Effect of carbachol in the absence and presence of phenylephrine on Rb+ efflux and tension in rabbit left atria

ELSEVIER

European Journal of Pharmacology 256 (1994) 311-319

Effect of carbachol in the absence and presence of phenylephrine on Rb + effiux and tension in rabbit left atria Abhijit Ray

1,

Kathleen M. MacLeod

*

Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, B.C. V6T1Z3, Canada (Received 13 October 1993; revised MS received 31 January 1994; accepted 8 February 1994)

Abstract

The muscarinic agonist carbachol produced a concentration-dependent increase in 86Rb+ efflux and decrease in tension in isolated, electrically stimulated rabbit left atria. However, the lowest concentration of carbachol tested produced only a very small increase in 86Rb+ efflux, while it caused a relatively greater decrease in tension. 4-Aminopyridine and pertussis toxin attenuated the carbachol-stimulated 86Rb+ efflux and negative inotropic effect. However, 4-aminopyridine had a greater inhibitory effect on carbachol-stimulated 86Rb+ efflux than on carbachol-induced decreases in tension. Pre-treatment of rabbits with pertussis toxin completely abolished the increase in 86Rb+ efflux and decrease in tension produced by carbachol in the presence of the a-adrenoceptor agonist phenylephrine. 4-Aminopyridine attenuated the negative inotropic response to carbachol in the presence of phenylephrine, but had less effect on the carbachol-induced increase in 86Rb+ effiux under these conditions. These results suggest that carbachol-induced increases in K ÷ effiux may contribute at least in part to the negative inotropic responses to carbachol in the presence and absence of phenylephrine. However, this may not be sufficient to explain the direct negative inotropic response of left atria to carbachol.

Key words: Carbachol; Phenylephrine; Atrium, left; Negative inotropy; 86Rb+ effiux; Pertussis toxin; 4-Aminopyridine

I. Introduction

In mammalian left atrium, muscarinic receptor stimulation results in a marked cyclic nucleotide-independent direct negative inotropic response which has been attributed, at least in part, to the ability of muscarinic agonists to open K + channels (Ten Eick et al., 1976; Cerbai et al., 1988; Ray and MacLeod, 1990; Urquhart et al., 1991). In addition, muscarinic receptor agonists have been shown to antagonize positive inotropic responses of rabbit left atrium to both a- and/3-adrenoceptor agonists (MacLeod, 1986; Ray and MacLeod, 1990). The ability of muscarinic agonists to inhibit /3-adrenoceptor agonist-stimulated cAMP generation appears to be largely responsible for the inhibitory effect of muscarinic agonists on positive inotropic re-

* Corresponding author. Tel. 604-822-3830, fax 604-822-3035. i Present address: Department of Pharmacology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00099-S

sponses to fl-adrenoceptor stimulation (Ray and MacLeod, 1990, 1992). However, it is not clear how muscarinic agonists antagonize positive inotropic responses to a-adrenoceptor stimulation, which are not associated with changes in intracellular cAMP levels (MacLeod, 1986; Ray and MacLeod, 1990). Recently, using K + channel agonists and an antagonist, and pertussis toxin, which uncouples muscarinic receptors from K + channels, we provided evidence suggesting that the inhibitory effect of the muscarinic agonist carbachol on positive inotropic responses to a-adrenoceptor stimulation may be related to the ability of carbachol to open K ÷ channels (Ray and MacLeod, 1990). The purpose of the present study was to obtain more direct evidence for the involvement of the muscarinic receptor-activated K + current in negative inotropic responses of left atria to carbachol in the presence and absence of the a-adrenoceptor agonist phenylephrine. Using S6rubidium (S6Rb+) as a tracer for K ÷ (Jahnel and Nawrath, 1989; Urquhart et al.,

312

.4. Ray, K.M. MacLeod / European Journal Of Pharmacology 256 (1994) 311-319

1991), we have simultaneously measured the effects of carbachol on K + channel activity and force of contraction of electrically stimulated rabbit left atrial strips in the presence and absence of phenylephrine. The effects of the K ~ channel blocker, 4-aminopyridine, and of pertussis toxin on these responses were also determined.

2. Materials and methods

2.1. Isolated tissue preparation Rabbits of either sex were housed individually in cages and had free access to food and water. For experiments involving pertussis toxin, animals were injected with pertussis toxin (0.5 /xg kg 1) through the ear vein 48 h before the experiments. Control rabbits were injected with normal saline. On the day of the experiment animals were killed by an injection of pentobarbitone sodium (65 mg kg -~) through the ear vein followed by exsanguination. Hearts were rapidly removed and placed in modified Chenoweth-Koelle solution (composition in raM: NaC1 120, KCl 5.7, CaCI 2 2.2, MgC12 0.9, N a H C O 3 25 and glucose 10) at room temperature, aerated with 95% 02-5% CO 2. Left atria were removed from the heart and were cut into half. One end of one half was attached to a bipolar platinum electrode which was placed in a 20 ml tissue bath containing modified Chenoweth-Koelle (C.K.) solution maintained at 35°C and aerated with 95% O 2 and 5% CO~. The other end of the muscle preparation was attached by means of a cotton thread to a Grass FT.03 force displacement transducer. Tissues were stimulated to contract with pulses of 5 ms duration at a frequency of 1 Hz and a voltage 2 times threshold. Responses were recorded on a Grass polygraph (model 7E). Atrial strips were placed under a resting tension of 0.5 g and the resting tension was adjusted throughout the 60 rain equilibrium period to give the optimal basal developed tension.

2.2. SORb+ efflux measurement After 1 h of equilibration, the atrial strip was exposed to 3-5 #Ci ml l of 86Rb+ (in a total volume of 17 ml) for another 2 h period, during which time the force of contraction was monitored. In preliminary experiments the rate constant of 86Rb+ effiux was found to decline from a very high value to a plateau of approximately 0.01 min -1 within the first 10 min and then remain stable for up to 1 h in untreated tissues. Therefore, tissues were washed with non-radioactive C.K. buffer every 2 min for 20 min before exposure to any drug. The atrial strip was exposed to drugs dissolved in C.K. buffer for various lengths of time during

which the tissue was washed every 2 min with non-radioactive C.K. buffer containing the drug, and the force of contraction was also monitored. In order for the contractile response of the atrial strip to stabilize to the pre-drug treatment level, the atrial strip was re-equilibrated for 40 min in 17 ml of non-radioactive C.K. buffer which was changed and collected for counting every 20 min. After the 40 rain washout period, the tissue was again washed every 2 min with non-radioactive C.K. buffer for another 10 min, followed by exposure to drugs as described above. The contents of the tissue bath were collected in polystyrene scintillation vials and ~ R b + was counted by scintillation counting in the Cerenkov mode without the addition of scintillation fluid (Urquhart et al., 1991). The radioactivity remaining in the atrial strip at the end of the experiment was obtained by dissolving the atrial strip in 2 ml of Protosol for 24 h followed by liquid scintillation counting after adding 11 ml of Aquasol. In order to permit comparison between aqueous counts (each 2 rain sample) and counts obtained in the presence of Aquasol, a non-radioactive atrial strip was spiked with a known amount of radioactivity and counted, after dissolution in Protosol and addition of scintillation fluid. At the same time, a 17 ml volume of C.K. solution was also spiked with the same amount of radioactivity and was counted without adding any scintillation fluid. The ratio of aqueous to organic counts was obtained (Urquhart et al., 1991). The counts present in the atrial strips were multiplied by the factor, which was 0.29 in our experiments, and compared with other aqueous counts. The efflux rate constant for ~6Rb+ was calculated using the formula:

k,=E,/(C, xt) where k, = efflux rate constant at time t, E, = efflux (measured as cpm) at time t, C t = total tissue counts (cpm) of 86Rb+ at time t, t = collection time in min. C t was calculated by back addition of the radioactivity remaining in the tissue at the end of the experiment plus all the radioactivity that was released during the experiment from time t to the end. Drugs were dissolved in 17 ml of warm C.K. solution containing 1 /xM timolol to block the /3-adrenoceptormediated component of the phenylephrine response. When the carbachol concentration-response curve was obtained, each concentration of carbachol was kept in contact with the tissue for an 8 min period, with the solution being changed every 2 min. When the carbachol response was measured in the presence of 4aminopyridine or phenylephrine, tissues were exposed to them for a period of 16 min, with carbachol being added for the final 8 min. When the carbachol response was measured in the presence of both 4aminopyridine and phenylephrine, tissues were ex-

A. Ray, K.M. MacLeod / European Journal of Pharmacology 256 (1994) 311-319

posed to 4-aminopyridine for 24 min with phenylephrine and carbachol being added for the last 16 and 8 min respectively. In each experiment, the average of the final four values of effiux rate constants or tension obtained immediately prior to addition of drug was considered the basal or initial response. In order to obtain the response to drug the four final values of rate constants and tension obtained in the presence of each concentration of drug were averaged. Average values from four or more such experiments were used to calculate mean_+ S.E.M. In some experiments data were expressed as a percent of the basal response using the formula: % of basal response

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Phenylephrine hydrochloride, timolol maleate and 4-aminopyridine were obtained from Sigma Chemical Co., St. Louis, USA. Carbamylcholine chloride was obtained from Aldrich Chemical Co., Wisconsin, USA and pertussis toxin from List Biological Laboratories, California, USA. [86]Rb+-labelled rubidium chloride was obtained from Amersham Canada, Ontario, Canada.

2.4. Statistics Mean values of tension and effiux rate constant obtained for different treatments within the same experiment were compared by paired t-test. Means obtained for the same treatments but from different experiments were analyzed by Student's unpaired t-test. A P < 0.05 was considered significantly different.

3. Results

3.1. Effect of carbachol on 86Rb + efflux and force of contraction The effect of increasing concentrations of carbachol on the the tension and rate constant of 86Rb+ effiux is illustrated in Fig. 1. Carbachol produced an increase in the rate constant of 86Rb+ effiux which reached a maximum (138 + 2% of basal) in the presence of 10 p.M carbachol (Fig. 2). Carbachol also produced a negative inotropic response in rabbit left atrial strips (Fig. 1) which reached a maximum at 1 ~ M carbachol (Fig. 2). A further increase in the carbachol concentration resulted in partial reversal of the negative inotropic response. The lowest concentration of carbachol used (100 nM) produced only a small increase in

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Fig. 1. Effect of carbachol on the S6Rb+ effiux rate constant (top, rain i) and contractile force (bottom) of an electrically stimulated rabbit left atrial strip. The atria was treated with 0.1 (A), 1 (B), and 10 (C, E) /~M carbachol alone, and with 10 /~M carbachol in the presence of 100 nM atropine (D) for 8 rain. Tissues were washed every 2 rain with either C.K. solution alone (B under the tracings), or C.K. solution containing carbachol, atropine, or atropine plus carbachol (C, A and A + C under the tracings, respectively). The C.K. solution removed was counted and used to calculate the S6Rb+ efflux rate constants as described in Materials and methods. The tissue was washed for 20 rain between each exposure to carbachol, and was pre-treated with atropine for 10 rain before exposure to carbachol (D).

86Rb+ effiux (to 105 _+ 3% of basal) but produced a larger decrease (45 +_ 7%) in basal developed force (Fig. 2). In the presence of 100 nM atropine, both the increase in 86Rb+ effiux and the reduction in tension produced by 10 ~ M carbachol were attenuated (Fig. 1). Overall, 86Rb+ effiux in response to 10/~M carbachol was reduced to 108 _+ 2% of basal by 100 nM atropine, while atropine alone slightly reduced the basal effiux

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rate constant, to 88 + 4% of basal (Fig. 3). In preliminary experiments, a higher concentration of atropine (1 /~M) was found to completely block the increase in 86Rb+ influx and decrease in tension produced by 10 tzM carbachol (data not shown).

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Fig. 4. Effect of carbachol on contractile force (A) and S6Rb+ efflux (B) in electrically stimulated rabbit left atrial strips in the absence (CON) and presence of 4-aminopyridine (4-AP, 50 and 500 ,~M). Open bars represent the basal response prior to addition of drug, cross-hatched bars represent the 4-aminopyridine response, and hatched bars represent the response to carbachol (10 #M). Atrial strips were exposed to carbachol for 8 rain. When carbachol was combined with 4-aminopyridine, atrial strips were exposed to 4aminopyridine for 16 rain with carbachol being added for the final 8 min. Contractile response is expressed as the attained tension in grams (g) and effiux is expressed as the effiux rate constant. Each bar represents mean _+S.E.M. of 6 - 8 experiments. * Significantly different from the corresponding response immediately prior to addition of 4-aminopyridine and carbachol ( P < 0.05, paired t-test). + Significantly different from carbachol response in the control group ( P < 0.05, t-test).

3.2. Effect of 4-aminopyridine on the carbachol-induced decrease in tension and increase in 86Rb + efflux 4-Aminopyridine alone (50 and 500 /xM) exerted a concentration-dependent positive inotropic effect in rabbit left atrium (Fig. 4A), but had no significant

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A B C Fig. 3. Effect of atropine on carbachol-stimulated S6Rb+ efflux in electrically stimulated rabbit left atrial strips. S6Rb+ efflux was monitored in the presence of 10 # M carbachol (A) alone, 10 izM carbachol plus 100 nM atropine (B) or 100 n M atropine alone (C). Atrial strips were exposed to atropine or carbachol alone for 8 min each. W h e n atropine was combined with carbachol, tissues were exposed to atropine for 16 min with carbachol being added for the final 8 min. Data are expressed as percent of initial efflux rate constant prior to addition of any drug. Data were analyzed by paired t-test. Each bar represents m e a n _+S.E.M. of 4 experiments. * Significantly different from carbachol alone.

effect on the 86Rb+ effiux rate constant (Fig. 4B). The same concentrations of 4-aminopyridine also attenuated the ability of carbachol to both reduce the tension (Fig. 4A) and to promote 86Rb+ effiux (Fig. 4B). In this set of experiments, carbachol alone (10 /xM) produced a significant increase in the effiux rate constant to 128 _+ 2% of basal and reduced the tension by 72 _+ 4%. In the presence of 50 tzM 4-aminopyridine, carbachol still produced a significant rise in the effiux rate constant to 120_+ 2%, while reducing the tension by 59 + 6%. In the presence of the higher concentration of 4-aminopyridine (500/zM), carbachol had no significant effect on the effiux rate constant (Fig. 4B), although it still produced a significant negative inotropic effect (Fig. 4A), reducing the tension by 45 _+ 7%.

3.3. Effect of carbachol on e6Rb + efflux and tension in the presence of phenylephrine Phenylephrine alone (100 tzM) had a positive inotropic effect in left atrial strips but had no significant effect on the S6Rb+ efflux rate constant (Table 1). In the presence of phenylephrine, 10 IzM carbachol produced a significant decrease in tension (Fig. 5A) and increase in 86Rb+ efflux (Fig. 5B). Overall, when expressed as a percentage of the corresponding values obtained in the presence of phenylephrine alone, carbachol produced a 65 _+ 5% decrease in the tension

A. Ray, K.M. MacLeod / European Journal of Pharmacology 256 (1994) 311-319 Table 1 Effect of phenylephrine and 4-aminopyridine, alone and in combination, on the contractile response and rubidium efflux rate constant in electrically stimulated rabbit left atrium Treatment

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Effiux rate constant ( X l 0 3)

Basal Phenylephrine (100/xM)

0.72 +-0.23 1.2 _+0.2 a

9.47 _+0.35 9.71 _+0.34

Basal 4-Aminopyridine (50 tz M) 4-Aminopyridine (50 tzM) + phenylephrine (100/xM)

0.54 -t-_0.24 0.56 +-0.05

10.04 _+0.48 9.95 _ 0.38

1.24_+0.18a

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Basal 4-Aminopyridine (500/zM) 4-Aminopyridine (500/xM) + phenylephrine (100/zM)

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9.91 +-0.49 9.74 +-0.45

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Each data point represents mean_+ S.E.M. of 9-11 experiments. a Significantly different from the corresponding basal response.

elevated by phenylephrine, and increased the S6Rb+ efflux rate constant to 123 + 4%. The increase in 86Rb+ effiux produced by carbachol in the presence of phenylephrine was not significantly different from that produced by carbachol alone (128 + 3%).

3.4. Effect of 4-aminopyridine on responses to carbachol in the presence of phenylephrine In this series of experiments, 4-aminopyridine alone (50 and 500 ~M) produced only a very small positive

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inotropic response and had little effect on the positive inotropic response of left atria to phenylephrine (Table 1). No significant effect on 86Rb+ effiux was detected when phenylephrine was administered in the presence of either concentration of 4-aminopyridine (Table 1). 4-aminopyridine attenuated the reversal by carbachol of the positive inotropic response to phenylephrine (Fig. 5A). In the presence of 50/zM 4-aminopyridine, carbachol inhibited the positive inotropic response to phenylephrine by 51 +_6%, while in the presence of 500 /zM 4-aminopyridine, carbachol reduced the positive inotropic response to phenylephrine by 25 _+5%. 4-Aminopyridine appeared to have somewhat less effect on the increase in 86Rb+ effiux produced by carbachol in the presence of phenylephrine (Fig. 5B). In the presence of the lower concentration of 4-aminopyridine (50 /~M) carbachol produced an increase of 117 + 3% in the effiux rate constant. The higher concentration of 4-aminopyridine (500/zM) reduced the magnitude of the carbachol-induced increase in the effiux rate constant to 113 _+3%. However, the increase in the 86Rb+ effiux rate constant produced by carbachol in the presence of the combination of phenylephrine and 500 p,M 4-aminopyridine was still significant (Fig. 5B).

3.5. Effect of pertussis toxin on the carbachol-induced increase in 86Rb + efflux and decrease in tension Pre-treatment of rabbits with 0.5/zg kg-1 pertussis toxin appeared to produce a rightward shift in the carbachol dose-response curve, while having little effect on the maximum stimulatory effect of carbachol on S6Rb+ efflux (Fig. 6B). In contrast, pertussis toxin pre-treatment inhibited the maximum negative inotropic response to carbachol, while having no effect

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Fig. 5. Effect of carbachol in the presence of phenylephrine on contractile force (A) and 86Rb+ efflux (B) in electrically stimulated rabbit left atrial strips. Open bars represent the response to phenylephrine (100 ~M) alone and hatched bars represent the response to carbachol (10 p,M) in combination with phenylephrine, in the absence (CON) and presence of 50 and 500/zM 4-aminopyridine (4-AP 50 and 500 /zM). Tissues were treated with 4-aminopyridine for 24 min, and phenylephrine for 16 min, with carbachol being added for the final 8 rain. Contractile response is expressed as the attained tension in grams (g) and effiux is expressed as the efflux rate constant. Each bar represents mean+S.E.M, of 9-11 experiments. * Significantly different from the corresponding response immediately prior to addition of carbachol (P < 0.05, paired t-test). ÷ Significantly different from carbachol response in the control group (P < 0.05, t-test).

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316

A. Ray, K.M. MacLeod / European Journal of Pharmacology 256 (1994) 311-319

on the reversal of the negative inotropic effect by 100 izM carbachol (Fig. 6A). Responses to 1 /~M carbachol, which produced a near-maximal increase in 86Rb + effiux and the maximum decrease in tension in tissues from saline-treated rabbits, were both markedly inhibited by pertussis toxin pre-treatment (Fig. 6A and B). However, while the increase in 86Rb+ effiux produced by 1 ~ M carbachol was almost abolished (Fig. 6B), this concentration of carbachol still reduced the tension by 36_+ 7% (Fig. 6A) in atria from pertussis toxin pretreated rabbits. No further reduction in tension of atria from pertussis toxin-treated rabbits was obtained by increasing the carbachol concentration to 10 IzM, despite the fact that this concentration of carbachol increased 86Rb+ efflux to almost the same extent as in atria from saline-treated rabbits (Fig. 6A and B).

3.6. Effect of pertussis toxin on responses to carbachol in the presence of phenylephrine Pre-treatment of rabbits with pertussis toxin had no significant effect on the magnitude of the positive inotropic response of left atria to 100 p.M phenylephrine. However, the ability of 10/xM carbachol both to reverse the positive inotropic response to phenylephrine and to increase the efflux rate constant in the presence of phenylephrine was essentially abolished by pertussis toxin pre-treatment (Fig. 7A and B). No carbachol-induced increase in 86Rb+ efflux could be detected in the presence of 100 /xM phenylephrine in

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Fig. 7. Effect of pre-treatment of rabbits with pertussis toxin on carbachol-induced changes in contractile force and 86Rb+ effiux in the presence of phenylephrine in electrically stimulated rabbit left atrial strips. Force of contraction (A) and 86Rb+ efflux (B) were monitored in left atrial strips from saline (CON) and pertussis toxin (PTX, 0.5 /xg k g - 1 ) pre-treated rabbits in the presence of 100 /xM phenylephrine alone (open bars) or 1 0 / x M carbachol in combination with phenylephrine (hatched bars). Atria were treated with phenylephrine for 16 min, with carbachol being added for the final 8 rain. Contractile response is expressed as the attained tension in grams (g) and 86Rb+ effiux is expressed as the efflux rate constant. Each bar represents mean ± S.E.M. of 4 - 6 experiments. * Significantly different from the response immediately prior to addition of carbachol ( P < 0.05, paired t-test).

atria from pertussis toxin pre-treated rabbits (Fig. 7B). While carbachol still produced a small reduction in the tension elevated by phenylephrine in atria from pertussis toxin-treated rabbits, this difference was not significant (Fig. 7A).

4. Discussion

In the present study, carbachol produced an increase in the effiux of 86Rb+ from rabbit left atrial strips, which was blocked by atropine and was attenuated by pre-treatment of rabbits with pertussis toxin. This is in agreement with previous reports in rat (Quast et al., 1988) and guinea pig (Urquhart et al., 1991) atria and suggests that in rabbit left atrium, muscarinic receptors are coupled to K + channels by means of a pertussis toxin-sensitive guanine nucleotide binding protein (G-protein). The ability of muscarinic agonists to open K + channels (Pfaffinger et al., 1985; Sorota et al., 1985) and to promote 42K+ and 86Rb+ efflux in mammalian atrial myocardium (Raynor and Weatherall, 1959; Quast et al., 1988; Jahnel and Nawrath, 1989; Urquhart et al., 199l) has been well-established. However, there have been a very few attempts made to correlate the opening of K + channels with negative inotropic responses to muscarinic receptor stimulation in the same, electrically stimulated, atria. In the present study, we found that the lowest concentration of carbachol used (0.1 ~ M ) caused a very marked decrease in left atrial tension, while producing only a very small increase in 86Rb+ effiux. Increasing the concentration of carbachol to 1 p~M resulted in in a near-maximal increase in 86Rb+ efflux and decrease in tension, while a further increase in carbachol concentration resulted in no further change in 86Rb+ effiux, and partial reversal of the negative inotropic response to carbachol. The relatively greater ability of 0.1 izM carbachol to decrease the contractile force than to increase S6Rb+ effiux in rabbit left atria could indicate that either a very small increase in K + effiux can produce a very marked effect on tension, or that changes in inotropic response can be measured with greater sensitivity than changes in K + effiux with the methods employed. Studies in vascular smooth muscle have shown that although 86Rb+ is an adequate marker for K +, effiux values for 86Rb+ underestimate changes in K + effiux (Smith et al., 1986), and the difference is more marked in response to low concentrations of agonist (Quast and Baumlin, 1988). However, Urquhart et al. (1991) have shown that there is a very good correlation between decreases in tension and increases in 86Rb+ effiux in response to adenosine receptor agonists over their entire concentration range in guinea pig left atria. A third possibility is that mechanisms other than increases in K + effiux con-

A. Ray, K.M. MacLeod / European Journal of Pharmacology 256 (1994) 311-319

tribute to the direct negative inotropic response to carbachol. To further investigate the relationship between increases in 86Rb+ efflux and decreases in tension in response to carbachol, we used 4-aminopyridine and pertussis toxin to interfere with the effects of carbachol on these processes. 4-Aminopyridine has been demonstrated to antagonize a number of different K ÷ currents, thereby acting as a relatively non-selective K + channel antagonist (reviewed in Rudy, 1988). It is well-established that 4-aminopyridine blocks the outward current in cardiac cells (Van Bogaert et al., 1982; Gilmour et al., 1986) and the positive inotropic effect of 4-aminopyridine in the concentration range used in the present investigation has beeen attributed to the resulting prolongation of the action potential (Wollmer et al., 1981). 4Aminopyridine has also been shown to block both the increase in action potential duration and negative inotropic effects of carbachol in guinea pig left atria, over the same concentration range (Freeman, 1979). Although 4-aminopyridine was found to release noradrenaline and acetylcholine from nerve terminals in various other cardiac preparations, this did not occur in rabbit left atria (Glover, 1981). 4-Aminopyridine has also been reported to displace muscarinic agonists from their binding sites (Lai et al., 1985; Drukarch et al., 1989). However, we have found that 500 tzM 4-aminopyridine (the highest concentration used in this investigation) had no effect on the ability of carbachol to inhibit isoproterenol-induced increases in cAMP levels in rabbit left atria (Ray and MacLeod, 1993), thus ruling out a direct effect of 4-aminopyridine at muscarinic receptors. Other actions of 4-aminopyridine, such as the inhibition of sarcoplasmic reticulum Ca 2÷ ATPase, are only seen at higher concentrations (1 mM or greater) (Ishida and Honda, 1993). In the present investigation, 4-aminopyridine alone had no significant effect on basal 86Rb+ efflux, in agreement with a previous report in guinea pig left atria (Urquhart et al., 1991). However, 4-aminopyridine concentration-dependently inhibited the increase in 86Rb+ efflux produced by carbachol in rabbit left atrial strips. At the same time, a concentration of 4-aminopyridine which completely blocked the carbachol-induced increase in 86Rb+ efflux, only partially attenuated the direct negative inotropic response to carbachol. This supports the suggestion that some process in addition to increased K ÷ conductance contributes to the direct negative inotropic response of rabbit left atria to carbachol. Pertussis toxin has been demonstrated in a number of studies to uncouple muscarinic receptors from K ÷ channels (Pfaffinger et al., 1985; Sorota et al., 1985). In the present investigation, pre-treatment of rabbits with 0.5/~g kg-1 pertussis toxin resulted in a marked reduction of the ability of 1 ~M carbachol to exert negative inotropic response and to promote 86Rb+ efflux. How-

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ever, pertussis toxin pre-treatment also appeared to cause relatively greater inhibition of the increase in 86Rb+ effiux than the negative inotropic effect produced by 1 /zM carbachol. These data are consistent with those obtained with 4-aminopyridine and further suggest that some mechanism in addition to increases in K + conductance contributes to the direct negative inotropic responses of left atria to muscarinic receptor stimulation. No further decrease in tension was obtained in response to 10 /xM carbachol in atria from pertussis toxin-treated rabbits, although this concentration of carbachol produced a much greater increase in 86Rb+ effiux than 1 /xM carbachol. Instead, 10 /zM carbachol had a tendency to reverse the negative inotropic response observed in the presence of 1 /zM carbachol in atria from both saline and pertussis toxin pre-treated rabbits. Increasing the concentration of carbachol to 100/zM produced an even greater reversal of the negative inotropic response to carbachol. It is well established that muscarinic agonists in concentrations of 10/zM and higher can promote phosphoinositide turnover and exert a positive inotropic response by a pertussis toxin-insensitive mechanism (Tajima et al., 1987). It is possible that this effect might have contributed to the reversal of the negative inotropic response seen with 10 and 100/zM carbachol despite the fact that increases in K + effiux were sustained in response to these concentrations of carbachol, in atria from both control and pertussis toxin pre-treated rabbits. The mechanism by which carbachol could produce a K + channel-independent direct negative inotropic response is not known. Previous studies seem to have ruled out the involvement of changes in cAMP (Endoh et al., 1985; MacLeod, 1986; Ray and MacLeod, 1990) and cGMP levels (MacLeod and Diamond, 1986) in this process. It is possible that the pertussis toxin and 4-aminopyridine-insensitive component of the direct negative inotropic response to carbachol may be related to the ability of muscarinic agonists to inhibit calcium influx and the calcium current directly (Ten Eick et al., 1976; Cerbai et al., 1988). The reversal by carbachol of the positive inotropic response of rabbit left atria to phenylephrine was also associated with an increase in S6Rb+ effiux. In this series of experiments, 4-aminopyridine produced a smaller magnitude of positive inotropic response and inhibited carbachol-stimulated S6Rb+ effiux to a lesser extent than in the experiments described above. Although the reason for this is unknown, 4-aminopyridine (50 and 500 /zM) significantly attenuated the negative inotropic response to carbachol in the presence of phenylephrine. In agreement with our previous report (Ray and MacLeod, 1990), the negative inotropic response to carbachol was antagonized to a greater extent in the presence of phenylephrine plus 4-amino-

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A. Ray, tC M. Mac Leod / European Journal qt" Pharmacology 250 (1994) 311-319

pyridine than in the presence of 4-aminopyridine alone. It is possible that an additive effect of 4-aminopyridine and phenylephrine in blocking the transient outward K + current (Braun et al., 1990), which in turn will prolong the action potential duration and the associated influx of calcium, produced greater opposition to the negative inotropic response to carbachol than either agent alone. The same concentration of pertussis toxin which had little effect on the increase in 86Rb+ effiux in response to 10 /xM carbachol alone, completely blocked the increase in 86Rb+ efflux produced by 10 p.M carbachol in the presence of phenylephrine. This was associated with almost complete loss of the reversal by carbachol of the positive inotropic response to phenylephrine. These results are consistent with a role for increases in K + effiux in the inotropic response to carbachol in the presence of phenylephrine. The greater antagonism by pertussis toxin of both the negative inotropic response and increase in S6Rb+ effiux produced by carbachol in the presence of phenylephrine than in its absence may be explained by an additive effect of phenylephrine and pertussis toxin in the inhibition of carbachol-induced increases in K + effiux, as previously suggested (Ray and MacLeod, 1990). Braun et al. (19921 have recently demonstrated that a-adrenoceptor stimulation can reduce acetylcholine-induced increases in K + current in rabbit atrial myocytes by a mechanism insensitive to pertussis toxin. Although phenylephrine appeared to have little effect on the maximum carbachol-induced increase in S6Rb+ effiux in atria from saline-treated rabbits in the present investigation, a greater inhibitory effect of phenylephrine on muscarinic receptor-mediated increases in K + effiux may have been revealed by the reduced efficiency of coupling between muscarinic receptors and K + channels in the presence of pertussis toxin. In summary, the results of the present investigation demonstrate that there is a relatively poor correlation between muscarinic receptor-mediated increases in 86Rb+ efflux and decreases in tension in rabbit left atria in the absence and presence of agents which block the ability of muscarinic agonists to increase K + conductance. These data are consistent with the hypothesis that increases in K + effiux alone are not sufficient to explain the direct negative inotropic responses of left atria to carbachol. On the other hand, there was a better correlation between negative inotropic responses and increases in S6Rb+ effiux produced by carbachol in the presence of phenylephrine in atria from saline and pertussis toxin-treated rabbits. This suggests that the ability of carbachol to increase K + conductance may be largely responsible for the reversal by carbachol of positive inotropic responses of rabbit left atria to c~-adrenoceptor stimulation.

Acknowledgements The authors gratefully acknowledge the financial support of the Heart and Stroke Foundation of B.C.& Yukon. A.R. was the recipient of a traineeship from the Canadian Heart Foundation during the time this work was done.

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