Pflfigers Arch (1992) 421:125-130

Joumal of Physiology 9 Springer-Verlag1992

Calcium-dependence of the calcium-activated chloride current in smooth muscle cells of rat portal vein P. Pacaud, G. Loirand, G. Gr6goire, C. Mironneau, and J. Mironneau Laboratoire de PhysiologicCellulaire et PharmacologicMol6culaire, C.N.R.S. URA 1489, 3 place de la Victoire, F-33076 Bordeaux, France Received December 20, 1991/Received after revision February 27, 1992/AcceptedMarch 6, 1992

Abstract. Ca2+-activated C1- current in freshly isolated smooth muscle cells from rat portal vein was studied using the whole-cell patch-clamp technique. Simultaneously, the free-cytosolic Ca 2§ concentration (Cai) was estimated using emission from the dye Indo-1. Pretreatment of the cells with amytal and carbonyl-cyanide-m-chlorophenylhydrazone, which reduced the intracellular adenosine triphosphate concentration, was used to weaken the cellular Ca 2+ homeostatic system. Ca i of treated cells slowly increased during perfusion with an external Ca 2§ solution. This rise in Ca~ gradually activated a Ca2+-dependent C1- current which allowed the study of the relationship between activation of this current and Ca i levels. The threshold Ca~ for activation of C1- channels was around 180 nM and full activation occurred at 600 nM. The Ca i dependence of the C1- channels was not changed during application of noradrenaline and did not depend on the membrane potential. The gating of Ca2+-dependent C1- channels of rat portal vein myocytes seems to be mainly controlled by intracellular Ca 2+ "

Key words: Free cytosalic calcium - Calcium-activated chloride current - Isolated smooth muscle cells - Noradrenaline

types of cation currents in jejunal and portal vein cells required the presence of an agonist of muscarinic or adreno-receptors and are modulated by Ca i [10, 17, 24]. During agonist stimulation, the physiological role of each of these current types should depend on the driving force (for the ion carried), on their voltage dependence and on their sensitivity to internal Ca 2+ ions. Little is known about the range of Ca~ at which these currents are activated. The cationic current induced by muscarinic receptor activation in jejunal cells seems to be very sensitive to Ca~, as the form of the current follows exactly the fluctuations of Cai [17]. Half-maximal and submaximal activation of the acetylcholine-induced non-selective cation current in ileum occurred at a Cai of about 200 nM and 1 gM respectively [10]. In portal vein myocytes, the large conductance non-selective cation channels seemed to be less sensitive to changes in Ca i than the C1- Current [15]. The Ca i required to open Ca2§ C1channels was not known but it has been suggested that concentrations higher than 0 . 1 - 0 . 5 gM could activate C I - current [18]. In the present study, the combination of patch-clamp technique and microspectrofluorimetry was used to examine the sensitivity of C1- channels to intracellular Ca 2+ ions.

Materials and methods Introduction Several classes of Ca 2+-activated membrane ion currents have been described in smooth muscle cells: those through K § channels [2, 22], C1- currents [3, 4, 12, 18] and those through nan-specific cation channels [15]. Ca2+-dependent K + channels and the Ca2+-dependent C1- currents were activated by a rise in intracellular Ca 2+ concentration (Cai), whatever the cause. Other

Offprint requests to: P. Pacaud

Cell preparation. Wistar rats (150g) were stunned and then killed by cervical dislocation. The portal vein was cut into several pieces, incubated for 10 min in low-Ca2+ (40 ~tM) physiologicalsaline solution (PSS, composition given below), and then incubated in low-Ca2+ PSS containing 1.4 mg/ml collagenase, 0.5 mg/ml pronase and 1 mg/ml bovine serum albumin at 37 ~ for 20 min. After this time, the solution was removed and the pieces of vein were incubated again in a fresh enzyme solution at 37 ~ for 20 min. Tissues were then placed in enzyme-free solution and triturated using a fire polished Pasteur pipette to release cells. Cells were stored on glass cover-slips at 4~ in PSS containing 0.8 mM Ca2+ and used on the same day. The cells were incubated during 30 min in PSS containing 3 mM amytal and 5 ~tM carbonyl-cyanide-m-chlorophenylhydrazone(CCCP) then, washed in normal PSS before starting the experiments.

126 Membrane current and fluorescence measurements. Voltage-clamp and membrane current recordings were made with standard patch-clamp techniques using a List EPC-7 patch-clamp amplifier (DarmstadtEberstadt, FRG). Whole-cell membrane currents were recorded with borosilicate patch pipettes of 1 - 4 Mf~ resistance. Membrane potential and current records were stored and analysed using an IBM-PC computer (P-clamp system, Axon, Foster City, CA, USA). Measurement of intracellular Ca 2+ concentration was carried out as described previously [17, 19]. Briefly, 50 ~tM Indo-1 was added to the pipette solution, and so entered cells following establishment of the whole-cell recording mode. The cell studied was illuminated at 360 nm. Emitted light from a window slightly larger than the cell was counted simultaneously at 405 nm and 480 nm by two photomultipliers (P 1, Nikon, Tokyo, Japan). Voltage signals at each wavelength were stored on an IBM-PC computer for subsequent analysis. Noradrenaline (NA) was applied to the recorded cell by pressure ejection from a glass pipette for the period indicated on the records. All experiments were done at room temperature. Estimation o f Cai. Background fluorescence was measured in cell-attached mode just prior to rupturing the membrane patch and cancelled by offsetting the output level of each photomultiplier. Cell fluorescence was then monitored in order to follow dye loading, which usually reached a steady state after 2 - 3 rain and recording was started as this point. Ca i was estimated from the 405/480 ratio [8] using a calibration for Indo-I determined within cells [19]. Results are expressed as the mean + SEM with n the sample size. Significance was tested by means of Student's t-test. Solutions. The normal PSS contained (in mM): 130NaC1, 5.6KC1, 1 MgClz, 2 CaC12, 11 glucose, 10 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid (HEPES), pH 7.4 with NaOH. The CaZ+-free solution was prepared by omitting CaC12 and by adding 1 mM ethylenebis(oxonitrilo)tetraacetate (EGTA); the MgC12 concentration was then increased to 3 mM. Low-C1- solutions were prepared by substituting sodium aspartate (100raM for experiments of Fig. 2 and 86 mM for experiments of Fig. 5) for NaC1. The basic pipette solution contained (in raM): 130 CsC1, 0.05 Indo-1, 10 HEPES and was buffered with the addition of approximately 5 mM NaOH. All experiments were done in the presence of 10 gM D-600 in the external solution. Chemicals and drugs. Collagenase (type XI), pronase (type E), bovine serum albumin and NA were purchased from Sigma (St. Louis, MO, USA). Indo-I (pentasodium salt) was from Calbiochem (San Diego, CA, USA) and D-600 (gallopamil-hydrochloride) from Knoll (Ludwigshafen, FRG).

Results

Assessment of the method used to activate C d +-dependent CI- currents Under control conditions, in normal PSS (2 mM Ca2+), isolated portal vein smooth muscle cells held at - 50 mV showed a stable resting Ca i of 60+_7 nM (n = 9) [19]. This value is the result of an equilibrium between Ca 2+ entry and Ca 2+ extrusion, which could be shifted by altering one of these two phenomena. It is well known that in smooth muscle cells CaZ+-transport adenosine triphosphatases (ATPase) play a predominant role in the extrusion of cytosolic Ca 2+ [6, 23, 25] and inhibition of the Ca2+-transport ATPases leads to an increased level of basal Ca i [9]. In portal vein smooth muscle cells pretreated for 30 rain, just before the experiments, with 3 mM amytal and 5 ~tM CCCP to deplete the intracellular ATP pool [13], Cai decreased when the external 2 mM Ca 2+ PSS was replaced by Ca 2+-free PSS, because of the reversed Ca 2+ gradient. When 2 mM Ca 2+ was readmit-

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Fig. 1A, B. Effects of change in external Ca 2+ concentration on free cytosolic Ca 2+ concentration (Cai) and on membrane current of a cell pretreated with amytal and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP). A Traces show membrane current (top) and Cai (middle). The lower trace represents the changes in extracellular Ca 2+ concentration. Depolarizing pulses to -20, 0 and + 20 mV were applied to the cell from a holding potential of -50mV. The removal of external Ca 2+ produced a decrease of Ca i. When 2 mM Ca 2+ was readmitted in the external solution, Ca i gradually increased and an inward current was activated at - 5 0 mV. Broken lines represent the current level and the Ca i obtained at a holding potential of - 5 0 mV in a Ca 2+ -free physiological saline solution (PSS). B Current/voltage relationship of the current activated by the rise in Cai showed in A. Current amplitude was determined by subtraction of current levels reached at each potential (-50, -20, 0 and +20 mV) in 2 mM Ca2+-PSS and in Ca2+-free PSS. The reversal potential was +2 mV

ted to the PSS, the Ca i gradually increased (Fig. 1A) and, sometimes, a stable value was reached. This might be due to a part of the Ca 2+ extruding mechanisms which remained functional and which could also account for the slower time course of Ca i changes associated with re-application of Ca 2§ than that observed upon Ca 2§ removal. The simultaneous recording of the membrane current showed that when the Ca i increased, an inward current was activated at a holding potential of - 5 0 m V (Fig. 1 A). To estimate the reversal potential of this current, three voltage jumps from - 5 0 mV to - 2 0 , 0, and +20 mV were applied to the cell. The amplitude of the current activated by the rise in Ca i was measured by subtraction of the current level reached at these potentials in Ca2+-free PSS. The reversal potential, determined from the current/voltage relationship (Fig. 1 B) was + 2 mV, a value close to the C1- equilibrium potential, which was - 1.8 mV under these experimental conditions. This suggests that the leak current component was weak, so that the current activated in the external Ca2+-containing solution was mainly a C1- current. When the same experiment was carried out in a low-C1- (31.6mM) PSS (Fig. 2A), the current/voltage relationship of the current activated by the rise in Ca~ was shifted toward positive potentials and the extrapolated reversal potential corresponded to the calculated C1- equilibrium potential

127

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the accumulation of Ca 2+ in the cytosol when the cells were perfused with a 2 mM CaZ+-containing solution. This rise in Cai gradually activates a membrane conductance mostly selective for C1- ions corresponding to the Ca 2+-activated C1- current previously described in these cells [18].

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The experimental protocol allowed the study of the Ca z+ dependence of the C1- current as it was possible to correlate directly the amplitude of the C1-current with the Ca i level. In the experiments shown Fig. 3, NA (10 ~tM) applied to the cell at a holding potential of - 5 0 mV in CaZ+-free PSS induced a transient increase in Ca i by releasing Ca z+ from intracellular stores [19]. After this rise, Cai returned to its initial value, probably because of the reversed Ca 2+ gradient. However, the duration of the transient increase in Ca~ was longer in the cells pretreated (27.8+1.7s, n = 7 ) than in control cells (12.5+0.6s, n = 9) suggesting that re-pumping of the released Ca 2+ into intracellular stores could account for a part of the decay of the transient Cai rise. The addition of 2 mM Ca 2+ to the PSS immediately produced a slow rise in Cai but there was no change in the current level until Ca i reached a value high enough to activate C1- channels (Fig. 3). Then, the amplitude of the CI- current and Ca i increased simultaneously, but the current amplitude reached a stable maximal value whereas the Cai went on increasing. The curves of the C1- current amplitude as a function of Cai corresponding to these two experiment~ (Fig. 3 A b, B b) showed that the current was nearly maximum between 420 and 500 nM. The threshold Ca i for the activation of CI- current was 177+3 nM (n = 11) and half-activation occurred for a Ca~ of 365+_13 nM

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p~O0 Fig. 2A, B. Effects of 4 h a n g e in the external C I - concentration on the current induced by the rise in Cap The external and internal C1- concentrations were 3 1 . 6 m M and 1 3 0 m M respectively. A Addition of 2 m M Ca 2+ in the external solution (lower trace) induced a rise in Ca i (middle trace) and the activation of an inward current at a holding potential o f - 5 0 m V (top trace). Depolarizing pulses to - 2 0 , 0 and + 2 0 mV from - 5 0 mV were applied to the cell in the absence and in the presence of extracellular Ca 2+ . Broken lines represent the current level a n d the Ca i obtained at a holding potential of - 5 0 m V in Ca2+-free PSS. B Current/voltage relationship corresponding to the current activated by the rise in Ca i showed in A. Current amplitude was determined by subtraction between current levels reached at each potential ( - 5 0 , - 2 0 , 0 and + 20 mV) in 2 m M Ca 2+ PSS and in Ca 2+-free PSS. The extrapolated reversal potential was + 37 mV

(+37 mV, Fig. 2B). In the experiment of Fig. 4 A where the external C1- concentration was 55.6mM, the C1equilibrium potential was then + 22 mV and the reversal potential of the Ca2+-activated current determined from the current/voltage relationship (not shown) was +24 mV. These results indicate that the pretreatment of the cells with the combination of amytal and CCCP leads to an inhibition of the Ca2+-transport ATPases allowing

A

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Effect of NA As described above, application of NA in Ca 2+-free PSS induced a transient rise in Ca i. Nevertheless, the mean

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Fig. 3 A , Bo Amplitude of the Ca 2+-activated C1- current as a function of Ca i in two different cells. Addition of external Ca 2+ (lower trace) gradually increased Ca i (middle trace) and activated an inward current at a holding potential of - 5 0 mV (top trace) A a , B a Application of noradrenaline (NA, 10 gM) in the absence of external Ca 2+ induced a transient rise in Ca i by releasing intracellularly stored Ca 2+ . This rise in Ca i activated a transient Ca2+-dependent C1- current in the cell B but not in the cell A. Broken lines represent the current level and the Ca i obtained at a holding potential of - 5 0 mV in Ca2+-free PSS. A b , B b For each cell, the amplitude of the C1- current was plotted as a function of the Ca i measured at the same time

128 maximal Ca i reached during the transient rise induced by N A was smaller ( 2 5 0 + 2 3 n M , n = 11) than in control cells (480_+43 nM, n = 9) [20]. In the example shown in Fig. 3 A a , the maximal value of Ca~ reached during the N A application was 170 nM. This value, reported on the curve representing the C1- current amplitude as a function of Ca~ (Fig. 3Ab), was just below the threshold of C1- activation, and in agreement with this, no or very small change in membrane current was observed during the NA-induced rise in Cai. In the experiment of Fig. 3 B a, the maximal Cai obtained during the transient increase induced by N A was around 300 nM. According to the curve (Fig. 3 B b), this Cai level would be able to activate a C1- current of about 450 pA. This amplitude was very close to that experimental obtained in this cell as the transient rise in Cai induced by N A activated a C1- current of 460 pA. A similar experiment was made in nine other ceils that gave maximal Cai during the transient rise induced by N A ranging between 142 and 387 nM. For all these cells, the amplitude of the NA-induced Ca2§ C1- current corresponded to that obtained for the same Cai level in the absence of NA. These results suggest that N A did not change the Ca 2+ dependence of the C1- current and it seems that the amplitude of the C1- current was only determined by Ca~.

Effect o f membrane potential on the Ca?+ dependence o f the Cl- current To study the potential dependence of the Ca 2§ dependence of the C1- current, the membrane potential was repetitively stepped for period of 2 s to - 3 0 and 0 mV from a holding potential of - 50 mV before and after the addition of 2 mM Ca 2+ in the PSS (Fig. 4A). The amplitude of the C1- current activated by the rise in Ca~ was determined as the difference between the current level reached, at each potential, in Ca2§ PSS and in 2 mM Ca 2+ PSS. In addition, a voltage jump to + 50 mV was applied to the cell before and after the addition of Ca 2+ to determine the reversal potential of the Ca2+-ac tivated current, as under these conditions (55.6 mM external C1-) the C1- equilibrium potential was + 22 mY. The current trace clearly showed that Ca2+-activated C1current was inward at - 50, - 30 and 0 mV, and outward at + 50 mV. To compare the Ca 2§ dependence of the C1current at these three membrane potentials, the amplitude of the C1- current was expressed as a fraction of its maximal value at each potential and was plotted against the Cai (Fig. 4B). The three curves obtained at membrane potentials of - 5 0 ( o ) , - 3 0 ( B ) and 0 (A) mV were superimposed, indicating that the Ca 2§ dependence of the C1- current was not dependent on the membrane potential.

Discussion In the present work, the Ca2+-transport ATPase by plete the cellular ATP pool mulation of Ca 2§ in the pretreatment of the cells

reduction of the activity of amytal and CCCP which dewas used to produce an accucytosol. In addition to the with these two compounds,

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Fig. 4A, B. Effect of membrane potential on the Ca2§ dependence of the CaE+-activated C1- current. A Addition of external Ca2+ (lower trace) gradually increased Cai (middle trace) and activated an inward current at a holding potential of -50 mV (top trace). Repetitive depolarizing pulses to -30 and 0 mV from -50 mV were applied to the cell in the absence and in the presence of extracellular Ca2+ . In addition, two pulses to + 50 mV ($) were applied just before and during the application of Ca2+ . Broken lines represent the current level and the Cai obtained at a holding potential of -50 mV in Ca2+-free PSS. B Amplitude of the C1- current plotted as a function of Cai measured at the same time. The C1- current was expressed as a fraction of its maximal value at each potential: -50 mV ( 9 -30 mV (I), 0 mV (A). The external and internal C1- concentrations were 55.6mM and 130 mM respectively

patch pipettes were filled with a 130 mM CsCl-containing solution since N a + - C a 2+ exchange was not functional under these conditions [20]. The Ca i was stable when the cells were perfused with a Ca2+-free PSS but increased when Ca 2+ was added to the bath solution. The rate of increase in Ca i varied from cell to cell, presumably because of a various degree of inhibition of the CaE+-transport ATPases. However, the increase of Ca i was always slower at a holding potential of + 50 mV (not shown) than at - 5 0 mV, suggesting that the rise in Ca i was produced by Ca 2+ entry through non-specific leak channels, down the Ca 2+ gradient. This experimental approach leads to a progressive increase of Ca i which produces gradual activation of Ca2+-dependent C l channels. The Ca i range for C I - current activation is reproducible from cell to cell and is between 177 and 600 nM, spanning a 3.3-fold factor. Such a variation is steeper than that predicted by a simple binding isotherm, and indicates that the Ca 2+ regulation mechanism most probably involves simultaneous binding of several Ca 2+ ions. A similar result was obtained for the Ca2+-dependent C1- current of lacrimal gland cells dialysed with CaE+-HEDTA buffers to maintain Cai at an abnormally high level [5]. Assuming that the Cai was the same as that calculated for the pipette solution, the threshold concentration for the activation of C1- channels was around 100 nM and full activation occurred only at 2 ~M

129 Ca 2+ . However, these authors t h o u g h t that the steepness o f the dose/response curve was p r o b a b l y larger than they found. It has been observed that Ca 2+-activated C I - current was enhanced by the non-hydrolysable analogue o f guanosine triphosphate, GTP-y-S [21]. One hypothesis m a d e to explain this action was a direct effect via an activated G-protein or an indirect effect via second messengers such as cyclic adenosine m o n o p h o s p h a t e (cAMP) or diacylglycerol. It seems that this was not true at least in portal vein myocytes. In these cells, it has been shown that N A activates a pertussis toxin insensitive G-protein which m a y stimulate the p r o d u c t i o n o f inositol, 1,4,5 trisphosphate (IP3) and diacylglycerol [14]. The amplitude o f the C1- current activated by the maximal Cai reached during the transient rise induced by N A t h r o u g h the action o f I P 3 on Ca 2+ stores was similar to that produced by a similar Ca~ in the absence o f NA. This observation was true for all Cai values, including the threshold concentration so that the dose/response curve was n o t shifted by N A . This suggests that the gating o f Ca 2+-dependent C I - channels was mainly controlled by Ca i. Thus, the regulation o f the NA-induced C1- current was different f r o m that o f the acetylcholine-induced cation current described in ileum which is under the control o f a pertussis toxin sensitive G-protein; Ca i has only a facilitating effect on the cation current [111. In addition, it seems that the amplitude o f the C I - current was determined by the Ca i level reached independently o f the rate o f Cai increase. The C1- current remained activated as long as the Cai was above the threshold concentration suggesting that the C I - channels had no intrinsic inactivation. Thus, the NA-induced C I - current was transient only because the NA-induced rise in Ca i was transient. This result agrees with the non-inactivating C I - current recorded in the presence o f high intracellular Ca 2+ concentration [5]. The decline o f the Ca2+-dependent C I current observed in the continuous presence o f ionomycin [24] probably occurred because the increase in Ca i was not sustained. However, the maximal Ca i obtained under our experimental conditions was a r o u n d 600 n M but this did not exclude the possibility that the C I - current inactivated at higher Cai values. The Ca 2+ dependence o f the C I - channels was not affected by the m e m b r a n e potential so that, at every potential value, the fraction o f C I - channels activated by a given Cai was similar. We have previously described that the rate o f deactivation o f the C I - current activated by Ca 2+ entry t h r o u g h voltage-dependent Ca 2§ channels is accelerated at hyperpolarized m e m b r a n e potential ( - 7 0 mV) c o m p a r e d to that obtained at - 3 0 mV [18]. It is possible that this difference m a y be due to a variation o f the rate o f decrease in Ca i which would be slower at - 3 0 m V . This could be explained by a Ca 2+ entry t h r o u g h voltage-dependent Ca 2+ channels that remained o p e n at - 30 mV [7]. In summary, the Ca2+-dependent C I - current o f portal vein myocytes has the following properties. First, it is activated in a Ca~ range between 170 and 600 nM. These concentrations are compatible with a physiological

role o f C1- channels as this spectrum o f Ca 2+ concentrations is generally obtained during agonist stimulation. Second, the CaZ+-dependent C1- channels remain open as long as the Cai remains higher than the threshold concentration. Thus, C1- current might be responsible for the maintained depolarization recorded during the effects o f agonists [16]. In s m o o t h muscle cells, at the resting m e m b r a n e potential the CaZ+-activated C1- channels can still generate an inward depolarizing current since the C1- equilibrium potential is less negative t h a n the resting potential [1]. Third, the C a 2+ dependence o f the C1channel is n o t affected by the m e m b r a n e potential value.

Acknowledgements. This work was supported by grants from Institut National de la Sant6 et de la Recherche M6dicale, R6gion Aquitaine, and Fondation pour la Recherche M6dicale, France.

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