Journal of Phy8iology (1992), 451, pp. 525-537 With 8 figure8 Printed in Great Britain
525
PROPERTIES OF SPONTANEOUS INWARD CURRENTS RECORDED IN SMOOTH MUSCLE CELLS ISOLATED FROM THE RABBIT PORTAL VEIN BY Q. WANG, R. C. HOGG AND W. A. LARGE* From the Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE
(Received 19 August 1991) SUMMARY
1. Characteristics of spontaneous transient inward currents (STICs) which produced membrane depolarization were analysed with the perforated patch technique in single smooth muscle cells isolated from the rabbit portal vein. 2. In K+-free solutions the amplitude of STICs was linearly related to membrane potential and the reversal potential (Er) was - 3 0 + 0 9 mV. Replacement of external NaCl with Nal shifted Er to - 40-0 +-0 mV. Substitution of external NaCl by NaSCN also moved Er to negative values but replacement of sodium with Tris and choline did not change Er. It is concluded that STICs are generated by an increase in chloride conductance. 3. STICs were abolished or reduced by the chloride channel antagonists anthracene-9-carboxylic acid (1 mM) and 4-acetamido-4'-isothiocyanato-2,2'-stilbene-disulphonic acid (2 mM). 4. STICs were blocked by noradrenaline and caffeine which deplete intracellular calcium stores. This effect was reversible and this result indicates that the primary trigger for STICs is calcium released from intracellular stores and therefore STICs are calcium-activated chloride currents (Icj(ca)X 5. Removal of calcium from the bathing solution abolished STICs in six out of seven cells but STICs persisted in Ca2+-free solution in one cell. When STICs were abolished in Ca2+-free external solution the size of the internal calcium store, as estimated from the noradrenaline-induced Ici(ca)' was not altered. It appears that an influx of calcium is usually necessary for STICs to be observed. 6. The frequency and amplitude of STICs were not altered by the voltagedependent calcium channel antagonist cadmium (1 mM). However, in some quiescent cells influx of calcium through voltage-dependent channels did activate STICs. 7. It was concluded that in isolated portal vein cells STICs represent a Ca21_ activated chloride current which leads to spontaneous depolarization of the membrane and may play an important physiological or pathophysiological role to produce smooth muscle contraction. *
MS 9659
Author for correspondence.
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Q. WANG, R. C. HOGG AND W. A. LARGE INTRODUCTION
Recently we have been investigating the electrophysiological actions of noradrenaline in freshly dispersed vascular smooth muscle cells with the 'perforated patch' method (Amedee, Large & Wang, 1990; Wang & Large, 1991 a, b). The major advantage of this technique compared to conventional whole-cell recording is that the degree of 'run-down' of agonist-evoked membrane currents is greatly reduced. During the course of these experiments with the 'perforated patch' recording in single rabbit portal vein cells we observed spontaneous transient inward currents (at negative holding potentials) which we had not observed previously with conventional whole-cell recording. Spontaneous transient outward currents (STOCs) have been described in both vascular and non-vascular smooth muscle. STOCs, which are sometimes called oscillatory outward currents, have been recorded in single cells isolated from rabbit ear artery and jejunum (Benham & Bolton, 1986) and portal vein (Ohya, Terada, Yamaguchi, Inoue, Okabe, Kitamura, Hirata & Kuriyama, 1988; Beech & Bolton, 1989; Hume & Leblanc, 1989). It has been shown that these outward currents represent the opening of Ca2+-activated potassium channels produced by the cyclical release of calcium from the sarcoplasmic reticulum. But there has been no previous report on spontaneous inward currents in isolated smooth muscle cells. Spontaneous depolarizations and inward currents have been observed in whole-tissue experiments. Van Helden (1991) described spontaneous depolarizations in the guinea-pig mesenteric vein and with a 'switching clamp' in short segments showed that spontaneous inward currents generated these depolarizations. These latter experiments yielded no clear-cut evidence on the ionic mechanisms of the spontaneous depolarizations. In this paper we have used the 'perforated patch' technique to analyse the ionic mechanisms, the role of calcium and some of the properties of spontaneous transient inward currents (STICs) in single cells isolated from the rabbit portal vein. These currents seem to be of special interest because, as will be shown, they can generate large depolarizations from the resting membrane potential and therefore may be involved in vascular spasm in various pathological conditions. METHODS
The experimental methods of the enzymatic dissociation of the rabbit portal vein cells were similar to those described previously (Wang & Large, 1991 a). However, a new dispersion technique (see Clapp & Gurney, 1991, for details) was also applied to isolate rabbit portal vein cells. Briefly, rabbits (2-2-5 kg) of either sex were killed by an overdose of i.v. sodium pentobarbitone. The portal vein was then dissected free of connective tissue and was cut into two parts, one of which was used on the same day and the other was maintained in an 'overnight enzyme solution' and dispersed for experimentation on the following day. The main advantage of this procedure was that tissue from one animal was used for two days of experimentation. On the first day, small pieces of portal vein (approximately 2 x 2 mm square) were incubated for 10 min at 37 °C in physiological salt solution containing 10 4uM-calcium after which the solution was replaced with a solution containing bovine albumin (5 mg/ml), collagenase (0 5 mg/ml), dithiothreitol (1-25 mM) and papain (4 mg/ml). The muscle pieces were incubated in the enzyme solution for 25-30 min at 37 °C and then washed twice with 10 ml low calcium solution at 37 'C. Single cells were dispersed by trituration of the pieces through a wide-bore siliconized Pasteur pipette at room temperature (20-25 IC). The subsequent cell suspension was centrifuged at 1000 r.p.m. for 2 min and the pellet was resuspended in 0 75 mM-Ca2+ solution. Cells were stored on cover-slips at 4 'C and were used on the same day.
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE
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The 'overnight enzyme solution' contained (mM): NaCl, 110; KCI, 5; CaCl2, 0A16; MgCl2, 2; HEPES, 10; NaHCO3, 10; KH2PO4, 05; NaH2PO4, 0-5; glucose, 10; EDTA, 0-49; taurine, 10 with papain (0 5 mg/ml). The pH was adjusted to 7 0 with NaOH. The rabbit portal vein was cut into strips ( 1 mm wide by 10 mm long) and put into 5 ml of the 'overnight enzyme solution' which was then maintained in the refrigerator (4 °C) overnight (about 15 h). The following morning, 0-1 mM-dithiothreitol was added to the solution which was incubated at 37 °C for 10 min prior to trituration of the tissue. Then the dispersion procedures were the same as 'the same day' dispersion. Whole-cell currents were measured with the perforated patch method as described previously (Amedee, Large & Wang, 1990; Wang & Large, 1991 a, b) with a patch-clamp amplifier (List EPC7; List-Electronic, Darmstadt, Germany) at room temperature (20-25 °C). In order to obtain a perforated patch nystatin (75-200 1g/ml) was contained in the patch pipette solution. The external salt solution contained (mM): NaCl, 126; KCl, 6; MgCl2, 1P2; CaCl2, 1P5; HEPES, 10 and glucose, 11. In potassium-free conditions 6 mM-KCl was omitted. The pipette solution contained (mM): KCl, 126; MgCl2, 1-2; HEPES, 10; glucose, 11 and EGTA, 0-2-1-0. In potassium-free conditions, 126 mM-KCl was replaced by 126 mM-NaCl. Propranolol (1 #M) was added to all external solutions to abolish any fi-adrenoceptor-mediated response and noradrenaline was applied by ionophoresis. The values given in the text are the means+ s.EM. and statistical significance was estimated with Student's t test. Chemicals used were: 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulphonic acid (SITS), anthracene-9-carboxylic acid (A-9-C), propranolol hydrochloride (all Aldrich); bovine serum albumin (fatty acid free), caffeine, dithiothreitol, noradrenaline bitartrate, nystatin, papain (type IV) (all Sigma); collagenase (CLS2 247 U/mg, Worthington). RESULTS
General characteristics of spontaneous inward currents In potassium-containing solutions at a holding potential of -50 mV the most common spontaneous current is an outward current (example is shown in Fig. lAa) which has been reported previously in rabbit portal vein (Ohya et al. 1988; Beech & Bolton, 1989; Hume & Leblanc, 1989). This outward current represents a Ca2+_ activated potassium conductance produced by the random release of calcium from intracellular stores (Benham & Bolton, 1986). In cells that were quiescent at -50 mV, STOCs were evoked when the holding potential was moved to more positive values (e.g. 0 mV). In a few cells the outward current was followed by an inward current (Fig. lAb). These events were normally biphasic in nature but interestingly in some cells spontaneous inward and outward currents occurred as independent responses. In other cells, spontaneous transient inward currents (STICs) were observed at -50 mV even in potassium-containing solutions (Fig. lAc). These responses became biphasic when the membrane potential was depolarized to between -20 and -30 mV. The simplest explanation for these observations is that in addition to the random opening of potassium channels there is spontaneous activation of another membrane mechanism which gives rise to inward currents. Moreover these STICs generate spontaneous depolarizations when current clamp mode of recording is used (Fig. l Ba and l Bb). In the cell shown in Fig. 1B the mean STIC amplitude at a holding potential of -50 mV was 12-1 + 1 1 pA (n = 35) and the mean amplitude of the associated spontaneous depolarizations at about -55 mV was 15-0 + 241 mV (n = 51). In order to study the properties of STICs we used K+-free external and pipette solutions to remove contamination of the prominent Ca2+activated potassium currents. In K+-free conditions, at a holding potential of -50 mV, STICs occurred randomly with a frequency of 0 44 + 007 Hz (range 0418-0-65, n = 15). The range of
Q. WANG, R. C. HOGG AND W. A. LARGE amplitude of STICs was 3-200 pA but typically we used cells where the amplitude was between 10-30 pA. 528
The ionic mechanism of the spontaneous transient inward current The observation that in potassium-containing solutions STOCs and STICs sometimes occurred as biphasic responses (e.g. Fig. 1 Ab) indicated that the A
B
a Voltage clamp
b
Current clamp
Fig. 1. Types of spontaneous currents and membrane potential fluctuations recorded in rabbit portal vein cells. A, voltage-clamp records in potassium-containing solutions at a holding potential of -50 mV. B, voltage-clamp (a) and current-clamp (b) records were obtained from another cell in potassium-free conditions. Vertical calibration: 10 pA for Aa, Ac and Ba, 20 pA for Ab and 20 mV for Bb. Outward and inward currents are represented as upward and downward deflections respectively.
spontaneous inward currents are also Ca2±-activated. This idea prompted us to think that the inward currents might be due to an increase in chloride conductance as it is now well established that there is a Ca2+-activated chloride conductance in rabbit portal vein cells (Byrne & Large, 1988; Wang & Large, 1991 a). The ionic mechanism of STICs was investigated in ion replacement experiments where the influence of various cations and anions on the reversal potential (Er) of STICs was studied. Figure 2 demonstrates the effect of clamp potential on STIC amplitude and it can be seen that reduction of the membrane potential from -50 mV decreased the amplitude of the currents which reverse to outward currents at potentials between -10 and + 10 mV (Fig. 2A). The plot of STIC amplitude against membrane potential between -50 and + 50 mV was linear and Er was + 2 mV (Fig. 2B). In these conditions in five cells Er was - 3-0 + 0 9 mV. Figure 3 illustrates an experiment in which 126 mM-NaCl in the bathing solution was replaced by an equimolar amount of Nal. The clamp potential was set at -30 mV where the spontaneous current is still inward in NaCl
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 529 (Fig. 3A). When NaCl was substituted with Nal the currents reversed to 'n outward direction (Fig 3A). In the continued presence of Nal, further depolarization increased the amplitude of the currents (Fig. 3B) and reversed to inward currents at more negative potentials (-50 and -70 mV, Fig. 3 C and D). At -30 mV, when Nal A -50 mV
-30 mV
+10mV
+30 mV
-10mV
+50 mV
JL
1J I-
40 pA
20 s
B
+80
-50
+50 Membrane potential (mV)
-80
STIC amplitude (pA)
Fig. 2. Relationship between STIC amplitude and membrane potential. In A are shown samples of STICs at the potentials indicated. B, the average amplitude is plotted against clamp potential. In this cell the reversal potential was +2 mV. K+-free conditiQns.
was replaced by NaCl there was reversal of the current (Fig. 3E). In the presence of the external Nal the amplitude of the spontaneous currents was linearly related to membrane potential and Er was -39 mV in this cell. The mean Er in Nal was - 40-0 +I 0 mV (n = 5). This result indicates that a chloride conductance increase contributes to the STIC and that iodide permeates the anion channel more readily than chloride. We have shown previously that thiocyanate is also very permeable through the chloride channel in the rabbit portal vein (Wang & Large, 1991 b) and therefore experiments were carried out when the external NaCl was replaced by NaSCN. In most experiments this manipulation resulted in STICs being abolished. We have no reasonable explanation for this result but in one experiment STICs did persist in the presence of NaSCN and Er was shifted to -65 mV.
Q. WANG, R. C. HOGG AND W. A. LARGE
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A -30 mV
I
NaCI
B -10
I
C -50 mV
Nal 20 pA
E -30 mV
D -70 mV
20 s
ILOlI
N1 r1T-
NaCI
Nal
_ __________
_
I
Fig. 3. Effect of external NaCl substitution on STICs. The holding potentials are given by each trace. In A, 126 mm external NaCl is replaced by an equimolar concentration of Nal as indicated by the bar under the experimental records. NaI is present in B, C and D and is replaced by NaCl in E. K+-free conditions. A
B
20 s
Fig. 4. The effect of chloride channel antagonists on STICs. In A the gap in the record was about 2 min. Vertical calibration: 20 pA in A and 40 pA in B. Holding potential, -50 mV in K+-free conditions.
In contrast, cation substitution did not alter Er of STICs. When external NaCl was replaced by Tris-Cl the mean Er was - 2-8 + 2-9 mV (n = 5). In one experiment sodium was replaced by choline and Er was + 4 mV. Overall these data show that the spontaneous inward currents result from an increase in chloride conductance with minimal contribution (probably none) from a cation conductance. Further evidence for this hypothesis was provided by experiments with chloride channel blocking agents. Figure 4 shows that anthracene-9-carboxylic acid (A-9-C, 1 mm) completely blocked STICs (four experiments) and 4-acetamido-4'-isothiocynato-2,2'-stilbenedisulphonic acid (SITS, 2 mM) reduced the STIC amplitude by
7~ ~ ~ 1 Rlf 1'JI-r
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 531 about 70 % (three experiments). The inhibitory effect of both these compounds was reversible as the spontaneous currents recovered in amplitude on wash-out of the blocking agents. Calcium dependence of spontaneous inward currents The similarity of the random nature of STICs and STOCs suggested that the inward currents also might be activated by the release of Ca2+ from internal stores. A
100 ms noradrenaline V
20 s
B
4Th7---s7 10 mM-caffeine
Fig. 5. The effect of noradrenaline and caffeine on STICs. In A, noradrenaline was applied by ionophoresis at the arrow-head (ionophoretic pulse, 50 nA for 100 ms). In B, 10 mMcaffeine was added to the bathing solution as indicated by the bar and the gap in the record represents about 2 min. Vertical calibration: 100 pA in A and 20 pA in B. Holding potential, -50 mV in K+-free conditions.
This possibility was tested by investigating the effect of drugs that deplete internal calcium stores. Figure 5A shows a cell in which STICs were temporarily abolished by a brief ionophoretic pulse of noradrenaline which itself activated a chloride current (Icl(ca)). About 1 min after the noradrenaline was applied a small STIC could be seen and then the amplitude and frequency of occurrence gradually recovered. A similar result was obtained with caffeine which also depletes calcium stores in high concentrations. Figure 5B demonstrates that bath-applied caffeine activated a chloride current and blocked STIC generation. No spontaneous currents were observed in the continued presence of caffeine but were recorded 2-3 min after
532
Q. WANG, R. C. HOGG AND W. A. LARGE
washing out of the drug (Fig. 5B). Further support for the calcium mediation of STICs was that we observed STICs in many more cells if the pipette EGTA solution was decreased from 1I0 to 0-1 or 0-2 mm. Thus, in one survey of 132 cells studied with 1-0 mM-EGTA in the pipette solution STICs were observed in only twenty cells. With 0-2 mM-EGTA in the pipette solution STICs were recorded from 73 of 137 cells. Consequently, since the percentage of cells which had STICs increased from 15 to 53 % we used 0-2 mM-EGTA in the pipette solution for the majority of experiments. One interpretation of this result is that EGTA enters the cell through the nystatininduced pores and therefore influences the intracellular calcium concentration. However, on theoretical grounds, the high molecular weight of EGTA should preclude permeation of the pores produced by nystatin. It is possible that the EGTA concentration in the pipette might influence calcium diffusion out of the cell into the pipette but divalent cations, also, are thought not to permeate the nystatin-induced channels. At present there appears to be no firm explanation for the influence of EGTA pipette concentration on STIC generation. However, it is difficult to side-step the notion that STICs are more likely to be observed when intracellular calcium is not buffered too strongly. It seemed that STICs were more likely to be recorded in cells which had been previously exposed to bath-applied caffeine (10 mM). Interestingly, a previous casual observation was that the noradrenaline-induced
ICi(ca) often was larger after the cell had been treated with caffeine. It is tempting to
speculate that the intracellular calcium store, which is depleted by caffeine, may be increased after caffeine treatment, but we have no direct evidence for this. These results with noradrenaline and caffeine indicated that STICs are produced by the spontaneous release of calcium from intracellular stores. However, in previous experiments we noticed that the frequency of STICs was reduced at positive potentials. Thus, in nine cells the STIC frequency at -50 mV was 0-43 + 0-02 Hz and at + 50 mV was 0-13 + 0-01 Hz. The most likely explanation appeared to be that there might be an influx of calcium down its electrochemical gradient which facilitates the generation of STICs. The effect of removing external calcium on STICs was investigated. Figure 6A shows that the usual response of Ca2+ removal (also 1 mM-EGTA was added to the bathing solution) was to abolish STICs within about 1 min. About 3 min after re-addition of ealcium to the external solution STICs reappeared (Fig. 6A). This result was obtained in six out of seven cells. However, in one experiment the removal of calcium had no significant effect on STICs either in amplitude or frequency, even after 5 min in Ca2+-free conditions (Fig. 6B). This last result does confirm the hypothesis that extracellular calcium is not essential for STIC generation and that calcium released from the caffeine- and noradrenaline-sensitive stores is the primary trigger for opening the chloride channels. There seem to be two possible explanations for the observation that calcium removal from the bathing solution usually abolished STICs. Firstly, the intracellular stores may be depleted when the extracellular calcium is removed. Secondly, influx of calcium might be necessary in most cells for STICs to be activated. The first possibility was tested by investigating the action of calcium removal on the chloride current activated by noradrenaline. Previously we have shown that the noradrenaline-induced ICi(ca) is mediated purely by calcium release from intracellular stores (Amedee et al. 1990; Wang & Large, 1991 a). In Fig. 7 it can be seen that in
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE A
_
533
1
0 Ca2+ + 1 mM-EGTA
3 min wash
20 pA 20 s
0 Ca2+ + 1 mM-EGTA
5 min 0 Ca2+ + EGTA
1 mM-Cd
Fig. 6. Effect of calcium-removal on STICs. A and B are two different cells. C is another cell in which it had been shown previously that calcium removal abolished STICs. Holding potential, -50 mV in K+-free conditions. 100 ms noradrenaline
tI~~~~
100 ms noradrenaline -- Pr ~~~~~
A
20 PA[
3 min wash 20 s
0 Ca2+ + 1 mM-EGTA
Fig. 7. Comparison of Ca2+ removal on STICs and noradrenaline-induced calciumactivated chloride current. Noradrenaline was applied as indicated by the arrow-head (ionophoretic pulse, 50 nA for 100 ms). Holding potential, -50 mV in K+-free conditions.
normal calcium solution the ionophoretic application of noradrenaline evoked ICl(ca) of 38 pA in amplitude, after which STICs were abolished for about 20 s. After the STICs had recovered the control amplitude and frequency calcium was removed from the bathing solution. In this cell this manipulation abolished STICs rather rapidly in about 10 s. After 1 min in Ca2+-free condition the same pulse of noradrenaline evoked Ic1(ca) with an amplitude of 64 pA. Thus, Ca2+ removal produced a marked potentiation of ICl(ca) rather than a diminution which would be expected if removal of calcium from the external solution abolished STICs due to depletion of intracellular calcium stores. In four cells with STICs the amplitude of the control noradrenaline-evoked ICi(ca) was 74+10 pA. When external calcium was
Q. WANG, R. C. HOGG AND W. A. LARGE
534
removed and STICs had been abolished the noradrenaline-induced current was 132 + 36 pA (n = 4, measured at 1-2 min in Ca2+-free solution). This potentiation of ICI(Ca) at early times in calcium-free solution is interesting and requires further investigation. Consequently it appears that influx of calcium is normally necessary for generation of STICs. B
A
2s
-50
-50 L
Fig. 8. STICs activated by depolarizing pulses. At the holding potential of -50 mV in both cells (A and B) there were no STICs but when depolarizing pulses (1 5 s) to O mV were applied (lower traces) STICs were generated. K+-free conditions.
STIC amplitude and frequency were not affected by chemical agents that block voltage-dependent calcium channels. Figure 6C shows an experiment in which cadmium (1 mM) was added to bathing solution and it can be seen clearly that cadmium had no effect on STICs. Nifedipine (1 ftM) equally did not attenuate STIC amplitude and frequency. Consequently, in terms of STIC production at -50 mV, calcium does not enter the cell through voltage-dependent calcium channels but presumably through some sort of leak pathway. However, in some cells it was quite obvious that calcium influx through voltage-dependent channels did activate spontaneous inward currents. Figure 8 shows the membrane current in two cells held at -50 mV. In both of the cells there were no STICs at this holding potential. A depolarizing step to 0 mV for 1-5 s evoked only a small calcium current but this increased the intracellular calcium concentration sufficiently to produce several inward currents in both cells (Fig. 8). Thus, although calcium entry through voltagedependent channels does not contribute to STIC generation usually, spontaneous inward currents, which resemble STICs, can be activated in quiescent cells by calcium entry through voltage-dependent channels when activated by depolarizing pulses. DISCUSSION
The purpose of the present work was to describe the properties of spontaneously occurring inward currents in rabbit portal vein cells. It appears that STICs are calcium-activated chloride currents which are triggered by transient increases in the intracellular calcium concentration. In this latter respect STICs resemble STOCs and presumably the outward currents are seen much more frequently than inward currents in dispersed cells because Ca2+-activated potassium channels are more sensitive to calcium than are calcium-activated chloride channels as occurs, for example, in rat lacrimal gland cells (Marty, Tan & Trautmann, 1984). Spontaneous
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 535 depolarizations, which are generated by STICs, have not only been seen in isolated cells (Hume & Leblanc, 1989, and present work) but also in whole tissue preparations of the rabbit portal vein (Holman, Kasby, Suthers & Wilson, 1968) and guinea-pig mesenteric vein (Van Helden, 1991). In these tissues sufficiently large spontaneous depolarizations produce action potentials and subsequent contraction and therefore calcium-activated chloride currents may be involved in the mechanism(s) which are responsible for spontaneous mechanical activity in the portal vein. In smooth muscle the chloride equilibrium potential is about -25 mV (Aickin & Brading, 1982) so that an increase in chloride conductance will produce depolarization. In whole tissue it is possible that a spontaneous current can spread electronically to surrounding cells and the consequent depolarization will open voltage-dependent calcium channels to produce contraction. An interesting question to be answered, however, is whether mechanisms exist to convert the apparent random occurrence of STICs into synchronous or propagating contraction. It is interesting that there have been no reports of spontaneous hyperpolarization in whole-tissue preparations of vascular smooth muscle although STOCs are readily seen in isolated cells from many smooth muscle types. A possible explanation is that the sensitivity of Ca2+-activated potassium channels to calcium may be abnormally high when patch pipette technqiues are used in dispersed smooth muscle cells as has been mooted earlier (Wang & Large, 1991a). The data from the ion substitution experiments suggest that there is no cation conductance contribution to the spontaneous inward currents. These results indicate that there might be different membrane mechanisms which generate STICs in rabbit portal vein and guinea-pig mesenteric vein. In the latter tissue, replacement of extracellular chloride or sodium did not produce changes of Er that would be expected if a pure cation or anion conductance mediated STIC generation. Van Helden (1991) concluded that STICs are comprised of more than one membrane conductance mechanism. In the present work the spontaneous inward current seems to be purely a calcium-activated chloride current. Substitution of sodium with Tris or choline produced no change in Er of STICs. Previously we have shown that replacement of sodium by Tris shifted Er of the cation conductance stimulated by adenosine triphosphate by about 35 mV to negative potentials in rabbit ear artery (Benham, Bolton, Byrne & Large, 1987). Also, in rabbit portal vein, Tris substitution altered Er of the noradrenaline-induced cation conductance by about -25 mV (Wang & Large, 1991 a). Thus if there was a cation conductance component of STICs it would be expected that Tris substitution experiments would have changed Er. In addition, it was shown that the chloride channel antagonist A-9-C blocked STICs. We have not investigated the selectivity of this agent rigorously but in the concentration used A-9-C does not block noradrenaline-activated cation current or potassium current (Ca2+-activated). Finally ErS of STICs in external solutions containing NaI and NaSCN agree very closely with values of Er of ICl(ca) activated by noradrenaline and caffeine in rabbit portal vein (Wang & Large, 1991 a). Therefore it seems that a single mechanism (Ca2+-activated chloride) is responsible for STICs observed in the present experiments. Since the Er values of STICs and the noradrenaline-induced IC1(Ca) are similar in external NaI and NaSCN (compare the values of the present work with data from Wang & Large, 1991 a) it might seem that 18
PHY 461
Q. WANG, R. C. HOGG AND W. A. LARGE the same chloride channels are used for both responses and that there are not subpopulations of calcium-activated chloride channels. However, we have pointed out the similar ionic selectivity of calcium-activated chloride channels in smooth muscle and y-aminobutyric acid operated channels in neurons (Amedee et al. 1990). These latter two types of channel are very different in many respects and thus the same 536
ionic selectivity does not necessarily mean that the agonist-evoked and spontaneous inward currents pass through the same channels. It was interesting that in a few cells STOCs and STICs occurred as independent events and the only reasonable explanation for this observation is that there is localization of potassium and chloride channels, at least in some cells. It seems that the primary trigger of STICs is calcium which is released from the intracellular stores. The major evidence for this assertion is that agents which are known to deplete internal stores abolished STICs. When noradrenaline was applied by a brief ionophoretic pulse the STICs were blocked and then recovered within 1-2 min. When caffeine, which also depletes internal calcium stores, was applied in the bathing solution STICs were abolished as long as caffeine was present but recovered within 1-4 min after wash-out. The recovery with both noradrenaline and caffeine presumably represents refilling of stores. Both noradrenaline and caffeine released Ca2+ from intracellular stores prior to abolition of STICs as evinced by large induced Ca2+-activated chloride currents. It is worth reiterating that in the conditions used the noradrenaline-induced chloride current is mediated purely by Ca2+ release from internal stores and therefore it seems the internal calcium responsible for generating STICs is derived from the same noradrenaline- and caffeine-sensitive stores. It seems, however, that extracellular calcium has an important facilitatory role in most cells because calcium removal from the bathing solution abolished STICs within 1-2 min in six out of seven cells. At the time when STICs were abolished in low calcium solution the internal stores did not seem to have been greatly depleted as the noradrenaline-induced ICI(Ca) was not reduced at all. (It should be noted that after 5-10 min in Ca2+-free solution the internal stores appear to be depleted because at these times the noradrenaline-evoked ICI(Ca) is reduced or abolished. For the purpose of the present discussion we are referring to early times in Ca2+-free solution.) It is known that substances which deplete or empty the internal stores (caffeine and papaverine) respectively reduce or abolish noradrenaline-induced IMc(ca) (Wang & Large, 1991 b). Moreover, reduction of the internal calcium stores by caffeine decrease STIC amplitude and thus we conclude that the abolition of STICs by zero external calcium concentration at times when the noradrenaline-evoked ICI(Ca) is unaffected indicates a genuine facilitatory role for extracellular calcium even though Ca21 from internal stores is the primary activator. The explanation that we favour is that there is an influx of calcium which provides a background of increased intracellular concentration upon which is superimposed sharp transient rises in concentration caused by sporadic quantal release of calcium from internal stores. The suggestion of a permissive role for calcium influx down its electrochemical gradient is supported by the observation that the STIC frequency is markedly reduced at a holding potential of + 50 mV compared to -50 mV. Since cadmium had no effect on STIC amplitude or frequency the inward movement of calcium does not
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 537 occur through voltage-dependent calcium channels and therefore some sort of leak pathway must be involved. The overall data suggest that STICs become more likely to be seen when intracellular calcium concentration rises which often happens when cellular metabolism is compromised by damage or disease. Since STICs produce depolarization and consequent action potential generation which leads to smooth muscle contraction, it is possible that STICs may have a pathophysiological role in contributing to vasospasm in pathological conditions. This work was supported by the Medical Research Council and the Wellcome Trust. REFERENCES
AICKIN, C. C. & BRADING, A. F. (1982). Measurement of intracellular chloride in guinea-pig vas deferens by ion analysis, 36chloride efflux and micro-electrodes. Journal of Physiology 326, 139-154. AMEDEE, T., LARGE, W. A. & WANG, Q. (1990). Characteristics of chloride currents activated by noradrenaline in rabbit ear artery cells. Journal of Physiology 428, 501-516. BEECH, D. J. & BOLTON, T. B. (1989). Two components of potassium currents activated by depolarization of single smooth muscle cells from the rabbit portal vein. Journal ofPhysiology 41, 293-309. BENHAM, C. D. & BOLTON, T. B. (1986). Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. Journal of Physiology 381, 385-406. BENHAM, C. D., BOLTON, T. B., BYRNE, N. G. & LARGE, W. A. (1987). Action of externally applied adenosine triphosphate on single smooth muscle cells dispersed from the rabbit ear artery. Journal of Physiology 387, 473-488. BYRNE, N. G. & LARGE, W. A. (1988). Membrane ionic mechanisms activated by noradrenaline in cells isolated from the rabbit portal vein. Journal of Physiology 404, 557-573. CLAPP, L. H. & GURNEY, A. M. (1991). Outward currents in rabbit pulmonary artery cells dissociated with a new technique. Experimental Physiology 76, 677-693. HOLMAN, M. E., KASBY, C. B., SUTHERS, M. D. & WILSON, J. A. F. (1968). Some properties of smooth muscle of the rabbit portal vein. Journal of Physiology 196, 111-132. HUME, J. R. & LEBLANC, N. (1989). Macroscopic K+ currents in single smooth muscle cells of the rabbit portal vein. Journal of Physiology 413, 49-73. MARTY, A., TAN, Y. P. & TRAUTMANN, A. (1984). Three types of calcium-dependent channel in rat lacrimal glands. Journal of Physiology 357, 293-325. OHYA, Y., TERADA, K., YAMAGACHI, K., INOUE, R., OKABE, K., KITAMURA, K., HRATA, M. & KURIYAMA, H. (1988). Effects of inositol phosphates on the membrane activity of smooth muscle cells of the rabbit portal vein. Pfluigers Archiv 412, 382-389. VAN HELDEN, D. F. (1991). Spontaneous and noradrenaline-induced transient depolarizations in the smooth muscle of guinea-pig mesenteric vein. Journal of Physiology 437, 511-541. WANG, Q. & LARGE, W. A. (1991 a). Noradrenaline-evoked cation conductance recorded with the nystatin whole-cell method in rabbit portal vein cells. Journal of Physiology 435, 21-39. WANG, Q. & LARGE, W. A. (1991 b). Modulation of noradrenaline-induced membrane currents by papaverine in rabbit vascular smooth muscle cells. Journal of Physiology 439, 501-512.
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PROPERTIES OF SPONTANEOUS INWARD CURRENTS RECORDED IN SMOOTH MUSCLE CELLS ISOLATED FROM THE RABBIT PORTAL VEIN BY Q. WANG, R. C. HOGG AND W. A. LARGE* From the Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE
(Received 19 August 1991) SUMMARY
1. Characteristics of spontaneous transient inward currents (STICs) which produced membrane depolarization were analysed with the perforated patch technique in single smooth muscle cells isolated from the rabbit portal vein. 2. In K+-free solutions the amplitude of STICs was linearly related to membrane potential and the reversal potential (Er) was - 3 0 + 0 9 mV. Replacement of external NaCl with Nal shifted Er to - 40-0 +-0 mV. Substitution of external NaCl by NaSCN also moved Er to negative values but replacement of sodium with Tris and choline did not change Er. It is concluded that STICs are generated by an increase in chloride conductance. 3. STICs were abolished or reduced by the chloride channel antagonists anthracene-9-carboxylic acid (1 mM) and 4-acetamido-4'-isothiocyanato-2,2'-stilbene-disulphonic acid (2 mM). 4. STICs were blocked by noradrenaline and caffeine which deplete intracellular calcium stores. This effect was reversible and this result indicates that the primary trigger for STICs is calcium released from intracellular stores and therefore STICs are calcium-activated chloride currents (Icj(ca)X 5. Removal of calcium from the bathing solution abolished STICs in six out of seven cells but STICs persisted in Ca2+-free solution in one cell. When STICs were abolished in Ca2+-free external solution the size of the internal calcium store, as estimated from the noradrenaline-induced Ici(ca)' was not altered. It appears that an influx of calcium is usually necessary for STICs to be observed. 6. The frequency and amplitude of STICs were not altered by the voltagedependent calcium channel antagonist cadmium (1 mM). However, in some quiescent cells influx of calcium through voltage-dependent channels did activate STICs. 7. It was concluded that in isolated portal vein cells STICs represent a Ca21_ activated chloride current which leads to spontaneous depolarization of the membrane and may play an important physiological or pathophysiological role to produce smooth muscle contraction. *
MS 9659
Author for correspondence.
526
Q. WANG, R. C. HOGG AND W. A. LARGE INTRODUCTION
Recently we have been investigating the electrophysiological actions of noradrenaline in freshly dispersed vascular smooth muscle cells with the 'perforated patch' method (Amedee, Large & Wang, 1990; Wang & Large, 1991 a, b). The major advantage of this technique compared to conventional whole-cell recording is that the degree of 'run-down' of agonist-evoked membrane currents is greatly reduced. During the course of these experiments with the 'perforated patch' recording in single rabbit portal vein cells we observed spontaneous transient inward currents (at negative holding potentials) which we had not observed previously with conventional whole-cell recording. Spontaneous transient outward currents (STOCs) have been described in both vascular and non-vascular smooth muscle. STOCs, which are sometimes called oscillatory outward currents, have been recorded in single cells isolated from rabbit ear artery and jejunum (Benham & Bolton, 1986) and portal vein (Ohya, Terada, Yamaguchi, Inoue, Okabe, Kitamura, Hirata & Kuriyama, 1988; Beech & Bolton, 1989; Hume & Leblanc, 1989). It has been shown that these outward currents represent the opening of Ca2+-activated potassium channels produced by the cyclical release of calcium from the sarcoplasmic reticulum. But there has been no previous report on spontaneous inward currents in isolated smooth muscle cells. Spontaneous depolarizations and inward currents have been observed in whole-tissue experiments. Van Helden (1991) described spontaneous depolarizations in the guinea-pig mesenteric vein and with a 'switching clamp' in short segments showed that spontaneous inward currents generated these depolarizations. These latter experiments yielded no clear-cut evidence on the ionic mechanisms of the spontaneous depolarizations. In this paper we have used the 'perforated patch' technique to analyse the ionic mechanisms, the role of calcium and some of the properties of spontaneous transient inward currents (STICs) in single cells isolated from the rabbit portal vein. These currents seem to be of special interest because, as will be shown, they can generate large depolarizations from the resting membrane potential and therefore may be involved in vascular spasm in various pathological conditions. METHODS
The experimental methods of the enzymatic dissociation of the rabbit portal vein cells were similar to those described previously (Wang & Large, 1991 a). However, a new dispersion technique (see Clapp & Gurney, 1991, for details) was also applied to isolate rabbit portal vein cells. Briefly, rabbits (2-2-5 kg) of either sex were killed by an overdose of i.v. sodium pentobarbitone. The portal vein was then dissected free of connective tissue and was cut into two parts, one of which was used on the same day and the other was maintained in an 'overnight enzyme solution' and dispersed for experimentation on the following day. The main advantage of this procedure was that tissue from one animal was used for two days of experimentation. On the first day, small pieces of portal vein (approximately 2 x 2 mm square) were incubated for 10 min at 37 °C in physiological salt solution containing 10 4uM-calcium after which the solution was replaced with a solution containing bovine albumin (5 mg/ml), collagenase (0 5 mg/ml), dithiothreitol (1-25 mM) and papain (4 mg/ml). The muscle pieces were incubated in the enzyme solution for 25-30 min at 37 °C and then washed twice with 10 ml low calcium solution at 37 'C. Single cells were dispersed by trituration of the pieces through a wide-bore siliconized Pasteur pipette at room temperature (20-25 IC). The subsequent cell suspension was centrifuged at 1000 r.p.m. for 2 min and the pellet was resuspended in 0 75 mM-Ca2+ solution. Cells were stored on cover-slips at 4 'C and were used on the same day.
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE
527
The 'overnight enzyme solution' contained (mM): NaCl, 110; KCI, 5; CaCl2, 0A16; MgCl2, 2; HEPES, 10; NaHCO3, 10; KH2PO4, 05; NaH2PO4, 0-5; glucose, 10; EDTA, 0-49; taurine, 10 with papain (0 5 mg/ml). The pH was adjusted to 7 0 with NaOH. The rabbit portal vein was cut into strips ( 1 mm wide by 10 mm long) and put into 5 ml of the 'overnight enzyme solution' which was then maintained in the refrigerator (4 °C) overnight (about 15 h). The following morning, 0-1 mM-dithiothreitol was added to the solution which was incubated at 37 °C for 10 min prior to trituration of the tissue. Then the dispersion procedures were the same as 'the same day' dispersion. Whole-cell currents were measured with the perforated patch method as described previously (Amedee, Large & Wang, 1990; Wang & Large, 1991 a, b) with a patch-clamp amplifier (List EPC7; List-Electronic, Darmstadt, Germany) at room temperature (20-25 °C). In order to obtain a perforated patch nystatin (75-200 1g/ml) was contained in the patch pipette solution. The external salt solution contained (mM): NaCl, 126; KCl, 6; MgCl2, 1P2; CaCl2, 1P5; HEPES, 10 and glucose, 11. In potassium-free conditions 6 mM-KCl was omitted. The pipette solution contained (mM): KCl, 126; MgCl2, 1-2; HEPES, 10; glucose, 11 and EGTA, 0-2-1-0. In potassium-free conditions, 126 mM-KCl was replaced by 126 mM-NaCl. Propranolol (1 #M) was added to all external solutions to abolish any fi-adrenoceptor-mediated response and noradrenaline was applied by ionophoresis. The values given in the text are the means+ s.EM. and statistical significance was estimated with Student's t test. Chemicals used were: 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulphonic acid (SITS), anthracene-9-carboxylic acid (A-9-C), propranolol hydrochloride (all Aldrich); bovine serum albumin (fatty acid free), caffeine, dithiothreitol, noradrenaline bitartrate, nystatin, papain (type IV) (all Sigma); collagenase (CLS2 247 U/mg, Worthington). RESULTS
General characteristics of spontaneous inward currents In potassium-containing solutions at a holding potential of -50 mV the most common spontaneous current is an outward current (example is shown in Fig. lAa) which has been reported previously in rabbit portal vein (Ohya et al. 1988; Beech & Bolton, 1989; Hume & Leblanc, 1989). This outward current represents a Ca2+_ activated potassium conductance produced by the random release of calcium from intracellular stores (Benham & Bolton, 1986). In cells that were quiescent at -50 mV, STOCs were evoked when the holding potential was moved to more positive values (e.g. 0 mV). In a few cells the outward current was followed by an inward current (Fig. lAb). These events were normally biphasic in nature but interestingly in some cells spontaneous inward and outward currents occurred as independent responses. In other cells, spontaneous transient inward currents (STICs) were observed at -50 mV even in potassium-containing solutions (Fig. lAc). These responses became biphasic when the membrane potential was depolarized to between -20 and -30 mV. The simplest explanation for these observations is that in addition to the random opening of potassium channels there is spontaneous activation of another membrane mechanism which gives rise to inward currents. Moreover these STICs generate spontaneous depolarizations when current clamp mode of recording is used (Fig. l Ba and l Bb). In the cell shown in Fig. 1B the mean STIC amplitude at a holding potential of -50 mV was 12-1 + 1 1 pA (n = 35) and the mean amplitude of the associated spontaneous depolarizations at about -55 mV was 15-0 + 241 mV (n = 51). In order to study the properties of STICs we used K+-free external and pipette solutions to remove contamination of the prominent Ca2+activated potassium currents. In K+-free conditions, at a holding potential of -50 mV, STICs occurred randomly with a frequency of 0 44 + 007 Hz (range 0418-0-65, n = 15). The range of
Q. WANG, R. C. HOGG AND W. A. LARGE amplitude of STICs was 3-200 pA but typically we used cells where the amplitude was between 10-30 pA. 528
The ionic mechanism of the spontaneous transient inward current The observation that in potassium-containing solutions STOCs and STICs sometimes occurred as biphasic responses (e.g. Fig. 1 Ab) indicated that the A
B
a Voltage clamp
b
Current clamp
Fig. 1. Types of spontaneous currents and membrane potential fluctuations recorded in rabbit portal vein cells. A, voltage-clamp records in potassium-containing solutions at a holding potential of -50 mV. B, voltage-clamp (a) and current-clamp (b) records were obtained from another cell in potassium-free conditions. Vertical calibration: 10 pA for Aa, Ac and Ba, 20 pA for Ab and 20 mV for Bb. Outward and inward currents are represented as upward and downward deflections respectively.
spontaneous inward currents are also Ca2±-activated. This idea prompted us to think that the inward currents might be due to an increase in chloride conductance as it is now well established that there is a Ca2+-activated chloride conductance in rabbit portal vein cells (Byrne & Large, 1988; Wang & Large, 1991 a). The ionic mechanism of STICs was investigated in ion replacement experiments where the influence of various cations and anions on the reversal potential (Er) of STICs was studied. Figure 2 demonstrates the effect of clamp potential on STIC amplitude and it can be seen that reduction of the membrane potential from -50 mV decreased the amplitude of the currents which reverse to outward currents at potentials between -10 and + 10 mV (Fig. 2A). The plot of STIC amplitude against membrane potential between -50 and + 50 mV was linear and Er was + 2 mV (Fig. 2B). In these conditions in five cells Er was - 3-0 + 0 9 mV. Figure 3 illustrates an experiment in which 126 mM-NaCl in the bathing solution was replaced by an equimolar amount of Nal. The clamp potential was set at -30 mV where the spontaneous current is still inward in NaCl
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 529 (Fig. 3A). When NaCl was substituted with Nal the currents reversed to 'n outward direction (Fig 3A). In the continued presence of Nal, further depolarization increased the amplitude of the currents (Fig. 3B) and reversed to inward currents at more negative potentials (-50 and -70 mV, Fig. 3 C and D). At -30 mV, when Nal A -50 mV
-30 mV
+10mV
+30 mV
-10mV
+50 mV
JL
1J I-
40 pA
20 s
B
+80
-50
+50 Membrane potential (mV)
-80
STIC amplitude (pA)
Fig. 2. Relationship between STIC amplitude and membrane potential. In A are shown samples of STICs at the potentials indicated. B, the average amplitude is plotted against clamp potential. In this cell the reversal potential was +2 mV. K+-free conditiQns.
was replaced by NaCl there was reversal of the current (Fig. 3E). In the presence of the external Nal the amplitude of the spontaneous currents was linearly related to membrane potential and Er was -39 mV in this cell. The mean Er in Nal was - 40-0 +I 0 mV (n = 5). This result indicates that a chloride conductance increase contributes to the STIC and that iodide permeates the anion channel more readily than chloride. We have shown previously that thiocyanate is also very permeable through the chloride channel in the rabbit portal vein (Wang & Large, 1991 b) and therefore experiments were carried out when the external NaCl was replaced by NaSCN. In most experiments this manipulation resulted in STICs being abolished. We have no reasonable explanation for this result but in one experiment STICs did persist in the presence of NaSCN and Er was shifted to -65 mV.
Q. WANG, R. C. HOGG AND W. A. LARGE
530
A -30 mV
I
NaCI
B -10
I
C -50 mV
Nal 20 pA
E -30 mV
D -70 mV
20 s
ILOlI
N1 r1T-
NaCI
Nal
_ __________
_
I
Fig. 3. Effect of external NaCl substitution on STICs. The holding potentials are given by each trace. In A, 126 mm external NaCl is replaced by an equimolar concentration of Nal as indicated by the bar under the experimental records. NaI is present in B, C and D and is replaced by NaCl in E. K+-free conditions. A
B
20 s
Fig. 4. The effect of chloride channel antagonists on STICs. In A the gap in the record was about 2 min. Vertical calibration: 20 pA in A and 40 pA in B. Holding potential, -50 mV in K+-free conditions.
In contrast, cation substitution did not alter Er of STICs. When external NaCl was replaced by Tris-Cl the mean Er was - 2-8 + 2-9 mV (n = 5). In one experiment sodium was replaced by choline and Er was + 4 mV. Overall these data show that the spontaneous inward currents result from an increase in chloride conductance with minimal contribution (probably none) from a cation conductance. Further evidence for this hypothesis was provided by experiments with chloride channel blocking agents. Figure 4 shows that anthracene-9-carboxylic acid (A-9-C, 1 mm) completely blocked STICs (four experiments) and 4-acetamido-4'-isothiocynato-2,2'-stilbenedisulphonic acid (SITS, 2 mM) reduced the STIC amplitude by
7~ ~ ~ 1 Rlf 1'JI-r
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 531 about 70 % (three experiments). The inhibitory effect of both these compounds was reversible as the spontaneous currents recovered in amplitude on wash-out of the blocking agents. Calcium dependence of spontaneous inward currents The similarity of the random nature of STICs and STOCs suggested that the inward currents also might be activated by the release of Ca2+ from internal stores. A
100 ms noradrenaline V
20 s
B
4Th7---s7 10 mM-caffeine
Fig. 5. The effect of noradrenaline and caffeine on STICs. In A, noradrenaline was applied by ionophoresis at the arrow-head (ionophoretic pulse, 50 nA for 100 ms). In B, 10 mMcaffeine was added to the bathing solution as indicated by the bar and the gap in the record represents about 2 min. Vertical calibration: 100 pA in A and 20 pA in B. Holding potential, -50 mV in K+-free conditions.
This possibility was tested by investigating the effect of drugs that deplete internal calcium stores. Figure 5A shows a cell in which STICs were temporarily abolished by a brief ionophoretic pulse of noradrenaline which itself activated a chloride current (Icl(ca)). About 1 min after the noradrenaline was applied a small STIC could be seen and then the amplitude and frequency of occurrence gradually recovered. A similar result was obtained with caffeine which also depletes calcium stores in high concentrations. Figure 5B demonstrates that bath-applied caffeine activated a chloride current and blocked STIC generation. No spontaneous currents were observed in the continued presence of caffeine but were recorded 2-3 min after
532
Q. WANG, R. C. HOGG AND W. A. LARGE
washing out of the drug (Fig. 5B). Further support for the calcium mediation of STICs was that we observed STICs in many more cells if the pipette EGTA solution was decreased from 1I0 to 0-1 or 0-2 mm. Thus, in one survey of 132 cells studied with 1-0 mM-EGTA in the pipette solution STICs were observed in only twenty cells. With 0-2 mM-EGTA in the pipette solution STICs were recorded from 73 of 137 cells. Consequently, since the percentage of cells which had STICs increased from 15 to 53 % we used 0-2 mM-EGTA in the pipette solution for the majority of experiments. One interpretation of this result is that EGTA enters the cell through the nystatininduced pores and therefore influences the intracellular calcium concentration. However, on theoretical grounds, the high molecular weight of EGTA should preclude permeation of the pores produced by nystatin. It is possible that the EGTA concentration in the pipette might influence calcium diffusion out of the cell into the pipette but divalent cations, also, are thought not to permeate the nystatin-induced channels. At present there appears to be no firm explanation for the influence of EGTA pipette concentration on STIC generation. However, it is difficult to side-step the notion that STICs are more likely to be observed when intracellular calcium is not buffered too strongly. It seemed that STICs were more likely to be recorded in cells which had been previously exposed to bath-applied caffeine (10 mM). Interestingly, a previous casual observation was that the noradrenaline-induced
ICi(ca) often was larger after the cell had been treated with caffeine. It is tempting to
speculate that the intracellular calcium store, which is depleted by caffeine, may be increased after caffeine treatment, but we have no direct evidence for this. These results with noradrenaline and caffeine indicated that STICs are produced by the spontaneous release of calcium from intracellular stores. However, in previous experiments we noticed that the frequency of STICs was reduced at positive potentials. Thus, in nine cells the STIC frequency at -50 mV was 0-43 + 0-02 Hz and at + 50 mV was 0-13 + 0-01 Hz. The most likely explanation appeared to be that there might be an influx of calcium down its electrochemical gradient which facilitates the generation of STICs. The effect of removing external calcium on STICs was investigated. Figure 6A shows that the usual response of Ca2+ removal (also 1 mM-EGTA was added to the bathing solution) was to abolish STICs within about 1 min. About 3 min after re-addition of ealcium to the external solution STICs reappeared (Fig. 6A). This result was obtained in six out of seven cells. However, in one experiment the removal of calcium had no significant effect on STICs either in amplitude or frequency, even after 5 min in Ca2+-free conditions (Fig. 6B). This last result does confirm the hypothesis that extracellular calcium is not essential for STIC generation and that calcium released from the caffeine- and noradrenaline-sensitive stores is the primary trigger for opening the chloride channels. There seem to be two possible explanations for the observation that calcium removal from the bathing solution usually abolished STICs. Firstly, the intracellular stores may be depleted when the extracellular calcium is removed. Secondly, influx of calcium might be necessary in most cells for STICs to be activated. The first possibility was tested by investigating the action of calcium removal on the chloride current activated by noradrenaline. Previously we have shown that the noradrenaline-induced ICi(ca) is mediated purely by calcium release from intracellular stores (Amedee et al. 1990; Wang & Large, 1991 a). In Fig. 7 it can be seen that in
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE A
_
533
1
0 Ca2+ + 1 mM-EGTA
3 min wash
20 pA 20 s
0 Ca2+ + 1 mM-EGTA
5 min 0 Ca2+ + EGTA
1 mM-Cd
Fig. 6. Effect of calcium-removal on STICs. A and B are two different cells. C is another cell in which it had been shown previously that calcium removal abolished STICs. Holding potential, -50 mV in K+-free conditions. 100 ms noradrenaline
tI~~~~
100 ms noradrenaline -- Pr ~~~~~
A
20 PA[
3 min wash 20 s
0 Ca2+ + 1 mM-EGTA
Fig. 7. Comparison of Ca2+ removal on STICs and noradrenaline-induced calciumactivated chloride current. Noradrenaline was applied as indicated by the arrow-head (ionophoretic pulse, 50 nA for 100 ms). Holding potential, -50 mV in K+-free conditions.
normal calcium solution the ionophoretic application of noradrenaline evoked ICl(ca) of 38 pA in amplitude, after which STICs were abolished for about 20 s. After the STICs had recovered the control amplitude and frequency calcium was removed from the bathing solution. In this cell this manipulation abolished STICs rather rapidly in about 10 s. After 1 min in Ca2+-free condition the same pulse of noradrenaline evoked Ic1(ca) with an amplitude of 64 pA. Thus, Ca2+ removal produced a marked potentiation of ICl(ca) rather than a diminution which would be expected if removal of calcium from the external solution abolished STICs due to depletion of intracellular calcium stores. In four cells with STICs the amplitude of the control noradrenaline-evoked ICi(ca) was 74+10 pA. When external calcium was
Q. WANG, R. C. HOGG AND W. A. LARGE
534
removed and STICs had been abolished the noradrenaline-induced current was 132 + 36 pA (n = 4, measured at 1-2 min in Ca2+-free solution). This potentiation of ICI(Ca) at early times in calcium-free solution is interesting and requires further investigation. Consequently it appears that influx of calcium is normally necessary for generation of STICs. B
A
2s
-50
-50 L
Fig. 8. STICs activated by depolarizing pulses. At the holding potential of -50 mV in both cells (A and B) there were no STICs but when depolarizing pulses (1 5 s) to O mV were applied (lower traces) STICs were generated. K+-free conditions.
STIC amplitude and frequency were not affected by chemical agents that block voltage-dependent calcium channels. Figure 6C shows an experiment in which cadmium (1 mM) was added to bathing solution and it can be seen clearly that cadmium had no effect on STICs. Nifedipine (1 ftM) equally did not attenuate STIC amplitude and frequency. Consequently, in terms of STIC production at -50 mV, calcium does not enter the cell through voltage-dependent calcium channels but presumably through some sort of leak pathway. However, in some cells it was quite obvious that calcium influx through voltage-dependent channels did activate spontaneous inward currents. Figure 8 shows the membrane current in two cells held at -50 mV. In both of the cells there were no STICs at this holding potential. A depolarizing step to 0 mV for 1-5 s evoked only a small calcium current but this increased the intracellular calcium concentration sufficiently to produce several inward currents in both cells (Fig. 8). Thus, although calcium entry through voltagedependent channels does not contribute to STIC generation usually, spontaneous inward currents, which resemble STICs, can be activated in quiescent cells by calcium entry through voltage-dependent channels when activated by depolarizing pulses. DISCUSSION
The purpose of the present work was to describe the properties of spontaneously occurring inward currents in rabbit portal vein cells. It appears that STICs are calcium-activated chloride currents which are triggered by transient increases in the intracellular calcium concentration. In this latter respect STICs resemble STOCs and presumably the outward currents are seen much more frequently than inward currents in dispersed cells because Ca2+-activated potassium channels are more sensitive to calcium than are calcium-activated chloride channels as occurs, for example, in rat lacrimal gland cells (Marty, Tan & Trautmann, 1984). Spontaneous
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 535 depolarizations, which are generated by STICs, have not only been seen in isolated cells (Hume & Leblanc, 1989, and present work) but also in whole tissue preparations of the rabbit portal vein (Holman, Kasby, Suthers & Wilson, 1968) and guinea-pig mesenteric vein (Van Helden, 1991). In these tissues sufficiently large spontaneous depolarizations produce action potentials and subsequent contraction and therefore calcium-activated chloride currents may be involved in the mechanism(s) which are responsible for spontaneous mechanical activity in the portal vein. In smooth muscle the chloride equilibrium potential is about -25 mV (Aickin & Brading, 1982) so that an increase in chloride conductance will produce depolarization. In whole tissue it is possible that a spontaneous current can spread electronically to surrounding cells and the consequent depolarization will open voltage-dependent calcium channels to produce contraction. An interesting question to be answered, however, is whether mechanisms exist to convert the apparent random occurrence of STICs into synchronous or propagating contraction. It is interesting that there have been no reports of spontaneous hyperpolarization in whole-tissue preparations of vascular smooth muscle although STOCs are readily seen in isolated cells from many smooth muscle types. A possible explanation is that the sensitivity of Ca2+-activated potassium channels to calcium may be abnormally high when patch pipette technqiues are used in dispersed smooth muscle cells as has been mooted earlier (Wang & Large, 1991a). The data from the ion substitution experiments suggest that there is no cation conductance contribution to the spontaneous inward currents. These results indicate that there might be different membrane mechanisms which generate STICs in rabbit portal vein and guinea-pig mesenteric vein. In the latter tissue, replacement of extracellular chloride or sodium did not produce changes of Er that would be expected if a pure cation or anion conductance mediated STIC generation. Van Helden (1991) concluded that STICs are comprised of more than one membrane conductance mechanism. In the present work the spontaneous inward current seems to be purely a calcium-activated chloride current. Substitution of sodium with Tris or choline produced no change in Er of STICs. Previously we have shown that replacement of sodium by Tris shifted Er of the cation conductance stimulated by adenosine triphosphate by about 35 mV to negative potentials in rabbit ear artery (Benham, Bolton, Byrne & Large, 1987). Also, in rabbit portal vein, Tris substitution altered Er of the noradrenaline-induced cation conductance by about -25 mV (Wang & Large, 1991 a). Thus if there was a cation conductance component of STICs it would be expected that Tris substitution experiments would have changed Er. In addition, it was shown that the chloride channel antagonist A-9-C blocked STICs. We have not investigated the selectivity of this agent rigorously but in the concentration used A-9-C does not block noradrenaline-activated cation current or potassium current (Ca2+-activated). Finally ErS of STICs in external solutions containing NaI and NaSCN agree very closely with values of Er of ICl(ca) activated by noradrenaline and caffeine in rabbit portal vein (Wang & Large, 1991 a). Therefore it seems that a single mechanism (Ca2+-activated chloride) is responsible for STICs observed in the present experiments. Since the Er values of STICs and the noradrenaline-induced IC1(Ca) are similar in external NaI and NaSCN (compare the values of the present work with data from Wang & Large, 1991 a) it might seem that 18
PHY 461
Q. WANG, R. C. HOGG AND W. A. LARGE the same chloride channels are used for both responses and that there are not subpopulations of calcium-activated chloride channels. However, we have pointed out the similar ionic selectivity of calcium-activated chloride channels in smooth muscle and y-aminobutyric acid operated channels in neurons (Amedee et al. 1990). These latter two types of channel are very different in many respects and thus the same 536
ionic selectivity does not necessarily mean that the agonist-evoked and spontaneous inward currents pass through the same channels. It was interesting that in a few cells STOCs and STICs occurred as independent events and the only reasonable explanation for this observation is that there is localization of potassium and chloride channels, at least in some cells. It seems that the primary trigger of STICs is calcium which is released from the intracellular stores. The major evidence for this assertion is that agents which are known to deplete internal stores abolished STICs. When noradrenaline was applied by a brief ionophoretic pulse the STICs were blocked and then recovered within 1-2 min. When caffeine, which also depletes internal calcium stores, was applied in the bathing solution STICs were abolished as long as caffeine was present but recovered within 1-4 min after wash-out. The recovery with both noradrenaline and caffeine presumably represents refilling of stores. Both noradrenaline and caffeine released Ca2+ from intracellular stores prior to abolition of STICs as evinced by large induced Ca2+-activated chloride currents. It is worth reiterating that in the conditions used the noradrenaline-induced chloride current is mediated purely by Ca2+ release from internal stores and therefore it seems the internal calcium responsible for generating STICs is derived from the same noradrenaline- and caffeine-sensitive stores. It seems, however, that extracellular calcium has an important facilitatory role in most cells because calcium removal from the bathing solution abolished STICs within 1-2 min in six out of seven cells. At the time when STICs were abolished in low calcium solution the internal stores did not seem to have been greatly depleted as the noradrenaline-induced ICI(Ca) was not reduced at all. (It should be noted that after 5-10 min in Ca2+-free solution the internal stores appear to be depleted because at these times the noradrenaline-evoked ICI(Ca) is reduced or abolished. For the purpose of the present discussion we are referring to early times in Ca2+-free solution.) It is known that substances which deplete or empty the internal stores (caffeine and papaverine) respectively reduce or abolish noradrenaline-induced IMc(ca) (Wang & Large, 1991 b). Moreover, reduction of the internal calcium stores by caffeine decrease STIC amplitude and thus we conclude that the abolition of STICs by zero external calcium concentration at times when the noradrenaline-evoked ICI(Ca) is unaffected indicates a genuine facilitatory role for extracellular calcium even though Ca21 from internal stores is the primary activator. The explanation that we favour is that there is an influx of calcium which provides a background of increased intracellular concentration upon which is superimposed sharp transient rises in concentration caused by sporadic quantal release of calcium from internal stores. The suggestion of a permissive role for calcium influx down its electrochemical gradient is supported by the observation that the STIC frequency is markedly reduced at a holding potential of + 50 mV compared to -50 mV. Since cadmium had no effect on STIC amplitude or frequency the inward movement of calcium does not
SPONTANEOUS INWARD CURRENTS IN SMOOTH MUSCLE 537 occur through voltage-dependent calcium channels and therefore some sort of leak pathway must be involved. The overall data suggest that STICs become more likely to be seen when intracellular calcium concentration rises which often happens when cellular metabolism is compromised by damage or disease. Since STICs produce depolarization and consequent action potential generation which leads to smooth muscle contraction, it is possible that STICs may have a pathophysiological role in contributing to vasospasm in pathological conditions. This work was supported by the Medical Research Council and the Wellcome Trust. REFERENCES
AICKIN, C. C. & BRADING, A. F. (1982). Measurement of intracellular chloride in guinea-pig vas deferens by ion analysis, 36chloride efflux and micro-electrodes. Journal of Physiology 326, 139-154. AMEDEE, T., LARGE, W. A. & WANG, Q. (1990). Characteristics of chloride currents activated by noradrenaline in rabbit ear artery cells. Journal of Physiology 428, 501-516. BEECH, D. J. & BOLTON, T. B. (1989). Two components of potassium currents activated by depolarization of single smooth muscle cells from the rabbit portal vein. Journal ofPhysiology 41, 293-309. BENHAM, C. D. & BOLTON, T. B. (1986). Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. Journal of Physiology 381, 385-406. BENHAM, C. D., BOLTON, T. B., BYRNE, N. G. & LARGE, W. A. (1987). Action of externally applied adenosine triphosphate on single smooth muscle cells dispersed from the rabbit ear artery. Journal of Physiology 387, 473-488. BYRNE, N. G. & LARGE, W. A. (1988). Membrane ionic mechanisms activated by noradrenaline in cells isolated from the rabbit portal vein. Journal of Physiology 404, 557-573. CLAPP, L. H. & GURNEY, A. M. (1991). Outward currents in rabbit pulmonary artery cells dissociated with a new technique. Experimental Physiology 76, 677-693. HOLMAN, M. E., KASBY, C. B., SUTHERS, M. D. & WILSON, J. A. F. (1968). Some properties of smooth muscle of the rabbit portal vein. Journal of Physiology 196, 111-132. HUME, J. R. & LEBLANC, N. (1989). Macroscopic K+ currents in single smooth muscle cells of the rabbit portal vein. Journal of Physiology 413, 49-73. MARTY, A., TAN, Y. P. & TRAUTMANN, A. (1984). Three types of calcium-dependent channel in rat lacrimal glands. Journal of Physiology 357, 293-325. OHYA, Y., TERADA, K., YAMAGACHI, K., INOUE, R., OKABE, K., KITAMURA, K., HRATA, M. & KURIYAMA, H. (1988). Effects of inositol phosphates on the membrane activity of smooth muscle cells of the rabbit portal vein. Pfluigers Archiv 412, 382-389. VAN HELDEN, D. F. (1991). Spontaneous and noradrenaline-induced transient depolarizations in the smooth muscle of guinea-pig mesenteric vein. Journal of Physiology 437, 511-541. WANG, Q. & LARGE, W. A. (1991 a). Noradrenaline-evoked cation conductance recorded with the nystatin whole-cell method in rabbit portal vein cells. Journal of Physiology 435, 21-39. WANG, Q. & LARGE, W. A. (1991 b). Modulation of noradrenaline-induced membrane currents by papaverine in rabbit vascular smooth muscle cells. Journal of Physiology 439, 501-512.
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