ax-adrenoceptor antagonist prazosin but was not affected by the ac2-adrenoceptor

Transcription

1 Journal of Physiology (1990), 428, pp With 9 ftgure8 Printed in Great Britain CHARACTERISTICS OF CHLORIDE CURRENTS ACTIVATED BY NORADRENALINE IN RABBIT EAR ARTERY CELLS BY T. AMEDEE*, W. A. LARGEt AND Q. WANG From the Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE (Received 8 December 1989) SUMMARY 1. Responses to noradrenaline were studied in isolated rabbit ear artery cells with the nystatin method of whole-cell patch-clamp recording. With this technique it was possible to obtain reproducible responses to noradrenaline which was not possible with traditional whole-cell recording. 2. With NaCl as the major constituent of the bathing solution (potassium-free pipette and external solutions) the reversal potential (Er) of the noradrenalineevoked current was about 0 mv. When external chloride was replaced by thiocyanate, iodide, nitrate and bromide, Er was shifted to more negative potentials which indicates that a chloride conductance increase contributes to the current activated by noradrenaline. 3. When sodium was substituted by Tris, N-methyl-D-glucamine, choline or barium, Er of the noradrenaline-evoked current did not alter. This result suggests that a cation conductance is not implicated in the response to noradrenaline recorded with the nystatin method of whole-cell recording. 4. The chloride current activated by noradrenaline was blocked by the selective ax-adrenoceptor antagonist prazosin but was not affected by the ac2-adrenoceptor antagonist yohimbine. 5. When cells were exposed to zero calcium bathing solutions the amplitude of the current elicited by noradrenaline was unaffected when measured within 1-2 min in zero calcium conditions. Continued exposure to 0 Ca+1 mm-egta solution reversibly abolished the chloride current to noradrenaline. 6. In the presence of caffeine, which releases Ca2+ from internal stores and itself induced an inward current (at a holding potential of -50 mv), noradrenaline did not elicit a current. These data suggest that the chloride current evoked by noradrenaline results from an increase in intracellular concentration of calcium derived from internal stores. 7. The chloride channel blocking agents 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 0 5 mm) and furosemide (0 5 mm) produced partial reduction of * Present address: Institut National de la Sante et la Recherche Medicale, Unite de Recherches de Neurobiologie des comportements, U.176, Rue Camille-Saint-Saens, Bordeaux Cedex, France. t Author for correspondence. MS 8132

2 502 T. AMEDEE, I1L. A. LARGE AND Q. WAXG the noradrenaline-evoked chloride current whereas 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), anthracene-9-carboxylic acid (A-9-C) and picrotoxin were ineffective in concentrations of up to 0 5 mm. However DIDS and furosemide were non-selective blockers as both agents were more effective against the adenosine triphosphate-induced cation current. INTRODUCTION Recently we have been studying the membrane action of noradrenaline in freshly dispersed smooth muscle cells with patch-clamp techniques (Byrne & Large, 1987; Byrne & Large, 1988; Amedee, Benham, Bolton, Byrne & Large, 1990). With the whole-cell mode of recording there were two major problems encountered in this work. Firstly, noradrenaline evoked a membrane current in only a few of the cells tested. Secondly, in those cells which did respond to noradrenaline it was usually only possible to record one or two currents before the cells became unresponsive. It was assumed that this insensitivity of the cells to noradrenaline could be attributed to wash-out of important intracellular mediators with the use of patch pipettes. This phenomenon of wash-out precluded the systematic study of the responses to noradrenaline in a single cell and therefore imposed severe limitations on the types of experiments that were carried out. It was possible to observe reproducible responses to noradrenaline when microelectrode recording was used (Amedee & Large, 1989). However a disadvantage of the latter technique is that it is not possible to dictate the ionic gradients across the cell membrane with the same precision that is possible with patch pipette techniques. It seemed that these handicaps might be overcome with the nystatin method of whole-cell recording which has been developed by Horn & Marty (1988). With this technique the patch pipette contains nystatin which, in the cell-attached mode, permeabilizes the membrane. The pores formed by nystatin are permeable to monovalent ions (mainly Na+, K+ and to a lesser extent Cl-) but are not permeable to larger organic substances that are likely to be intracellular mediators of agonist-induced responses (see Horn & Marty, 1988 for greater detail). We have used the nystatin method of whole-cell recording in isolated rabbit ear artery smooth muscle cells. With this method of recording it was possible to obtain reproducible responses to noradrenaline and it was found that in potassium-free conditions the response to noradrenaline was purely an increase in membrane chloride conductance. Therefore in the present experiments we have studied the relative permeability of anions and the calcium-dependence of the chloride conductance. In addition we have investigated the actions of suggested chloride channel blocking agents and have identified the nature of the pharmacological receptor activated by noradrenaline. METHODS The experimental methods of the enzymatic dissociation of the rabbit ear artery cells were identical to those described previously (Amedee et al. 1990). Whole-cell currents were measured with the nystatin method of Horn & Marty (1988) with a patch-clamp amplifier (List EPC7; List- Electronic, Darmstadt, FRG) at room temperature (20-24 C). The patch-pipette resistance ranged

3 SMOOTH MUSCLE CHLORIDE CURRENTS from 3 to 6 MQl. In our experiments nystatin was freshly dissolved by sonication in absolute methanol (5 mg/ml) and included in the pipette solution at a final concentration of ,ug/ml. The potassium-free pipette solution contained (mm): NaCl 126, MgCl2 1-2, HEPES 10, glucose 11 and EGTA 1. The potassium-free external solution contained (mm): NaCl 126, MgCl2 1P2, CaCl2 1.5, HEPES 10 and glucose 11. All solutions were adjusted to ph 7-2 with NaOH. In potassiumcontaining conditions 6-0 mm-kcl was added to the external solution and 126 mm-nacl was replaced by 126 mm-kcl in the pipette solution. In order to identify the ionic mechanism of the noradrenaline-induced inward current the reversal potential of the response was measured in solutions where the external NaCl was replaced with various salts (see Table 1). All external solutions contained 10-6 M-propranolol to block any fl-adrenoceptor-mediated responses. Noradrenaline was applied by ionophoresis from micropipettes filled with 0-2 M-noradrenaline bitartrate with resistance of MQl. The ionophoretic electrode was placed within 5,um of the cell, and ionophoretic pulses of na for s were used. The values given in the text are the mean + s.e. of mean and statistical significance was estimated with Student's t test. Chemicals used were: bovine serum albumin (fatty acid free), dithiothreitol, noradrenaline bitartrate, papain type IV, nystatin, picrotoxin (all Sigma), collagenase CLS2 190 U/mg (Worthington), propranolol hydrochloride (ICI), furosemide sodium (Antigen), anthracene-9-carboxylic acid (A-9-C), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanatostilbene- 2,2'-disulphonic acid (DIDS; all from Aldrich Chemical Co.). RESULTS Responses recorded with the nystatin whole-cell recording method In the present experiments the whole-cell configuration with nystatin in the pipette was often achieved 1-2 min after formation of a gigaseal and the access resistance was usually MQ. In many cells the access resistance was similar to the value obtained with normal whole-cell recording (Amedee et al. 1990). Some characteristics of the membrane responses observed with nystatin whole-cell recording in rabbit ear artery smooth muscle cells are illustrated in Fig. 1. When potassium-containing pipette and bathing solutions were used, noradrenaline evoked an outward current (holding potential -50 mv, Fig. laa). Noradrenaline was applied every minute and after 25 min the amplitude of the response had not declined (Fig. lab). It is not evident why there is such a pronounced calcium-activated potassium conductance increase to noradrenaline in freshly dispersed ear artery cells, as in whole tissue preparations noradrenaline produces membrane depolarization (e.g. Suzuki & Kou, 1983). Consequently all the experiments to be described were carried out in potassium-free conditions (potassium was excluded from both bathing and pipette solutions, see Methods for details) in order to study the noradrenaline-evoked inward current. In potassium-free conditions noradrenaline activated a biphasic current at early times (Fig. lba) after obtaining whole-cell recording (with nystatin) but after 4-6 min only an inward current was observed (Fig. lbb). Since we were interested in studying the inward current, after achieving whole-cell recording at least 6 min were allowed for dialysis of the cell before the experiment was started. In most cells the amplitude of the inward current was sustained over min. One example is illustrated in Fig. 1 C where the amplitude of the current had decreased by only a small amount after 25 min. For comparison, the amplitude of the current stimulated by noradrenaline with normal whole-cell recording from a cell on the same cover-slip is also shown in the graph in Fig. 1 C. It was evident that in rabbit ear artery cells the nystatin method of whole-cell recording could be used and that this prevented the run-down of noradrenaline-induced currents. 503

4 504 T. AMEDEE, W. A. LARGE AND Q. WANG A a A 5 min b4a, 30 min 100 pa L 4 s B a b C 1st response 2 min 1 6 min 20 pal 4 s 0 14th response 20 pa 'a E 4 s %M Time (min) Fig. 1. Membrane responses to noradrenaline with the nystatin method of whole-cell recording. A, response to ionophoretically applied noradrenaline (at arrow-heads) at 5 (Aa) and 30 (Ab) min after whole-cell recording was obtained in K+-containing solutions. B, action of noradrenaline in K+-free conditions at 2 and 6 min after whole-cell configuration was achieved. After 2 min noradrenaline evoked a biphasic response but after 5-6 min with whole-cell recording there was no outward current to noradrenaline which suggests that wash-out of potassium was effectively complete. C, reproducibility of noradrenalineevoked inward currents with nystatin whole-cell recording (left-hand traces and in graph). Amplitude refers to normalized value and first application of noradrenaline was given at time 0 min. With normal whole-cell recording (0) two responses were obtained to noradrenaline from a cell on the same cover-slip as used for the nystatin recording (@). In most records, in this and subsequent records, the ionophoretic artifacts can be seen quite clearly on the current traces and normally the start of the ionophoretic pulse precedes the inward current by 05-OsO. Holding potential in all records is -50 mv. lonophoretic pulse: 50 na for 100 ms (A), 200 ms (B) and 500 ms (C).

5 SMOOTH MUSCLE CHLORIDE CURRENTS 505 Ionic mechanism underlying the inward current elicited by noradrenaline Previously it has been shown in ear artery cells that in potassium-free conditions noradrenaline increases membrane conductance to both chloride and cations (Amedee et al. 1990). Experiments were carried out to see if both of these NaCI B Nal 2s D Membrane current 1 NaSCN Cf]0JIiu' L60LiJL < Membrane potential (mv) Fig. 2. Reversal potential of the response to noradrenaline in various external anion solutions. In all experiments the membrane potential was held at -50 mv and the potential was stepped for 100 ms to -30, -10, + 10 and + 30 mv. The amplitude of the agonist-induced current was calculated by subtracting the membrane current in the absence of noradrenaline from the value obtained in the presence of the agonist. The amplitude of the agonist-induced current as a normalized value (the current at -50 mv is -1 for NaCl and NaI and + 1 for NaSCN) is plotted against membrane potential to obtain Er of the noradrenaline-evoked membrane current in D. In order to fit all the data on the graph in D, the vertical scale for the results with Nal (A) and NaSCN (V) is one tenth of that for NaCl (*). The vertical bar represents 40 pa in A and 20 pa in B and C. lonophoretic pulse was 50 na for 200 ms in all records. conductance mechanisms contributed to the current recorded with the nystatin whole-cell method. Figure 2A shows a response to noradrenaline with external 126 mm-nacl with a voltage-jump protocol. The normalized agonist-induced current is plotted in Fig. 2D and the reversal potential (Er) was 7 mv. In thirteen experiments with external NaCl, Er was mv (Table 1) which is similar to the value obtained with normal whole-cell recording (0-63 mv, Amedee et al. 1990). Figure 2B shows the response to noradrenaline when external NaCl was replaced by Nal and Er was more negative (-42 mv, Fig. 2D). In thirteen experiments with external Nal, Er was mv (Table 1) which implicated an anion conductance in the current stimulated by noradrenaline and also that iodide is more permeable than chloride through the anion conductance pathway. Substitution of NaCl with NaSCN produced an even greater shift of Er to more negative values.

6 506 T. AMEDEE, W. A. LARGE AND Q. WANG A B C A Control 10 nm-prazosin (3 min) 5 min wash 10 pa 2 s D E F Control 1,uM-yohimbine (5 min) 5 min wash Fig. 3. Pharmacological identification of the receptor which mediates the response to noradrenaline. A-C illustrates that 10 nm-prazosin produces a reversible block of the noradrenaline-induced current whereas yohimbine (1,UM) was without effect (D-F). lonophoretic pulse was 50 na for 200 ms in all records. Holding potential was -50 mv. TABLE 1. Reversal potential of the noradrenaline-evoked current in various external solutions Reversal potential External solutiont (mv) n A Anions altered NaCl NaSCN * 10 NaI * 13 NaNO * 8 NaBr * 5 Sodium glutamate * 7 B Cations altered Tris Cl Choline chloride N-Methyl-D-glucamine chloride BaCl t All salts at a concentration of 126 mm except for 89 mm-bacl2. * Statistically different from control value with external NaCl solution, P < Figure 2C illustrates the response in a cell bathed in 126 mm-nascn where noradrenaline produced an outward current at -50 mv and the extrapolated value of Er was -60 mv (Fig. 2D). In NaSCN the mean Er of the conductance increase to noradrenaline was mv (Table 1 A). Replacement of external NaCl with either NaNO3 or NaBr also moved Er to more hyperpolarized potentials (Table 1 A). When glutamate, usually considered to be a relatively impermeant anion, was the major anion in the bath solution Er was at a more positive value ( mv, Table 1 A). These data strongly indicate that noradrenaline causes an increase in chloride conductance in these conditions.

7 SMOOTH MUSCLE CHLORIDE CURREXTS In contrast when sodium was replaced by different cations there was no change in the Er value of the noradrenaline-evoked current. When external sodium was replaced by Tris, choline and N-methyl-D-glucamine (which are usually impermeant through cation channels) the reversal potential of the noradrenaline-evoked current was respectively , and mv (Table 1 B). It has been shown that barium is more permeable than sodium through the cation conductance activated by noradrenaline in rabbit portal vein (Byrne & Large, 1988). However when barium was substituted for sodium, Er of the current to noradrenaline was similar to the control value (Table 1 B). Thus it appears that with the nystatin method of whole-cell recording the inward current appears to be due solely to an increase in chloride conductance with negligible cation contribution. Thus this method of recording provides an opportunity for studying chloride currents in arterial smooth muscle cells. Identity of the pharmacological receptor which mediates the increase in chloride conductance Previously it has been shown in normal whole-cell recording that the non-selective a-adrenoceptor antagonist phentolamine blocks the noradrenaline-evoked inward current (a mixture of anionic and cationic mechanisms). It was of interest to investigate the type of a-adrenoceptor which activates the chloride current in ear artery muscle cells. Figure 3A-C shows that 10 nm-prazosin, an a1-adrenoceptor antagonist, reversibly blocks the chloride current. In contrast 1 /am-yohimbine, an a2-adrenoceptor antagonist, has no effect on the response (Fig. 3D-F). The concentrations used of prazosin and yohimbine are about the same multiples of their relative equilibrium dissociation constants for respectively a1- and a2-adrenoceptors. These data suggest that noradrenaline stimulates a1-adrenoceptors to produce an increase in membrane chloride conductance. Ca2' dependence of the chloride current Earlier work has shown that caffeine, which releases calcium from intracellular stores, induces a chloride current in the rat anococcygeus (Byrne & Large, 1987), rabbit portal vein (Byrne & Large, 1988) and the rabbit ear artery (Amedee et al. 1990). This suggested that the increase in chloride conductance to noradrenaline might also result from an increase in intracellular calcium concentration. We have tested this possibility further in the present experiments. Figure 4 shows the result from one experiment in which the normal bathing solution was replaced by one containing 0 Ca21 +1 mm-egta. It can be seen that when noradrenaline was applied soon after the cell was exposed to 0 Ca2+ conditions the response to noradrenaline was unaltered (Fig. 4A and B). In seven cells the amplitude of the noradrenaline-induced current after s in 0 Ca2+ solution was pa which was similar to the control value of pa. However in the continued presence of 0 Ca2+ solution the noradrenaline-evoked response progressively declined (Fig. 4C-E). When calcium was readmitted to the bathing solution noradrenaline did not elicit a current when applied very soon after the inclusion of calcium (Fig. 4F) but the current returned on continued presence of external calcium (Fig. 4G-I). These data suggest that external calcium is not immediately important for the generation of the chloride current but prolonged soaking in 0 Ca2+ solution leads to loss of responsiveness. A 507

8 508 T. AMEDEE, W. A. LARGE ANVD Q. WANG A B CL D E 4 Control 1.5 mm-ca2, 1 40s 3 min 5 min 8 min 0 Ca mm-egta F1_GmMa,I" HI 1.5 mm-ca2+ 20 pa 40 s 3 min 5 min 7 min Fig. 4. Effect of calcium-free solution on the chloride current elicited by noradrenaline. The salient points are that the current evoked by noradrenaline is not affected soon after going into Ca-free conditions (compare B with A) but that the response declines after several minutes in 0 Ca2" solution (C-E). Also, noradrenaline did not produce a response 40 s after returning to solution containing Ca2" (F), but the amplitude attained the control value after about 7 min in normal solution (record I). lonophoretic pulse was 50 na for 100 ms and the holding potential was -50 mv. A _5 # _ B 4 s Control 10 mm-carffeine 10 pa 2 s c rl 3 ff eine D 3 min caffeine 4 min wash Fig. 5. Effect of caffeine on the response to noradrenaline. Bath-applied caffeine (10 mm) produced an inward current (B) and 3 min in the continued presence of caffeine blocked the response to noradrenaline (C), and this effect was reversible (D). lonophoretic pulse was 50 na for 200 ms and holding potential was -50 mv.

9 SMOOTH MUSCLE CHLORIDE CUTRRENTS possibility might be that internal calcium is required for the initiation of the chloride current and that these stores might decline in 0 Ca2+ bathing solutions. We tested the hypothesis that internal Ca2+ stores may be involved in the response to noradrenaline by experiments with caffeine. Figure 5 shows that 10 mm-caffeine induces an inward A B C 50I9 Control 0-5 mm-dids Wash 20 pa 4 s D E F Control 0-5 mm-dids Wash 100 pa 4 s Fig. 6. The action of DIDS (0-5 mm) on the inward (A-C; NaCl pipette) and outward current (D-F; KCl pipette) elicited by noradrenaline. A-C were carried out in K+-free conditions whereas D-F were recorded from a cell in which 6 and 126 mm-kcl was present in the external and pipette solutions, respectively. Note that the chloride current is reduced by DIDS whereas the potassium current is enhanced. The arrow-heads indicate the time at which noradrenaline was applied in D-F as the ionophoretic artifacts cannot be seen. The ionophoretic pulse was 50 na for 200 ms (A-C) or 100 ms (D-F). Holding potential was -50 mv. current (Fig. 5B) and after 3 min in the continued presence of caffeine, noradrenaline' failed to evoke a current (Fig. 5C). Presumably caffeine released the intracellular calcium stores which could not be refilled in the presence of caffeine even with the presence of calcium in the bathing solution. On washing out caffeine the application of noradrenaline produced an inward current (Fig. 5D). This result was obtained in six cells tested. Overall these data indicate that intracellular stores of calcium are required to activate the chloride current. Action of chloride channel antagonists on the current to noradrenaline It seemed worthwhile to study the action of chloride channel antagonists on the noradrenaline-induced current as little is known about the action of such drugs in smooth muscle. Figure 6A-C illustrates the effect of DIDS on the chloride current evoked by noradrenaline. The amplitude of the current was reduced by about 50 % by 0-5 mm-dids and this was reversed on washing out the antagonist. It should be noted that in this experiment DIDS was present for only 2 min but after longer exposure to DIDS the blocking action was only poorly reversible. This antagonism

10 510 T. AMEDEE, W. A. LARGE AND Q. WANG Control 0.1 mm-dids (5 min) 5 min wash A kij i B 1 X C Control 0.2 mm-dids (3 min) D j L E lljll A 100 pa Fig. 7. Dose-dependent reduction of the inward cation current to ATP by DIDS. With 01 mm-dids the reduction of the response was partially reversible (A-C) but with 0-2 mm-dids the effect could not be reversed. In this experiment K+-containing external and pipette solutions were used but similar results were obtained in K+-free conditions. lonophoretic pulse was 50 na for 100 ms and the holding potential was -50 mv. 4 s A B C Control 0.5 mm-furosemide (5 min) 5 min wash 50 pa D E F 4s Control 1.0 mm-furosemide (5 min) 5 min wash Fig. 8. Action of furosemide on the noradrenaline-induced chloride current. All records were obtained from the same cell and illustrate that 1 mm-furosemide produced only a marginally greater effect than 0-5 mm-furosemide. The ionophoretic pulse was 50 na for 200 ms and the holding potential was -50 mv.

11 SMOOTH MUSCLE CHLORIDE CURRENTS might be due to blockade of the a1-adrenoceptor rather than the ion channel. This possibility was tested by investigating the action of DIDS against the increase in potassium conductance which results from a-adrenoceptor stimulation. Figure 6D-F demonstrates that 05 mm-dids increased the amplitude of the noradrenaline- 511 TABLE 2. Effect of chloride channel blocking drugs on inward currents induced by noradrenaline and ATP Reduction in current amplitude (%) DIDS (mm) Furosemide (mm) '5 10 Noradrenaline 28± (n = 5) (n =5) (n =7) (n =4) ATP ±2 (n =4) (n= 5) (n = 13) (n = 3) A B C D Control 1 min 3 min 3 min wash 0.5 mm-furosemide (5 min) 50 pa 4 s Fig. 9. The action of furosemide on the ATP-induced cation current. Note that 1 min after furosemide was included in the bathing solution the response to ATP was potentiated but had been blocked after 3 min in furosemide. This potentiation was seen regularly. The ionophoretic pulse was 50 na for 100 ms and the holding potential was -50 mv. activated potassium current. This enhancement may be due to removal of some inward current that is masked by the outward current. Nevertheless the result indicates that the reduction of the chloride current to noradrenaline is not due to blockade of acz-adrenoceptors. It has been shown in rabbit ear artery cells that adenosine triphosphate (ATP) evokes an inward current which is mediated by an increase in the membrane cation conductance with no chloride contribution (Benham, Bolton, Byrne & Large, 1987). In the present experiments Er of the ATPinduced current was not changed by anion substitution of the external solution but

12 512 T. AMEDEE, W. A. LARGE AND Q. WANG Er was shifted to more negative values when sodium was replaced by Tris. These data suggest that ATP activates only a cation conductance in ear artery cells with the nystatin method of recording. The action of DIDS was tested against the ATP current to investigate the selectivity of the antagonist. Figure 7A-C illustrates that 0-1 mm-dids reduced the amplitude of the ATP-induced cation current by about 65 %. The response was almost abolished by 0-2 mm-dids (Fig. 7D and E). It should be noted that the blocking effect of DIDS against ATP was also poorly reversible. Quantitative data are given in Table 2 which shows that DIDS is a more effective antagonist of the ATP-induced cation current than of the noradrenaline-evoked chloride current. It has been shown that the loop diuretic furosemide is an antagonist of the muscarinic receptor-mediated chloride current in rat lacrimal glands (Evans, Marty, Tan & Trautmann, 1986) and we investigated the action of this drug in ear artery cells. Figure 8A-C shows that 0 5 mm-furosemide decreased the amplitude of the chloride current to noradrenaline and that 1 mm-furosemide produced only a slightly greater effect (Fig. 8D-F). The effect of furosemide against ATP was somewhat variable. After 1 min in the presence of furosemide the amplitude of the ATP-induced current was increased (Fig. 9B) but after 3 min the current was blocked (Fig. 9C). Therefore, as with DIDS, furosemide seemed to be more effective against ATP than noradrenaline (Table 2). The blocking action of furosemide was usually reversible after removal of the antagonist from the bathing solution (e.g. Fig. 9D). The chloride channel antagonists SITS and anthracene-9-carboxylic acid in concentrations of up to 0 5 mm were ineffective against the noradrenaline-induced current. Also the y-aminobutyric acid (GABA) activated chloride channel blocker, picrotoxin (5 x 10-5 M) did not reduce the chloride current activated by noradrenaline. DISCUSSION The present experiments demonstrate that the nystatin method of whole-cell recording can be applied successfully to smooth muscle cells and this permits the recording of reproducible responses to pharmacological receptor agonists. An interesting question concerned the ionic mechanism which underlies the inward current activated by noradrenaline with the nystatin technique. In potassium-free conditions with conventional whole-cell recording and with the same pipette solution (minus nystatin) we have shown that noradrenaline increases membrane cation and anion conductance mechanisms (Amedee et al. 1990). However in the present experiments it appeared that the current evoked by noradrenaline was purely due to an increase in chloride conductance because anion substitution of chloride in the external solution altered Er of the response to noradrenaline whereas cation substitution had no effect on Er. In rabbit ear artery cells the equilibrium potential of the cation conductance activated by ATP is shifted by almost 35 mv to a more hyperpolarized value when external sodium is replaced by Tris (Benham et al. 1987). In cells isolated from the pregnant rat myometrium replacement of sodium by N-methyl-D-glucamine moved Er of the response to ATP by about 50 mv to more hyperpolarized values (Honore, Martin, Mironneau & Mironneau, 1989). Also replacement of sodium by Tris, choline and barium have revealed a cation current to

13 SMOOTH MUTSCLE CHLORIDE CURRENTS noradrenaline in vascular smooth muscle (Amedee & Large, 1989; Amedee et al. 1990). None of these solution changes altered the equilibrium potential of the response to noradrenaline in this study. In contrast anion substitution of the external solution produced marked changes in Er of the noradrenaline-induced current. It can be concluded that with nystatin whole-cell recording the membrane TABLE 3. Relative permeabilities of anions through membrane chloride conductance mechanisms SCN- I- N03- Br- C1- Rabbit ear artery*, Ca2+ -activated (a-adrenoceptor) Rat lacrimal glandt, Ca2+-activated (muscarinic receptor) Mouse spinal neuronel, y-aminobutyric acid-activated All values are relative to P,1. * Present study; t Evans & Marty, 1986; t Bormann, Hamill & Sakmann, current activated by noradrenaline appeared to be solely due to an increase in chloride conductance with no perceptible contribution from an increase in cation conductance. We have no firm explanation why the cation conductance is suppressed and the chloride mechanism is so dominant when the nystatin technique is used. It is possible that the degree of calcium buffering with the two techniques may be important. EGTA is included in patch pipettes for both normal and nystatin whole-cell recording methods but little EGTA is expected to permeate the perforations produced by nystatin. Consequently with normal whole-cell recording the calcium concentration may not be able to reach those values obtained with the nystatin recording technique. However the ability to obtain reproducible responses to noradrenaline does suggest that the conditions achieved with the nystatin method appear to be more physiological than those obtained with normal whole-cell recording. From the values of Er in anion substitution experiments it is possible to estimate with the Goldman-Hodgkin-Katz equation the relative permeabilities of the various anions through the chloride channel. The permeability sequence is compared with values from two other studies in Table 3 in which a chloride current is activated in response to pharmacological receptor stimulation. It can be seen that the relative sequence found in the present study is the same as in rat lacrimal glands and mouse neurones although the absolute values for SCN-, I-, NO3- and Br- are slightly greater in ear artery smooth muscle cells. Despite the similarity of the relative permeabilities of anions through the noradrenaline-activated and GABA-stimulated channel in smooth muscle and neurones respectively it appears that this does not mean that the same drugs will block the channel. Thus picrotoxin which is a wellestablished and potent blocker of GABA receptor-operated chloride channels, was ineffective against the chloride current studied in smooth muscle in the present experiments. However furosemide produced a similar reduction in the amplitude of the response to noradrenaline in the present work and the calcium-activated chloride current in rat lacrimal glands which represents another similarity between 17 PH Y

14 514 T. AMEDEE, W. A. LARGE AND Q. W11AATG these two membrane mechanisms which has been noted before (e.g. Amedee & Large, 1989). In most records the noradrenaline-induced inward currents were associated with little perceptible increase in the membrane noise. Moreover in many experiments the background noise was sufficiently low that a single channel opening of 0 5 pa in amplitude would have been detected. Since the driving force of the chloride current was 50 mv the upper limit of the single chloride channel conductance is 10 ps. It is likely that the actual conductance is much lower than this upper value and may be closer to the 1-2 ps value for the calcium-activated chloride channel described in the rat lacrimal gland (Marty, Tan & Trautmann, 1984). The results from the present experiments provide further support for the idea that the chloride conductance opens as a consequence of an increase in the intracellular calcium concentration. The response to noradrenaline was abolished reversibly in calcium-free conditions and in the presence of caffeine, which itself induced a current, noradrenaline did not elicit a membrane current. The results with caffeine indicate that the chloride current is activated by calcium released from internal stores. The data also suggest that the calcium which is necessary to activate the chloride current derives purely from internal stores because when the response was abolished in calcium-free conditions noradrenaline did not elicit a current when applied soon after calcium was readmitted to the bathing solution. The amplitude of the current returned to control values with continued presence of calcium, probably because the internal stores of calcium were replenished. However it is possible that external calcium can stimulate the chloride response but that internal calcium is required to produce an influx of calcium into the cell. It would seem likely that the chloride current is activated by calcium acting on the internal surface of the membrane. It is interesting that there was no evidence of the calcium-activated chloride current during the depolarizing voltage-jump protocol. It might be expected that this procedure might lead to entry of calcium through voltage-operated calcium channels into the cell to activate chloride channels. It is possible that calcium from extracellular sources is not as effective as calcium released from internal stores in the ability to activate the chloride current. It is also possible that insufficient calcium enters the cells during the voltage steps. This latter explanation is supported by the observation that there are no obvious inward currents evoked by the depolarizing steps in the absence of noradrenaline (see Fig. 2). Also the current-voltage relationship is linear between -50 and +30 mv which would not be expected if the depolarizing jumps had evoked significant voltage-dependent calcium current. The only report on the effects of chloride channel blocking agents on chloride currents in smooth muscle has stated that DIDS (10-3 M) reduced a calciumactivated chloride current in cultured rat portal vein cells (Pacaud, Loirand, Lavie, Mironneau & Mironneau, 1989) and therefore our results are in agreement with that report. However it seemed that high concentrations of DIDS were required to produce a partial blockade of the response to noradrenaline. Thus 0 5 mm-dids reduced the chloride current by 50 % in our experiments whereas in cultured rat astrocytes 2 x 10-5 M-DIDS reduced a voltage-gated chloride current by over 90 % (Gray & Ritchie, 1986). In the latter study 10-4 M-SITS was also an effective channel blocker whereas SITS in concentrations of up to 5 x 10-4 M had no effect in our experiments. In guinea-pig ventricular muscle it has been shown that a,-

15 SMOOTH MUSCLE CHLORIDE CURRENTS adrenoceptor-mediated chloride current was reduced by 10' M-A-9-C (Harvey & Hume, 1989) which was without effect in our experiments at higher concentrations. It is evident that there is quite a large disparity of the sensitivity of chloride currents to putative channel blocking agents. It seems that the chloride channel antagonists studied are not as effective against calcium-activated chloride currents in smooth muscle as on some other types of chloride currents described in other tissues. It is also evident that even with very high concentrations of channel antagonists (up to 10- M) it was not possible to obtain total block of the chloride currents. An interesting finding was that both DIDS and furosemide were even more effective against the ATP-induced cation current than against the noradrenaline-activated chloride current. The lack of selectivity of these compounds should be taken into account when they are used as pharmacological tools to provide evidence that a membrane current is carried by chloride ions. Our results do not indicate whether the blockers antagonize the cation channel or the purinoceptor itself. Finally, it is worth noting that it has been argued previously that an increase in membrane chloride conductance may be an important trigger for contraction in smooth muscle (Amedee & Large, 1989). Thus, Wahlstrom & Svennerholm (1974) demonstrated that when external chloride ions were replaced by more permeant anions (e.g. NO3-) noradrenaline was more effective in evoking contractions in rat portal vein. In contrast, substitution of external chloride ions with less permeant anions (e.g. benzenesulphonate) to reduce the membrane chloride conductance markedly suppressed the noradrenaline-induced contractions. Similar results were obtained with a-adrenoceptor activation in non-vascular smooth muscle (Large, 1984). Previously it has been suggested that furosemide is a venodilator in man (Dikshit, Vyden, Forrester, Chatterjee, Prakesh & Swan, 1973). Since it has been shown that rabbit portal vein cells possess both chloride and cation currents (Byrne & Large, 1988) it may be that the venodilator activity of furosemide is due to blockade of these conductances activated by noradrenaline released from sympathetic nerves. This work was supported by the Medical Research Council. 515 REFERENCES AMEDIEE, T., BENHAM, C. D., BOLTON, T. B., BYRNE, N. G. & LARGE, W. A. (1990). Potassium, chloride and non-selective cation conductances opened by noradrenaline in rabbit ear artery cells. Journal of Physiology 423, AM9DJE, T. & LARGE, W. A. (1989). Microelectrode study on the ionic mechanisms which contribute to the noradrenaline-induced depolarization in isolated cells of the rabbit portal vein. British Journal of Pharmacology 97, 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 rabbit ear artery. Journal of Physiology 387, BORMANN, J., HAMILL, 0. P. & SAKMANN, B. (1987). Mechanism of anion permeation through channels gated by glycine and y-aminobutyric acid in mouse cultured spinal neurones. Journal of Physiology 385, BYRNE, N. G. & LARGE, W. A. (1987). Action of noradrenaline on single smooth muscle cells freshly dispersed from the rat anococcygeus muscle. Journal of Physiology 389, 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,

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