0*1

E 4-

003

2-

1-1

z

0-01 II

0

4

8

Time (ins)

12

-40

I

-20 20 0 Membrane potential (mV)

Fig. 7. Voltage dependence of INa. A, INa at different membrane potentials as indicated (in mV) near each current race. Holding potential -80 mV. Pulse duration 12 ms. Current calibration was 200 pA; note the different calibration scales. B. average I-V relationship for INa' Each point and vertical bar represents mean and S.E.M. for eleven ileal cells. Currents were normalized to the peak of 4Na in each cell studied. C, semilogarithmic plot of INa at different membrane potentials against the time of current development. Numbers near each symbol indicate the membrane potential in mV. The time constants were calculated to be 5-18, 2-00, 1-02, 0-67, and 0 35 ms for test potentials of -30, -20, -10, 0 and 10 mV, respectively. The same cell as shown in A. D, dependence of time constant of INa(Th) decay versus membrane potential. Each point and vertical bar represents mean and S.E.M. for six to eleven cells studied except for -35 mV obtained from two cells. The continuous line was drawn by eye.

INa decay (time constant was smaller then 0 5 ms) was comparable with a duration of capacitive current in our experiments. This circumstance also made it difficult to analyse the activation kinetics of INa. A cumulative current-voltage (I-V) relationship for normalized INa obtained from eleven cells is shown in Fig. 7B. INa

562

S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA

peak amplitude (measured at -10 mV) varied from 33 to 1370 pA with a mean value of 636+ 110 pA. As can be seen from Fig. 7A INa measured at the peak of its I-V relationship decayed completely during the 12 ms depolarizing pulse. INa decay was well fitted by

or~~~r

(2)

1.o

(5)

30 60 _ 90~ 120 150 -180 210

1200 pA

4m

~0.5 (6)

(4)

~~~~~~~~~~~~~~~~(4) 0

01

05 1

5

10

50 100

500 1000

[TTX] (nM) Fig. 8. Dose dependence of blockade of INa by external TTX ([TTX]). INa was measured at -10 mV. ITTX/Ic is the ratio of peak INa in the presence and absence of appropriate

[TTX] respectively. Each point and vertical bar represents mean and S.E.M. for the number of cells indicated in parentheses near each point. The smooth curve was fitted according to the following equation:

TTX/c

=

1+ [TTX]/AK

where Kd is the dissociation constant of 4-5 nm. The inset shows IN. 20 s after addition of 10 nm-TTX (marked by arrow-head) and at different times (indicated in seconds) after removal of the drug from the external solution.

a single exponential in the whole voltage range studied (Fig. 7 C). Time constants of INa decay decreased monotinically with increasing membrane depolarization and were 4-7+002 ms (n = 6) at -30 mV, 1-45+0-13 ms (n = 11) at -10 mV, and 0-51+0.03 ms (n = 7) at 20 mV (Fig. 7D).

Sensitivity to TTX TTX a well-known blocker of potential-dependent Na+ channels in most tissues, reduced INa in a concentration-dependent manner. Application of 01 nm-TTX only slightly reduced 'Na amplitude, whereas, TTX at a concentration of 1 /LM blocked Na+ channels in the SMC membrane of the rat ileum almost completely. The apparent dissociation constant was determined as 4-5 nM (Fig. 8). The TTX-induced block developed quickly in about 15-20 s. However, restoration of INa amplitude

Ca2+ AND Nat CHANNELS IN SMOOTH MUSCLE 563 after removal of TTX from the external solution was slow and complete recovery was observed 3-5-4 min after removal of TTX (Fig. 8, inset). Steady-state inactivation and recovery from inactivation of INa It was found that a prolongation of depolarization resulted in a further suppression of INa (slow inactivation). The slow inactivation of INa was fully completed after A TT# rp

rC

t=10ms 30ms

t-.L-

50 ms

M

I

100 ms

w

150 ms

250 ms

r i-s uor =I=r*

=6

-10 mV 80

t

200 pA

mV

50 ms

-100 mV B {V.

1.0

1 0

-lO0mV

-JL 880 mV

150 ms

hx

05

-130

-100

-60

-20

0

(mV) Fig. 9. A, slow development of INa inactivation. Current traces were obtained in the same cell using a two-pulse protocol with conditioning prepulses of variable duration (indicated near each record) applied to -100 mV (top row) or -60 mV (bottom row). Test pulse of 10 ms duration was -10 mV. Interpulse interval 3 ms. Experimental protocol shown in the bottom of the figure. Note the interruption in records with 150 and 250 ms prepulses. B, steady-state inactivation of INa Prepulse and test pulse were 150 and 10 ms, correspondingly. Interpulse interval 3 ms. The scheme of experiments is shown in the inset. ho: was determined as a ratio of INa peaks with a given conditioning prepulse (Vc) to that obtained with a prepulse to - 130 mV. Each point and vertical bar represents mean and S.E.M. for six cells studied. The continuous line was drawn in accordance to eqn (1) with V0.5 = -60-3 mV and k = 9-84 mV.

about 150 ms of depolarization to -60 mV (Fig. 9A). Therefore, steady-state inactivation for INawas studied in two-pulse experiments with a 150 ms prepulse to different voltages (Fig. 9B). It can be seen from the figure that inactivation of INa

|~ ~ ~ 0XpA

S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA becomes visible between -100 and -90 mV and that INa is completely inactivated near -20 mV. The dependence of steady-state inactivation of INa on the conditioning potential was well fitted to the Boltzmann equation with a half-inactivation potential of -60-3 mV and a slope of 9-8 mV (Fig. 9B). 564

A

Q'~ffjf

f f

C -10 mV -60 mV -80

f j200 pA 50 ms

t

Fig. 10. Recovery of INa from inactivation. A and B, INa elicited by a 50 ms prepulse and superimposed current recordings elicited by 10 ms pulses after different interpulse intervals (5, 10, 20, 30, 50, 80, 150, 200 and 300 ms) at two different holding potentials -60 (A) and -80 (B) mV. The continuous lines were drawn according to a single exponential with a time constant of 57 (A) and 14 (B) ms. A and B obtained from the same cell. C, the scheme of the experiment.

Recovery of INa from inactivation was studied after a 50 ms depolarization to -10 mV at two different holding potentials. Recovery of INa from inactivation was fast, monoexponential, and depended on the membrane potential (Fig. 10). The average time constant of INa recovery measured at the holding potential of -90 mV was 18-1 + 1-4 ms (n = 5), whereas increasing the holding potential to -60 mV resulted in a more than twofold slowing of INa recovery. In this case the average time constant was equal to 46-5 + 3 9 ms (n = 5).

Influence of external Ca2+ ions8 The properties of INa in the newborn rat SMC have been studied mainly in the Ca2+-free external solution. Therefore, it is important to know the effect of external Ca2+ ions on the inactivation properties of I... To study this phenomenon currents were elicited by 50 ms conditioning pulses to different voltages and test pulses to 0 and -10 mV (which corresponded to the peak of the I-V relationship for IN. in each solution) in the presence and absence of 2-5 mm-Ca2+ ions respectively. The relative IN. amplitude was plotted against the conditioning potentials and was fitted according to the Boltzmann equation with VO5 = -562 mV and k = 10-8 mV (n = 6) in the Ca2+-free solution and VO.5 = - 43.7 mV and k = 12-96 mV (n = 7) in the presence of 2-5 mm-Ca2+ ions (Fig. 11). In general, the ratio of IN. peak amplitude

565 Ca2+ AND Na+ CHANNELS IN SMOOTH MUSCLE measured at 0 mV from the holding potential of -50 mV to that from -80 mV was found to be 0'45 + 0-02 in sixty-five cells studied in the presence of 2-5 mM-Ca2+ ions. If the 10 mV shift in the steady-state inactivation of INa shown in Fig. I is assumed, then a ratio of the fraction of available Na+ channels at -60 mV to that at -90 mV was calculated to be 0-52, which is close to the experimental finding. A

B

-80

-80

1200 pA

|400 pA

i

-50 _20 ms

X

-30

I

-50w m _

r

20 ms

r

-30

c

1*0

° mv v °R -10

-

c

-80 mV

50 ms

h50 05-

0- I

0 -20 -60 Vc (mV) Fig. 11. Effect of external Ca2+ ions on IN.. A and B, current traces in the presence of 2 5 mM-Ca2+ ions and in Ca2+-free external solution respectively. Conditioning depolarization (VI') indicated (in mV) near each trace. C, dependence of the relative IN amplitude on VT' in Ca2+-containing (0) and Ca2+-free (0) solutions. h6o was determined as a ratio of INa peak amplitude with a 50 ms conditioning pulse to that with a prepulse to - 130 mV. Each point and vertical bar shows mean and S.E.M. for seven (0) and six (0) ileal cells studied. Continuous lines were drawn in accordance to eqn (1) with parameters described in the text. The inset shows experimental protocol. Duration of the test pulse was 10 ms; interpulse interval 1 ms.

-130

-100

566

S. V. SMIRNOV, A. V. ZHOLOS AND M. F. SHUBA

INa in the adult rat ileal cell membrane was not studied in detail in the absence of external Ca2+ ions. However, in the absence of K+ ions in the external and pipette solutions and the presence of 2-5 mM-Ca2+ both fast ('Na) and sustained (ICa) components of inward current were observed in 12/19 (63 %) cell studied. The peak of INa was more negative than that in newborn rat ileal cells (about -10 mV) and varied between 400 and 4 nA in the presence of external Ca2+ ions. However, the ratio of the IN. peak at the holding potential of -50 to that at -80 mV was 0 45 + 0X08 (n = 5), which is close to that found in neonatal rats. DISCUSSION

The present study has shown that rat ileal smooth muscle cells possess three main components of potential-dependent inward current: LVA and HVA calcium currents and tetrodotoxin-sensitive sodium current.

LVA Ca2+ current, ICalow In ileal SMCs isolated from newborn animals a rapidly inactivating component of Ica in the voltage range between -40 and -30 mV in the presence of physiological Ca2+ concentration was observed. Its amplitude was increased when Ca2+ was elevated to 10 mm and was similar at this concentration of Ba2+. Steady-state inactivation of Ica low occurred in a rather negative voltage range (parameters of the Boltzmann equation were V.5 = - 60-6 mV and k = 8-8 mV at 10 mm-Ca2+). This value of VO.5 close to that found for Ica low in vascular SMC (Yatani, Seidel, Allen & Brown, 1987; Aaronson et al. 1988; Loirand, Mironneau, Mironneau & Pacaud, 1989), but is positive to values found in rat aorta SMC in primary culture (VO.5 = -80 mV, k = 4 mV at 20 mm-Ca2+; Akaike, Kanaide, Kuga, Nakamura, Sadoshima & Tomoike, 1989). This current was insensitive to a high dose of nifedipine and to a low concentration of Cd2+ ions, but it was significantly suppressed by 0 5 mm-Ni2+ ions. All these findings, as well as its relative stability during cell perfusion, suggest that the LVA Ca2+ channel current described in newborn rat ileal SMC is quite similar to the LVA or T-type Ca2+ channel current previously described in a variety of tissues (e.g. Bean, 1985; Fedulova et al. 1985; Nowycky, Fox & Tsien, 1985; Carbone & Lux, 1987; Fox et al. 1987 a, b; Gonoi & Hasegawa, 1988), including SMC (Sturek & Hermsmeyer, 1986; Friedman et al. 1986; Yatani et al. 1987; Aaronson et at. 1988; Akaike et al. 1989; Loirand et al. 1989; Ganitkevich & Isenberg, 1990). However, these channels have not been found in most visceral SMCs studied previously with the whole-cell voltage-clamp technique (Ganitkevich et at. 1985, 1986; Kldckner & Isenberg, 1985; Mitra & Morad, 1985; Ohya et al. 1986; Amedee, Mironneau & Mironneau, 1987; Yamamoto et al. 1989), although multiple types of single Ca2+ channel conductances have recently been observed in visceral SMC (Yoshino et al. 1989). HVA Ca2+ current HVA Caa2+ channels have been found in all SMCs studied previously (Kldckner & Isenberg, 1985; Ganitkevich, Shuba & Smirnov, 1986, 1987; Caffrey et al. 1986; Jmari et al. 1986; Ohya et al. 1986; Ame'dee et al. 1987; Benham et al. 1987; Aaronson

567 Ca2+ AND Nat CHANNELS IN SMOOTH MUSCLE et al. 1988; Loirand et al. 1989; Yamamoto et al. 1989; Hisada, Kurachi & Sugimoto, 1990; Aaronson & Russell, 1991). They possess the following main features: (1) an apparent activation threshold usually in the voltage range between -40 and -20 mV in the presence of a physiological Ca2+ concentration; (2) dependence of the inactivation process both on Ca2+ ions and the membrane potential; (3) larger wholecell current and unitary conductances in the presence of Ba2+ rather than Ca2+ as a current carrier; and (4) high sensitivity to dihydropyridine antagonists. All these characteristics of HVA Ca21 channels are mainly related to L-type Ca2+ channels according to the classification originally proposed by Tsien and colleagues (Nowycky et al. 1985). In rat ileal SMC the apparent threshold of activation of ICa high was close to -30 mV at 2-5 mm-external Ca2+ and was shifted to about -20 at elevated Ca2+ concentration. The amplitude of IBa through HVA Ca2+ channels was doubled and its inactivation time course was slower in comparison to that of ICa. The ICa,high was nearly completely blocked by 100 gm-Cd2+ and suppressed by about 50% by Ni2+ ions. Also, when the availability of ICa was studied using a two-pulse protocol, about 20% of the current could still be activated even following 10 s depolarization to 100 mV (not shown), which may indicate that at least some part of the Ca2+ current inactivation depends on Caa2+ entry into the cell. HVA Ca2+ channels found in rat ileal cells do demonstrate two strikingly unusual features which contrast them to other SMCs studied previously. Firstly, these HVA Ca2+ channels possess a quite low sensitivity to high concentration of nifedipine. Even at holding potential of -50 mV, 30 jrm-nifedipine blocked this current by only 33%. Secondly, we observed a strong dependence of 'Cahigh peak on the holding potential. ICa was about two times smaller at a holding potential of -50 mV than at -80 mV, which was also seen from the dependence of steady-state inactivation on the holding potential; a half-inactivation potential (VO.5) of -49 mV was observed in the presence of 10 mM-external Ca2+ ions. This dependence is arranged about 20-30 mV negative of that described in other SMCs (Ohya, Kitamura & Kuriyama, 1987; Loirand et al. 1989; Kldckner & Isenberg, 1989; Buryi, Gordienko & Shuba, 1989; Yamamoto et al. 1989; Matsuda et al. 1990). However, in the guinea-pig urinary bladder (Kldckner & Isenberg, 1985) and pregnant rat uterus (Ohya & Sperelakis, 1989) a value of V0.5 was found at about -40 mV in 3-6 mM-Ca2 , which is still more positive than that for rat ileal SMC if an approximately 10 mV shift in the presence of 10 mm-Ca2+ ions is taken into account. Also, the slope of inactivation dependence for 'Ca high in rat ileal cells was about two times less steep than that in different SMCs (compare 13-6 mV in the presence work with 5-8 mV found in studies mentioned above). One possible explanation for such differences could be some contribution of LVA ICa to the net ICa in the rat ileal cell membrane at positive voltages. However, this suggestion seems to be unlikely for the following reasons. Firstly, ICalow and ICahigh have quite different steady-state inactivation dependencies and different time courses of recovery from inactivation. ICalow is suppressed significantly by Ni2+, whereas ICahigh is more sensitive to Cd2+. Secondly, in some newborn ileal cells (about 18% of ninety-four cells studied), as well as in single SMC isolated from adult rat ileum, where the rapidly inactivating component of Ica was not observed in the 19

PHY 454

S. 1V. SMIRNOYl A. V. ZHOLOS AND M. F. SHUBA negative potential range, the dependence of peak 'Ca on the holding potential was similar to that in cells with a prominent 'Ca low The properties of HVA Ca21 channels, in particular that the steady-state inactivation curve is arranged in a more negative voltage range in contrast to the positive membrane potential range of its activation and is much less steep than that usually observed in other SMCs, and also the very weak sensitivity of these HVA Ca21 channels to high doses of nifedipine, are quite similar to those of potentialdependent Ca21 channels which have been recently described in neurones (Nowycky et al. 1985; Fox et al. 1987 a, b; see for review Pelzer. Pelzer & McDonald, 1990) and have been called N-type Ca21 channels. However, difficulties in separation of N- and L types of Ca21 channels, which mainly arise from their similar ranges of activation and partially overlapping ranges of inactivation, exist (see e.g. Fox et al. 1987a, b). Additional studies including patch-clamp experiments are necessary to fully elucidate the contribution and physiological role of different types of Ca2" channels in the newborn rat ileal cell membrane. -568

Comparison of Ca21 currents in the newborn and adult rat ileal cell The properties of Ca2+ currents, as well as other conductances, have not been studied previously in single SMC during post-natal development. Recently, Gonoi & Hasegawa (1988) have shown that in mouse skeletal fibres 'Ca low was more prominent immediately after birth and became undetectable by day 17. Our comparison of some of the properties of ICa in ileal SMC from newborn and adult rats suggest that this may also be the case in intestinal smooth muscle. It is also consistent with other whole-cell studies carried out on a single visceral SMC obtained as a rule from the adult animals and where ICa low has not been found (Kliekner & Isenberg, 1985; Ganitkevich et al. 1986; Jmari et al. 1986; Ohya et al. 1986; Amede'e et al. 1987; Ohya & Sperelakis, 1989; Yamamoto et al. 1989). In conclusion, we speculate that the disappearance of 'Ca low in the adult rat SMC and the comparatively small contribution of these Ca2" channels to the whole-cell ICa in newborn rat ileal cells indicates that LVA Ca2" channels probably do not play a significant physiological role in intestinal SMC during postnatal development. However, the role of these Ca2+ channels may be important during the early phase of ontogenesis. The important and interesting question needs to be studied further in these as well as in other SMCs.

Potential-dependent sodium channel Our results have shown the presence of potential-dependent tetrodotoxinsensitive Na+ channels in the rat ileal smooth muscle cell membrane. It should be noted that in general Na' channels are not widely present in SMC and have been described only in rat azygous vein (Sturek & Hermsmeyer, 1986), and rabbit pulmonary artery (Okabe, Kitamura & Kuriyama, 1988; however, Clapp & Gurney (1991) did not observe these channels in the same tissue) as well as in pregnant rat myometrial cells (Ohya & Sperelakis, 1989). They have not been observed previously in mammalian intestinal SMC. 'Na in rat ileal cells is activated in a membrane potential range near -40 mV, which is close to the range of activation of Ca2+ currents. INa reached its peak at

Ca2` AND Xra+ CHANN.VELS IN SMOOTH MUSCLE

569

about 0 mV, which is similar to the value found in other SMCs (Sturek & Hermsmeyer, 1986; Okabe et al. 1988; Ohya & Sperelakis, 1989). Moreover, the kinetic properties of INa in rat ileal cells seem to be close to those found in rabbit pulmonary artery and rat myometrial SMC. The sensitivity to TTX of the Na' channels in rat ileal cells (A = 45 nm) differs significantly from that in rat azygous vein cells (Kd about 30 /M, Sturek & Hermsmeyer, 1986), and in rat myometrial cells (Kd = 27 nm, Ohya & Sperelakis. 1989) and is close to that found in rabbit pulmonary artery (K1 = 8&7 nm, Okabe et al. 1988). It should be noted that, in general, the properties of 'Na found in rat ileal SMC (potential dependence of inactivation, kinetics of 'Na decay and their dependence on the membrane potential, sensitivity to TTX) are quite similar to those observed in giant axons (reviewed by Hille, 1984), frog node of Ranvier (Benoit, Corbier & Dubois, 1985), and single myocardial cells (Benndorf, Beldt & Nilius, 1985). Our results show that at physiological Ca2+ concentration about 50% of Na+ channels are available at a membrane potential of -50 mV. which is close to or even positive to reliably measured membrane potentials in the gastrointestinal tracts of various animal species (see e.g. Szurszewski, 1981; Hara, Kubota & Szurszewski, 1986). It may indicate that Na+ channels together with Ca21 ones play an important physiological role in excitation-contraction coupling in rat ileal smooth muscle. XWe are indebted to Professor P. G. Kostyuk for valuable comments on the manuscript. We are very grateful to Dr P. I. Aaronson for critical reading, helpful suggestions, and the great work of improving the language of the manuscript. We would like to thank Mr V. I. Demchenko for technical help with preparation of the figures. REFERENCES

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AM1EDfE, T., MIRONNEAU, C. & MIRONNEAU, J. (1987). The calcium channel current of pregnant rat single myometrial cells in short-term primary culture. Journal of Physiology 392, 253-272. BEAN, B. P. (1985). Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity and pharmacology. Journal of General Physiology 86, 1-30. BEAN, B. P., STUREK, M.. PUGA, A. & HERMSMEYER, K. (1986). Calcium channels in muscle cells isolated from rat mesenteric arteries: modulation by dihydropyridine drugs. Circulation Research 59, 229-235. BENHAM, C. D.. HESS. P. & TSIEN, R. XV. (1987). Two types of calcium channels in single smooth muscle cells from rabbit ear artery studied with whole-cell and single channel recordings. Circulation Research 61, suppl. 1, 110-11 6. BENNDORF, K., BELDT, W. & NILIUS, B. (1985). Sodium current in single myocardial mouse cells. Pfuigers Archiv 404, 190-196. BENOIT, E., CORBIER, A. & DUBOIS, J.-M. (1985). Evidence for two transient sodium currents in the frog node of Ranvier. Journal of Physiology 361, 339-360. Bossu, J. L., FELTZ, A. & THOMANN, J. M. (1985). Depolarization elicits two distinct calcium currents in vertebrate sensory neurones. Pfligers Archiv 403, 360-368. 19- 2

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BURYI, A. V., GORDIENKO, D. V. & SI1UBA, MI. F. (1989). On inhomogeneity of voltage-sensitive calcium channel population in smooth muscle cells from guinea-pig mesenteric artery. Biologicheskie Membrany 6, 740-748. CAFFREY, J. M., JOSEPHSON, I. R & BROWN, A. AM. (1986). Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells. Biophysical Journal 49, 1237-1242. CARBONE, E. & Lux, H. D. (1987). Kinetics and selectivity of a low-voltage-activated calcium current in chick and rat sensory neurones. Journal of Physiology 386. 547-570. CLAPP, L. H. & GURNEY, A. M. (1991). Modulation of calcium movements by nitruprusside in isolated vascular smooth muscle cells. Pfliigers Archiv 418, 462-470. FEDULOVA, S. A., KOSTYUK, P. G. & VESELOVSKY, N. S. (1985). Two types of calcium channels in the somatic membrane of new-born dorsal root ganglion neurones. Journal of Physiology 359, 431-436. Fox, A. P., NOVYCKY. AM. C. & TSIEN, R. W. (1987a). Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones. Journal of Physiology 394, 149-172. Fox, A. P., NOVYCKY, M. C. & TSIEN, R. W. (1987b). Single-channel recordings of three types of calcium channels in chick sensory neurones. Journal of Physiology 394, 173-200. FRIEDMAN, M. E., SUAREZ-KURTZ, G., KACZOROWSKI, G. J., KATZ, G. N1. & REUBEN, J. P. (1986). Two calcium currents in a smooth muscle cell line. American Journal of Physiology 250, H699-703. GANITKEVICH, V. YA. & ISENBERG, G. (1990). Contribution of two types of calcium channels to membrane conductance of single myocytes from guinea-pig coronary artery. Journal of Physiology 426, 19-42. GANITKEVICH, V. YA., SHUBA, M. F. & SMIRNOV, S. V. (1986). Potential-dependent calcium inward current in a single isolated smooth muscle cell of the guinea-pig taenia caeci. Journal of Physiology 380, 1-16. GANITKEVICH, V. YA., SHUBA, M. F. & SMIIRNOV, S. V. (1987). Calcium-dependent inactivation of potential-dependent calcium inward current in an isolated guinea-pig smooth muscle cell. Journal of Physiology 392, 431-449. GANITKEVICH, V. YA., SMIRNOV, S. V. & SHUBA, AM. F. (1985). Separation of calcium current in isolated smooth muscle cells. Doklady Akademiia Nauk SSSR 282, 717-720. GONOI, T. & HASEGAWA, S. (1988). Post-natal disappearance of transient calcium channels in mouse skeletal muscle: effects of denervation and culture. Journal of Physiology 401, 617-637. HARA, Y., KUBOTA, M. & SZURSZEWSKI, J. H. (1986). Electrophysiology of smooth muscle of the small intestine of some mammals. Journal of Physiology 372, 510-520. HILLE, B. (1984). Ionic Channels of Excitable Membranes. Sinauer Inc., Sunderland, MA, USA. HIRST, G. D. C., SILVERBERG. G. D. & VAN HELDEN, D. F. (1986). The action potential and underlying ionic currents in proximal rat middle cerebral arterioles. Journal of Physiology 371, 289-304. HISADA, T., KURACHI, Y. & SUGIMOTO. T. (1990). Properties of membrane currents in isolated smooth muscle cells from guinea pig trachea. Pfiuigers Archiv 416, 151-161. JMARI, K., MIRONNEAU, C. & MIRONNEAU, J. (1986). Inactivation of calcium channel current in rat uterine smooth muscle: evidence for calcium and voltage-mediated mechanisms. Journal of Physiology 380, 111-126. KL6CKNER, U. & ISENBERG, G. (1985). Calcium currents of caesium loaded isolated smooth muscle cell (urinary bladder of the guinea-pig). Pflidgers Archiv 405, 340-348. KL6CKNER, U. & ISENBERG, G. (1989). The dihydropyridine niguldipine modulates calcium and potassium currents in vascular smooth muscle cells. British Journal of Pharmacology 97, 957-967. LOIRAND, G., MIRONNEAU, C., MIRONNEAU, J. & PACAUD, P. (1989). Two types of calcium currents in single smooth muscle cells from rat portal vein. Journal of Physiology 412, 333-349. LOIRAND, G., PACAUD, P., MIRONNEAU, C. & MIRONNEAU, J. (1986). Evidence for two distinct calcium channels in rat vascular smooth muscle cells in short-term primary culture. Pfluigers Archiv 407, 566-568. MCCARTHY, R. T. & COHEN, C. J. (1989). Nimodipine block of calcium channels in rat vascular smooth muscle cell lines. Journal of General Physiology 94, 669-692. MATSUDA, J. J., VOLK, K. A. & SHIBATA, E. F. (1990). Calcium currents in isolated rabbit coronary arterial smooth muscle myocytes. Journal of Physiology 427, 657-680.

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