517

Journal of Physiology (1990), 428, pp. 517-529 With 5 figures Printed in Great Britain

GTP-BINDING PROTEINS MEDIATE NORADRENALINE EFFECTS ON CALCIUM AND CHLORIDE CURRENTS IN RAT PORTAL VEIN MYOCYTES

BY G. LOIRAND, P. PACAUD, C. MIRONNEAU AND J. MIRONNEAU* From the Laboratoire de Physiologie Cellulaire et Pharmacologie Moleculaire, INSERM JF 88-13, 3 place de la Victoire, 33076 Bordeaux, France

(Received 27 October 1989) SUMMARY

1. Membrane currents were recorded by a patch-clamp pipette technique in cultured cells from rat portal vein using the whole-cell mode. 2. Noradrenaline (NA, 10-5 M) and phorbol-12,13-dibutyrate (PDBu, 10-7 M) produced an increase in voltage-dependent inward current carried by barium (5 mM), but their effects were not additive. Calcium-activated chloride current was evoked by NA but not by PDBu. 3. The NA-induced increase in peak voltage-dependent inward current was inhibited by intracellular application of GDP-,f-S (10-3 M) while the effect of PDBu was unchanged. GDP-,f-S blocked the NA-induced chloride current but had no effect on the caffeine-induced chloride current. 4. Inclusion of GTP-y-S (10-5-10-4 M) in the pipette solution increased the voltage-dependent inward current and inhibited the NA- or PDBu-induced increase in peak current. GTP-y-S potentiated the effect of NA on calcium-activated chloride current. At higher concentrations (10-3 M), GTP-y-S activated the chloride current and prevented the effects of NA or caffeine on this current. 5. The combination of 10-5 M-aluminium chloride and 10-2 M-sodium fluoride had an effect similar to that of high concentrations of GTP-y-S on both inward current and calcium-activated chloride current. In contrast, arachidonic acid (10-3 M) had no effect on calcium and chloride conductances activated by NA. 6. Cells responded normally to NA after pre-treatment for 4-30 h with 10 ,g ml-' pertussis toxin (PTx). 7. It is concluded that the stimulation of calcium and chloride conductances by NA is mediated through activation of a PTx-insensitive GTP-binding protein. This effect may involve activation of phospholipase C enzyme and production of both D-myo-inositol 1,4,5-trisphosphate which depletes calcium stores and diacylglycerol which activates protein kinase C.

* To whom correspondence should be sent. .MS 6

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INTRODUCTION

Stimulation of a1-adrenoceptors in vascular smooth muscle produces contractions that can be dependent on an increase in both calcium influx through voltage-

dependent calcium channels and release of calcium from intracellular stores (Mironneau & Gargouil, 1979; Dacquet, Mironneau & Mironneau, 1987; Pacaud, Loirand, Mironneau & Mironneau, 1987; Nelson, Standen, Brayden & Worley; 1988). Although little is known about the transduction mechanism for agonist-induced increase in calcium current, agonist-stimulated release of calcium from intracellular stores is attributed to increased production of D-myo-inositol 1,4,5-trisphosphate (IP3) due to the accelerated activity of a triphosphoinositide phosphodiesterase (phospholipase C; Berridge, 1984). Activation of this enzyme also leads to production of diacylglycerol (DAG) which increases the activity of the calcium-dependent protein kinase C (Nishizuka, 1984; Exton, 1985) and may be responsible for the sustained phase of the cellular response in vascular smooth muscle (Danthuluri & Deth, 1984; Forder, Scriabine & Rasmussen, 1985; Griendling, Rittenhouse, Brock, Powers, Gimbrone & Alexander, 1986). Control of the phospholipase C activity may involve a guanine nucleotide-binding protein (G protein; Sasaguri, Hirata & Kuriyama, 1985; Cockcroft, 1987). G proteins have been implicated in the transduction of signals from several types of receptor (Dolphin, 1987), includinga1-adrenoceptors. Activation of x1-adrenoceptors causes opening of several types of calcium-dependent channels: chloride channels in mesenteric and portal vein cells (Byrne & Large, 1988; Van Helden, 1988; Pacaud, Loirand, Mironneau & Mironneau, 1989a), potassium channels in arterial (Benham & Bolton, 1986) and venous smooth muscle cells (Komori & Bolton, 1989). In the latter example, a G protein step seems to be involved between adrenoceptor activation and calcium store release. The present study investigates the role of G proteins in intracellular mechanisms involving activation of calcium-dependent chloride current and stimulation of voltage-dependent inward current in response to noradrenaline. Accordingly, we have examined whether intracellular applications of substances stimulating or inhibiting G protein activity or extracellular applications of phorbol dibutyrate (an activator of protein kinase C) modulated the action of

noradrenaline. METHODS Wistar rats (150 g) were stunned and then killed by cervical dislocation. Portal veins were dissected free of connective tissue and single cells were obtained by a dispersal procedure similar to that described previously (Loirand, Pacaud, Mironneau & Mironneau, 1986). Whole-cell membrane currents were measured in single cells in short-term primary culture for 4-36 h using the standard patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Patch pipettes had resistances of 2-5 Data were analysed with a Plessey 6220 microcomputer. Calcium channel currents were digitally corrected for leakage currents by subtraction of scaled current traces obtained when hyperpolarizing or small depolarizing pulses from the holding potential were applied to the cells. The extracellular medium (reference solution) contained (mm): NaCl 130, 56, CaC12 5, MgC22024, glucose I1, HEPES 8, brought to pH with NaOH. The patch pipette solution had the following composition (mm): CsCl 130, Na2ATP 5, MgCl2 5. HEPES 10, brought 7-3 to pH with CsCI

MQ.

KCl

7-4

CsOH.

currents.

was

used

of

KCl

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order to

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outward

potassium

G PROTEINS AND IONVIC CHANNELS

519

Chemicals used were: noradrenaline (NA); prazosin; phenylephrine; propranolol; phorbol-12,13dibutyrate (PDBu); phorbol- 13,20-diacetate (PDA); arachidonic acid; dithiothreitol; ethylene glycol bis-(,/-aminoethylether) N.N.N.'KV tetraacetic acid (EGTA) (all from Sigma); caffeine (Merck); guanosine 5'-O-(y-thio)triphosphate (GTP-y-S); guanosine 5'-O-(,-thio)diphosphate (GDP-,-S) (Boehringer); pertussis toxin (PTx) (List Biological Laboratories). Phorbol esters were diluted from a stock solution (10-2 M) in dimethylsulphoxide (DMS0). Thus, at the maximal phorbol ester concentration used (10-v M), the DMSO concentration was 10' M and had no effect on the membrane currents. For the studies of calcium-activated chloride current, noradrenaline and caffeine were applied with a pressure ejector from a glass pipette for 200 ms and 1 s, respectively. Effects on calcium channel currents were recorded during bath perfusion of noradrenaline. The values given in the text are the means+ S.E. of the mean with n the sample size. Significance was tested by means of Student's t test. RESULTS

Voltage-dependent inward current Effects of noradrenaline (NA) and phorbol-12, 13-dibutyrate (PDBu) were studied on calcium channels by using 5 mM-barium as the charge carrier. Experiments were started after perfusion of the cells with a barium-containing solution for more than 30 min. Under these conditions, NA or PDBu stimulated both peak and steady inward currents (Fig. 1). The current-voltage relationships reveal that the peak inward current was increased at any given voltage pulse without any change in the voltage threshold, the potential for the maximal current and the apparent reversal potential. In all experiments, the measurements were made only when cells reached steady state, i.e. within 4-5 min at a stimulation frequency of 0.05 Hz. The maximal peak inward current was increased by 68-3 ±6 % (n = 12, P < 00001) in 10- M-NA and by 65-9 ±3A4 % (n = 11, P < 0-001) in 10-7 M-PDBu. The effects of NA were completely reversed within 5-7 min in reference solution while those of PDBu were less readily reversible on washing within 10-15 min. The action of NA was mimicked by 10-5 M-phenylephrine while 10-5 M-isoprenaline was ineffective. Furthermore, the stimulatory effect of NA was blocked in 10-7 M-prazosin while it remained unchanged in 10- M-propranolol. These results suggest that the responses to NA are mediated by az-adrenoceptors. In order to determine whether NA and PDBu acted via the same membrane transduction process, NA was applied in the presence of PDBu and vice versa. As illustrated in Fig. 2A, when the peak inward current elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV was maximally stimulated by 10 MPDBu, a further application of 10-5 M-NA was ineffective (-1P3 11 %; P > 0-05, n = 4). In the presence of 10-5 M-NA (Fig. 2B), application of 10-7 M-PDBu produced a decrease of the inward current by 54 ± 1-5 % (P < 0-05, n = 6). Furthermore, when the inactive form of the phorbol ester PDA was included in the external solution the inward current was not noticeably changed and the NA- or PDBu-induced increase in inward current was normally observed (Fig. 2A, n = 4). These observations favour the idea that calcium channels are stimulated through the same membrane transduction process by NA and PDBu and that effects of PDBu may involve the activation of protein kinase C. With 10-3 M-GDP-,/-S in the pipette solution, applications of 10-5 M-NA failed to produce a significant change in peak inward current (+ 2-0 + 1 3 %; P > 0 05, n = 11) elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV (Fig. 3A).

G. LOIRAND AND OTHERS

520

In contrast, 1O' M-PDBu still induced an increase in inward current reaching 59-8+7-6% (P < 001, n = 5) suggesting that GDP-fl-S did not act directly on calcium channels. When GTP-y-S (10-5-10-3 M) was added to the pipette solution, the NA-induced increase in peak inward current was reduced in a concentrationA

B mV

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Fig. 1. Effects of noradrenaline (NA) and phorbol 12,13-dibutyrate (PDBu) on inward currents recorded in a single vascular smooth muscle cell, isolated from rat portal vein, with the whole-cell patch-clamp technique. A, currents evoked by successive depolarizations from a holding potential of -60 mV in the absence (0) and presence (@) of 10`5 M-NA, and the corresponding plots of peak current against voltage. B, currents evoked by successive depolarizations from a holding potential of -60 mV in the absence (0) and presence (0) of 10- M-PDBu, and the corresponding plots of peak current against voltage. External solution contained 5 mM-barium.

dependent manner (Fig. 3C). However, it should be noted that greater inward currents were recorded in the presence of GTP-y-S than in control conditions (Fig. 3B). For example, the maximal peak inward current was increased from 152+29 pA (n = 12, control) to 380+41 and 460+45 pA (n = 6) in 10-4 M- and 10-3 M-GTP-y-S, respectively. With 10-3 M-GTP-y-S, 10-5 M-NA failed to significantly enhance the inward current (+ 2X5 + 2 %; P > 0-05, n = 5) while the increase in current induced by 10' M-PDBu was only 10-7 + 1 9 % (P < 0.01, n = 7, not shown). These results suggest that with high concentrations of GTP-y-S in the pipette solution a maximal activation of phospholipase C can occur in response to binding of GTP-y-S to G proteins.

G PROTEINS AND IONIC CHANNELS

521

B A 1

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Fig. 2. Effect of externally applied phorbol esters and noradrenaline on inward currents evoked by depolarizations to 0 mV from a holding potential of -60 mV. A, time course of the peak inward current during cumulative applications of phorbol 13, 20-diacetate (PDA, 10-6 M), phorbol 12,13-dibutyrate (PDBu, 10-7 M) and noradrenaline (NA, 10-5 M). PDBu induced a sustained increase in inward current while PDA and NA were ineffective. B, time course of the peak inward current during cumulative applications of NA and PDBu. NA induced an increase in inward current while PDBu reduced the current by about 10 %. Currents are expressed as a fraction of their maximal values. Inset, current traces corresponding to numbers on the curves. External solution contained 5 mmbarium. C

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Fig. 3. Effects of guanine nucleotides included in the pipette on inward currents evoked by depolarizations to 0 mV from a holding potential of -60 mV. A, use of a pipette filled with guanosine 5-0-(,-thio)diphosphate (GDP-,f-S, 10-3 M) abolished the stimulatory effect of NA while PDBu induced a large increase in peak current amplitude. Currents are expressed as a fraction of their maximal values. Inset, current traces corresponding to numbers on the curve. B, current traces obtained with different concentrations (10-5-10-3 M) of guanosine 5-O-(y-thio)triphosphate (GTP-y-S), in the pipette solution in the absence (a) and presence (b) of 10-5 M-NA. The NA-evoked increase in inward current was progressively abolished as the internal GTP-y-S concentration was augmented. C, concentration-response curve obtained by plotting fractional increases in peak current evoked by 10-5 M-NA against GTP-y-S concentration in the pipette. At A, control experiments with no GTP-y-S in the pipette solution. Each point represents the mean of three to twelve cells (numbers beside points); vertical lines show S.E. of the mean. External solution contained 5 mM-barium.

G. LOIRAND AN2D OTHERS

522

Combination of aluminium and fluoride is believed to stimulate G proteins by mimicking the effect of GTP-y-S on the a-subunit (Cockeroft, 1987). Inclusion of 10-5 M-aluminium chloride and 10-2 M-sodium fluoride in the pipette solution (Fig. 4A) had an effect similar to that of GTP-y-S in that the stimulatory effect of NA B

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0

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15

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(10-5 M)

10 Time (min)

15

Fig. 4. Effects of intracellular applications of the combination of aluminium and fluoride and of arachidonic acid. A, in the presence of AlCl3 (10- M)+ NaF (10-2 M) in the pipette solution, the stimulatory effect of 10-5 M-NA on peak inward currents evoked by depolarizations to 0 mV from a holding potential of -60 mV was suppressed. The 10-7 MPDBu-induced increase in current was reduced to about 10%. B, with 10-3 M-arachidOnic acid in the pipette solution the NA-evoked increase in peak inward current was normally observed. Currents are expressed as a fraction of their maximal values. Inset, current traces corresponding to numbers on the curves. External solution contained 5 mM-barium.

on the maximal inward current was suppressed (- 1- + 0-8 %; P > 0.05, n = 5). PDBu (0M- M) remained effective but the increase in current was 10-5 + 2-5 % (P < 0.01, n = 8). Pertussis toxin (PTx) ADP ribosylates the G protein inhibiting adenylate cyclase, thus preventing its activity (Gilman, 1987). Cells were incubated in culture in the presence of 10 ,ug ml-' PTx with and without 10 mM-dithiothreitol for 4-30 h. These cells exhibited normal inward currents, which were increased by 65 + 7 % (P < 0-001, n = 12) in 10-5 M-NA, i.e. indistinguishable from controls. Arachidonic acid can be considered as a possible intracellular second messenger since it is released in the cell in response to various chemical stimulations including adrenergic activation (Axelrod, Burch & Jelsema, 1988). Inclusion of 10-3 Marachidonic acid in the pipette solution did not produce a significant effect on the maximal inward current (+ 2-0 + 1-7 %; P > 0-05, n = 6). This current could still be increased by 54-5+6-5% (P < 0 01, n = 4) in the presence of 10-5 M-NA (Fig. 4B).

Calcium-activated chloride current In 5 mM-calcium-containing solutions, microejections of NA (200 ms) or caffeine (1 s) induced calcium-activated chloride currents while bath application of PDBu (10-7 M) was ineffective. When the cell was held at a holding potential of -60 mV the

G PROTEINS ANVD IONIC CHANNELS

523

agonist-activated inward current occurs as a consequence of a rise in intracellular calcium concentration depending on release of calcium from intracellular stores (Byrne & Large, 1988; Pacaud et al. 1989 a). Figure 5A shows an experiment in which depolarizations to -10 mV from a holding potential of -60 mV evoked the B

A

C

-10 mV -60 n

Caffeine(102M) NA(105M) V

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9-

4 NA(3x10m) (10 jM)

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8o

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(0)

5 4

pA 2s

-log [NA] (m)

Fig. 5. Effects of guanine nucleotides included in the pipette on calcium-activated chloride current in cells held at a holding potential of -60 mV. A, with 10-3 M-GDP-/-S in the pipette solution both calcium current and calcium-activated chloride current were recorded in response to depolarizations to -10 mV from a holding potential of -60 mV. At this holding potential external applications of 10-5 M-NA had no effect, while 10-2 M-caffeine evoked a noticeable current. B, concentration-response curve (@) obtained by plotting the amplitude of NA-evoked chloride currents (as a percentage of the maximal current in response to 10' M-NA) against NA concentration. The slope factor of the curve is 1. With 10-4 M-GTP-y-S in the pipette solution, a subthreshold concentration of NA (10-8 M) produced a response reaching 92 + 4 % (n = 4) of the maximal current (U). Each point represents the mean of three to twenty-three cells (numbers beside points); vertical lines show S.E. of the mean. C, examples of NA-activated chloride currents elicited from a holding potential of -60 mV in the absence (top traces, control) and presence (bottom traces) of 10-4 M-GTP-y-S in the pipette solution. D, high concentrations of GTP-y-S (10-3 M) in the pipette solution elicited a large transient inward current followed by abolition of both NA- and caffeine-evoked responses. External solution contained 5 mM-calcium and there was no EGTA in the pipette solution.

peak and sustained components of inward current. The current is followed by a large inward tail current at -60 mV corresponding to the deactivation of the chloride current (Pacaud, Loirand, Lavie, Mironneau & Mironneau, 1989b). With 10-3MGDP-,3-S in the pipette solution, NA (10-5 M) was unable to induce a noticeable response when the cell was held at -60 mV (n = 18). In contrast, 10-2 M-caffeine induced an inward current whose amplitude (1076 + 67 pA) was similar to that of control experiments obtained in the absence of internal GDP-/3-S (1 12-9 + 83 pA; n = 18). As caffeine is still effective in the presence of intracellular GDP-,/-S these results suggest that the blockade of the NA-induced response is not due to depletion of the internal calcium store or direct inhibition of calcium-activated chloride

524

G. LOIRAND AND OTHERS

current. Figure 5B and C illustrates the effects of inclusion of GTP-y-S in the pipette solution on the NA-induced response at -60 mV. In control conditions, the NAevoked current increased in a concentration-dependent manner. The NA concentration required to produce 50 % of the maximal response was estimated to be 2 10-7 M. When 10-4 M-GTP-y-S was added to the pipette solution, a subthreshold concentration of NA (10-8 M) induced an inward current reaching 92 + 4 % (n = 4) of the maximal response. At higher concentrations (10-3 m), GTP-y-S induced both a transient and a sustained inward current when the cell was held at -60 mV (Fig. 5D). The amplitude of the peak inward current evoked by 10-3 M-GTP-y-S was 141+19 pA (n = 5). The GTP-y-S-induced current displayed a dependence on membrane potential (reversal potential near 0 mV) similar to that obtained with NA or caffeine and it was never observed in barium-containing solution, when 10-2 MEGTA was added to the pipette solution, or in the continuous presence of 10-2 Mexternal caffeine (Pacaud et al. 1989a). As shown in Fig. 5D, after 5 min of dialysis with 10-3 M-GTP-y-S, external applications of 10-5 M-NA or 10-2 M-caffeine failed to induce any transient responses (n = 5). A similar absence of effect was observed when the combination of aluminium chloride and sodium fluoride was added to the pipette solution (n = 4). In contrast, inclusion of 10-3 M-arachidonic acid in the pipette solution had no effect on the NA- or caffeine-evoked responses. Incubation of the cells in the presence of 10 ,tg ml-' PTx for 30 h did not affect the amplitude of the NAinduced chloride current at -60 mV (-1 6 + 0 9 %, P > 0-05, n = 10). In contrast, bath application of 10-7 M-PDBu for 10 min produced a small decrease of the NAinduced chloride current (- 13-1 + 1-8 %; P < 0-01, n = 7). Taken together, these observations suggest that activation of G proteins by GTP-y-S or the combination of aluminium and fluoride may produce an increase in intracellular calcium by release of calcium from intracellular stores and thus, in turn, activate the chloride current. DISCUSSION

The present experiments suggest that the stimulatory effects of noradrenaline (NA) on both inward current and calcium-activated chloride current in smooth muscle cells from rat portal vein are mediated by a GTP-binding protein. Three main pieces of evidence support this view: first, addition of GTP-y-S or the combination of aluminium and fluoride to the cell interior increased the voltage-dependent inward current and activated the calcium-dependent chloride current in the absence of added agonist; second, these substances suppressed the NA- and caffeine-induced responses and strongly reduced the PDBu-evoked response; third, the effects of NA were suppressed on adding GDP-,-S to the cell interior while those of PDBu and caffeine were not significantly modified.

Voltage-dependent inward current We report that in barium-containing solutions NA increased inward channel current. A similar effect of NA in arterial myocytes has been briefly reported (Aaronson, Benham, Bolton, Hess, Lang & Tsien, 1986), while Benham & Tsien (1988) have shown that NA stimulates only the L-type calcium channel through interaction with an adrenergic receptor different from a- and fl-receptors. On the other hand, Droogmans, Declerck & Casteels (1987) have reported that a-adreno-

G PROTEINVS ANVD IONIC CHANNELS

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ceptor activation induces a decrease in inward channel current. We observed the NA-induced inhibition of inward channel current during the first 30 min of perfusion of the cells in a barium-containing solution with 5 mM-intracellular EGTA. Increase of inward current was seen only after this perfusion period. This observation indicates that a residual release of calcium from the stores may occur during short perfusion periods in barium-containing solutions and block the calcium channels by a calcium-dependent inactivation process (Pacaud et al. 1987). To determine whether a G protein-mediated stimulation of the phospholipase C activity may underlie the stimulatory effects of NA on inward channel current, we have tested the effects of GTP and GDP analogues. As a stable analogue of GDP, GDP-,f-S acts as a competitive inhibitor of G protein activation by GTP, and thereby inhibits agonist-induced activation (Eckstein, Cassel, Levkovitz, Lowe & Selinger, 1979). In the present study, GDP-,-S inhibited the NA-induced increase in inward channel current while the effect of PDBu was unchanged. In contrast, both GTP-y-S and the combination of aluminium and fluoride trap the G protein in the activated state (Eckstein et al. 1979; Sternweis & Gilman, 1982). Internal dialysis of GTP-y-S (10-5-10' M) or a combination of aluminium and fluoride increased the inward channel current and decreased the NA- or PDBu-induced enhancement of this current. High concentrations of GTP-y-S increased the phospholipase C activity to a level which prevented further important effect of NA or PDBu. Thus, our results strongly suggest that G protein activation is involved in the NA activation of calcium channels. However, steps subsequent to G protein activation remain uncertain. In view of the PTx insensitivity of G proteins, indicating that the G protein involved in calcium channel stimulation is neither Go nor Gi (Gilman, 1987), it seems unlikely that G proteins control calcium channels through an interaction with adenylate cyclase. This proposal is supported by the absence of any clear effect of cyclic AMP derivatives on calcium current (Kl6ckner & Isenberg, 1985; Ohya, Kitamura & Kuriyama, 1987; G. Loirand, P. Pacaud, C. Mironneau & J. Mironneau, unpublished experiments). The most plausible intermediary step is the activation of phosphatidylinositide hydrolysis leading to generation of two substances which might act as second messengers; D-myo-inositol 1,4,5-trisphosphate (JP3) and diacylglycerol (Berridge, 1984). Although calcium channels may be regulated by 'P3, as recently reported in lymphocytes and mast cells (Kuno & Gardner, 1987; Penner, Matthews & Neher, 1989), there is no clear evidence to substantiate a similar role for IP3 in smooth muscle cells. Diacylglycerol (DAG) provides an alternative possibility as a prospective second messenger since inward channel current is increased by phorbol dibutyrate (PDBu), which replicates the effect of diacylglycerol in activating protein kinase C (Nishizuka, 1984). Our results are in good agreement with the data showing that, in vascular smooth muscle, phorbol esters cause a slowly developing contraction and associated transmembrane calcium influx sensitive to dihydropyridine derivatives (Danthuluri & Deth, 1984; Rasmussen, Forder, Kojima & Scriabine, 1984; Forder et al. 1985; Chiu, Tetzloff, Chatterjee & Sybertz, 1988; Fish, Sperti, Colucci & Clapham, 1988). The absence of an intracellular effect of arachidonic acid on inward channel current suggests that generation of biologically active metabolites (that is, products of cyclooxygenase, lipoxygenase and cytochrome P450 metabolic pathways) is not involved in calcium channel modulation. As the effects of NA and PDBu on inward channel current were not additive and the inactive

526

G. LOIRAND AND OTHERS

phorbol diacetate (PDA) had no effect, our results suggest that modulation of calcium channels by NA may possibly involve the generation of DAG and activation of protein kinase C. Calcium-activated chloride current It has been previously shown that at resting membrane potentials, activation of chloride current induced by noradrenaline or caffeine is mediated by an increase in intracellular calcium concentration depending on release of calcium from intracellular stores (Byrne & Large, 1988; Pacaud et al. 1989 a). The present study supports a role for G proteins in mobilizing internal calcium. Internal application of GDP-,6-S inhibited the noradrenaline-evoked chloride current while GTP-y-S potentiated the effects of a-adrenergic stimulation. At high concentrations (10' M), GTP-y-S induced opening of calcium-activated chloride channels, and completely inhibited noradrenaline or caffeine effects. Combination of aluminium and fluoride has the same effect. These results suggest that both GTP-y-S and the combination of aluminium plus fluoride, by activating the G protein, induce the release of calcium from internal stores, allowing an increase in cytoplasmic calcium concentration sufficient for the activation of calcium-dependent chloride current. Our data are in good agreement with contraction experiments in smooth muscle skinned fibres (Kobayashi, Somlyo & Somlyo, 1988) but differ from those obtained by Bolton & Lim (1989) who found that GTP-y-S, as ryanodine, slowly depleted calcium stores and did not increase the cytoplasmic calcium concentration sufficiently for calciumactivated potassium channels to be opened. This may be attributed to the temperature at which experiments were done, as they worked at room temperature while our experiments were done at 30 'C. Phorbol esters Since PDBu diminishes the responses to NA, i.e. increased inward channel current and NA-activated chloride current, the present results are compatible with regulatory sites which are presumably phosphorylated by protein kinase C. The protein kinase C has been shown to interfere with IP3 generation in different cellular agonist-stimulated systems (Caramelo, Tsai & Schrier, 1988; Itoh, Kubota & Kuriyama, 1988; Sugiya & Putney, 1988; Maruyama, 1989) suggesting that the sites that are phosphorylated by protein kinase C are probably located in the activation step prior to second messenger formation. In this regard phosphorylation of the a1adrenergic receptor by protein kinase C has been proposed to be involved in negative regulation of these receptors (Leeb-Lundberg, Cotecchia, Lomasney, DeBernardis, Lefkowitz & Caron, 1985). A second possible site for protein kinase C-mediated phosphorylation may involve regulatory G proteins. Protein kinase C phosphorylates G proteins of the adenylate cyclase mechanism and modifies their activity (Katada, Gilman, Watanabe, Baver & Jacobs, 1985). Finally, a third possible site for action of protein kinase C is a phosphorylation-mediated, direct decrease in phospholipase C activity (Caramelo et al. 1988). Our results suggest the existence of a blocking site of phosphorylation for protein kinase C located on the z1-adrenoceptor because PDBu application has no inhibitory effect on the response induced by internal application of the combination of aluminium plus fluoride (Fig. 4A). In contrast, PDBu stimulated the GTP-y-S-activated inward channel current and this effect may

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be dependent on either a phosphorylation of G proteins producing a stimulation of the phospholipase C activity or a direct stimulatory effect of PDBu on calcium channels, independent of its effect as activator of protein kinase C. This latter possibility seems unlikely as a direct effect of phorbol esters on calcium channels (Hockberger, Toselli, Swandulla & Lux, 1989) has been observed at concentrations higher (1-5 x 10' M) than those used in the present study (10' M). Further experiments are required to clarify the multiple sites of action of phorbol esters on receptor signalling pathways. In conclusion, this study suggests the involvement of a G protein in regulating the phospholipase C activity in the electrophysiological effects of noradrenaline in vascular smooth muscle cells. IP3 may be responsible for calcium release and subsequent activation of chloride current, while DAG may be responsible for the stimulation of inward channel current, probably by activation of protein kinase C. Further tests with suitable blocking agents will be necessary to evaluate precisely the respective role of 1P3 and DAG in NA-induced stimulation in smooth muscle cells. This work was supported by grants from Ministere de la Recherche et de la Technologie (89. H.0082), Centre National des Etudes Spatiales, Fondation pour la Recherche Medicale, Association Francaise contres les Myopathies, and Conseil Regional d'Aquitaine, France. We thank Ms Biendon for her secretarial assistance. REFERENCES

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GTP-binding proteins mediate noradrenaline effects on calcium and chloride currents in rat portal vein myocytes.

1. Membrane currents were recorded by a patch-clamp pipette technique in cultured cells from rat portal vein using the whole-cell mode. 2. Noradrenali...
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