Carbachol and acetylcholine delay the early postdenervation depolarization of muscle fibres through M1-cholinergic receptors

Neuroscience Research 37 (2000) 255 – 263 www.elsevier.com/locate/neures

Carbachol and acetylcholine delay the early postdenervation depolarization of muscle fibres through M1-cholinergic receptors Albert Urazaev a, Nikolay Naumenko a, Artem Malomough a, Eugeny Nikolsky a,b, Frantisˇek Vyskocˇil c,d,* a Kazan State Medical Uni6ersity, Butlero6 st. 49, Kazan, Russian Federation Kazan Institute of Biology, Academy of Sciences, Kazan, PO Box 30, Russian Federation c Department of Animal Physiology and De6elopmental Biology, Faculty of Sciences, Charles Uni6ersity, Vinicˇna´ 4, Prague 2, Czech Republic d Institute of Physiology, Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, Prague 4, Czech Republic b

Received 11 October 1999; accepted 13 April 2000

Abstract The resting membrane potential (RMP) of denervated muscle fibres of rat diaphragm muscle is depolarized by  8 – 10 mV during the first 3 h after nerve section and this early postdenervation depolarization is reduced substantially by the presence of 5× 10 − 8 M acetylcholine (ACh) or carbachol (CB). The muscarinic antagonist atropine (Atr; 5 × 10 − 9 to 5 ×10 − 6 M) reduced the effect of CB in a dose-dependent manner (Ki = 7×10 − 8 M) and increased the rate of the early postdenervation depolarization. In lower doses (5×10 − 7 M), Atr acted only in the presence of an allosteric stabilizator hexamethylene-bis[dimethyl-(3-phtalimidopropyl)ammonium] (W-84). Also pirenzepine, a specific inhibitor of the M1 subtype of muscarinic receptor, blocked the action of CB in a dose-dependent manner with an apparent inhibition constant Ki = 1×10 − 7 mM. DAMP, a specific M3 antagonist, was without effect on the muscle hyperpolarization induced by CB. CB also hyperpolarized the membrane potentials of muscles which were denervated for 1 – 3 days. It is concluded that ACh and CB protect the muscle fibres from early depolarization through M1-cholinergic receptors on the muscle membrane. These particular receptors can apparently mediate the ‘trophic’, non-impulse regulation of RMP in skeletal muscles when they are activated by acetylcholine released non-quantally. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Acetylcholine; Carbachol; Muscarinic receptors; Membrane potential; Denervated skeletal muscle

1. Introduction Acetylcholine (ACh) is released at the neuromuscular junction by two mechanisms. There is quantal, probably vesicular, release, which can be evoked by stimulation or can occur spontaneously, and, particularly in the rat and mouse, there is non-quantal release, which is due to leakage of transmitter from the nerve terminal (Katz and Miledi, 1977; Vyskocˇil and Illes, 1977). In Abbre6iations: ABET, arecaidine but-2-ynyl ester tosylate; ACh, acetylcholine; APET, arecaidine propargyl ester tosylate; Atr, atropine; CB, carbachol; Oxo, oxotremorine sesquifumarate; Pir, pirenzepine; RMP(s), resting membrane potential(s); W-84, hexamethylene-bis-[dimethyl-(3-phtalimidopropyl)ammonium]; 4DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide. * Corresponding author. Tel.: + 420-2-4752529. E-mail address: [email protected] (F. Vyskocˇil).

the absence of neural stimulation, much of the ACh which accumulates in the synaptic cleft is due to the non-quantal release of neurotransmitter (Mitchell and Silver, 1963; Vizi and Vyskocˇil, 1979; Dolezˇal and Tucˇek, 1983). After motor nerve section the non-quantal release decreases gradually and disappears completely within 4 h (Zemkova´ et al., 1987; Nikolsky et al., 1996). Nerve section also causes an early depolarization of the resting membrane potential (RMP) in muscle fibres within similar time interval of 3–4 h (Albuquerque et al., 1971; Urazaev et al., 1987a). This depolarization appears to be the result of increased active chloride transport across the membrane into the sarcoplasm (Betz et al., 1986; Urazaev et al., 1987b). We have suggested that there might be a correlation between the decrease of non-quantal release of transmitter and de-

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polarization of the RMP in the denervated muscle membrane (Urazaev et al., 1997). Indeed, ACh and its non-hydrolysable analogue carbachol (CB) in concentrations of 5× 10 − 8 M, similar to the concentration of ACh expected to be present in the synaptic cleft of the neuromuscular junction due to non-quantal ACh release (Bray et al., 1982; Vyskocˇil et al., 1983), delay the development of depolarization of denervated muscle fibres (Urazaev et al., 1987a,b). It has been suggested that ACh released non-quantally could be involved in the neural control of ion transport systems in the membrane which set the levels of ions inside the muscle fibre and the RMP (Urazaev et al., 1997). In agreement with this idea it has been found that ACh and CB inhibit the early postdenervation depolarization of the muscle membrane by activating Ca2 + -dependent NO-synthase; the release of NO probably acts as a retrograde signal at the neuromuscular junction. To determine the type of receptor responsible for the inhibitory action of ACh and CB on early postdenervation depolarization the selective nicotinic and muscarinic drugs were studied. While the nicotinic antagonists (+)tubocurarine and a-bungarotoxin (and the Na+/K+-ATPase inhibitor ouabain as well) had no effect on the action of CB (Urazaev et al., 1996, 1997), we report here that it is the muscarinic M1 subtype of cholinergic receptor which apparently mediates the protecting effects of cholinergic drugs on the denervated muscle membrane. Thus these particular receptors may be involved in the neural regulation of RMP in skeletal muscles.

2. Materials and methods

2.1. Preparations Under ether anaesthesia, diaphragms were excised from male 180–200-g Wistar rats. We used 3 – 4-mm wide strips of parallel intact muscle fibres of the diaphragm with no extramuscular nerve stump. The muscle strips were pinned with glass needles to the silicon rubber bottom of transparent glass dishes containing 12 ml of glutamic acid-free medium No. 199 (Hank’s salts with total 22 mM NaHCO3 for stabilizing pH at 7.2–7.4), and placed in a moist atmosphere of 5% CO2 and 95% O2 at 37°C under sterile conditions for 180–200 min. When muscles were incubated at 37°C for longer periods (up to 3 days), 5% embryonic calf serum, 2 mM L-glutamine and 100 U/ml penicillin were added to the culture medium which was replaced every day in a sterile laminal flow box (Bevan and Steinbach,

1983; Gonoi et al., 1983; Hollingworth et al., 1984; Urazaev et al., 1987a,b).

2.2. Electrophysiology Standard glass microelectrodes (tip resistance 15–20 MV, filled with 2.5 M KCl) were used for recording of the resting membrane potential (RMP) of 25–30 superficial muscle fibres of each strip within 5–7 min. RMPs from three to four strips were compared and when the variance among strips was insignificant, the data were pooled and analyzed. The input resistances of the muscle membrane were measured using two intracellular electrodes. The distance between electrodes was 20–50 mm. One electrode was used for applying a hyperpolarizing current (100 ms) and the second electrode registered the changes of RMP. Currents of amplitudes which evoked a potential changes across the fibre membrane of less than 5 mV were applied. The membrane resistances were calculated by Ohm’s law from the currents and induced potential changes. Temperature of the bath was 20°C.

2.3. Chemicals Acetylcholine chloride, oxotremorine sesquifumarate, atropine sulphate and pirenzepine were from Sigma (St. Louis, MO, USA), carbachol (carbamylcholine chloride) was from Koch-Light (UK), hexamethylene-bis[dimethyl-(3-phtalimidopropyl)ammonium] (W-84), 1,1dimethyl-4-diphenylacetoxypiperidinium (4-DAMP), arecaidine but-2-ynyl ester tosylate, and arecaidine propargyl ester tosylate were from Tocris Cookson (UK).

2.4. Statistics SigmaStat version for Windows 3.1 (Jandel Corporation 1992–1994) was used for statistical calculations. Analysis of variances of the experimental groups versus the control group were made by oneway comparison using the analysis of variation (ANOVA). The pairwise comparisons were done with t-tests, then the P-values were multiplied by the number of comparisons that were made to account for the compounding risk of doing many statistical comparisons (Bonferroni t-test). Throughout the text, statistically significant differences between mean9 S.E.M. of two groups are indicated at given levels of probability by PB , non-significant differences by P\ . All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the protocol of the experiments was approved by the Animal Care and Use Committee of The Institute of Physiology, Czech Academy of Sciences.

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3. Results

3.1. Carbachol and oxotremorine The mean RMP of control muscles measured 5–10 min after dissection was −74.5 90.4 mV (Table 1). The RMP depolarized to −66.6 mV after 3 h of incubation (PB0.001; see Tables for S.E.M. and n from now on, if not indicated in text). The presence of CB in the bath reduced this early postdenervation depolarization (Fig. 1). In concentrations of 5×10 − 8 Table 1 Resting membrane potential (RMP) of rat diaphragm fibres measured 5–10 min after muscle dissection and after 3-h incubation in the culture medium with selected concentrations of various drugsa Conditions

RMP (mV)

5–10 min after dissection

−74.59 0.4

After 3 h of incubation in culture medium (dener6ated muscle) a) Control denervated −66.69 0.4 b) Carbachol (5×10−8 M) −72.890.4 c) Carbachol (5×10−8 M)+atropine −66.090.4 (1×10−6 ?) d) Carbachol (5×10−8 M)+ pirenzepine −65.39 0.5 (1×10−7 M) e) Oxotremorine (1×10−7 M) −69.49 0.4 f) Arecaidine butynyl ester, ABET −69.19 0.2 (5×10−5 M) g) Arecaidine propargyl ester, APET −65.99 0.3 (5×10−5 M) h) Carbachol (5×10−8 M)+DAMP −70.59 0.3 (1×10−5 M) i) Carbachol (5×10−8 M)+DAMP −68.19 0.2 (1×10−4 M)

n 150 100 115 85 90 60 166 130 60 60

a Data are the means9 S.E.M., n= number of muscle fibres recorded as taken and pooled from three to six animals. Statistical significance is given in the text.

Fig. 2. Effect of different concentrations of atropine (in M/l: 0, 5 ×10 − 9, 1 × 10 − 8, 5 × 10 − 8, 1 × 10 − 7 and 1 ×10 − 6) on the resting membrane potential (RMP) of 3-h denervated diaphragm fibres in the presence of 5 ×10 − 8 M carbachol. Each point is the mean 9 S.E.M. of 55 – 120 RMP values measured from three to five animals. Temperature: 20°C.

to 1 × 10 − 7 M, the muscle fibres depolarized to only − 71 to −72 mV (PB 0.001; Table 1). Oxotremorine (Oxo), a highly specific muscarinic agonist which does not have a nicotinic effect, diminished the early postdenervation depolarization in similar way as CB (Fig. 1). Oxo 1× 10 − 8 M increased the absolute value of the RMP by  2.3 mV as compared with the control postdenervation depolarization. The maximal RMP increase of  6 mV was observed in concentrations of 5× 10 − 8 and 1×10 − 7 M. A comparison of the actions of Oxo and CB (Fig. 1) suggests that Oxo was slightly but significantly more potent in hyperpolarizing the muscle membrane than was CB at least at concentrations of 1× 10 − 8 and 1× 10 − 7 M. The effects of 5 × 10 − 7 M CB and Oxo were lower: −68.9 and −69.6 mV, respectively.

3.2. Atropine

Fig. 1. Effect of different concentrations of carbachol (CB, open circles) and oxotremorine (OXO, filled circles) on the resting membrane potential (RMP) of 3-h denervated diaphragm fibres. Each point is the mean 9 S.E.M. of 60–150 RMP values measured from three to five animals. Temperature: 20°C.

Atropine (Atr) blocked the protective effect of CB in a concentration-dependent manner (Fig. 2). This inhibition was significant in the presence of Atr 5× 10 − 9 M, which shifted the RMP from about −72 mV in 5 × 10 − 8 M CB alone to − 70 mV when both drugs were present. Higher concentrations of Atr (1 and 5×10 − 6 M) blocked the CB-induced protection of the RMP completely at all CB concentrations tested (Table 1, Figs. 2 and 3). The final RMP values in 5× 10 − 8 to 1× 10 − 7 CB and 5×10 − 6 M Atr of about −64 to −65 mV were even more depolarized (PB 0.05; Fig. 2,C,D) than the RMP in muscles incubated without any drug. The standard dose-response ratio for Atr was obtained with 5× 10 − 8 M CB in the muscle bath (Fig. 2 and Fig. 3C) and the estimated Ki of Atr competition for CB in this concentration was 1× 10 − 7 M.

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The action of Atr in the presence of some CB concentrations was complicated by the ‘agonist-like effect of the antagonist’ phenomenon. When 1× 10 − 8 M CB was present in the bath, the addition of 1 × 10 − 7 and 5× 10 − 7 M Atr produced a hyperpolarization (Fig. 3B). In the presence of 1 × 10 − 7 M CB (Fig. 3D), 1×10 − 7 M Atr blocked the RMP protection by CB and the RMP depolarized to the level of − 66 mV. However, a somewhat higher Atr concentration (5× 10 − 7 M) hyperpolarized RMP by  3 to − 69.3 mV (Fig. 3D). Further increases of the Atr concentrations to 1 and 5× 10 − 6 M again blocked the protection of RMP by CB and the fibres became depolarized. W-84, which stabilizes cholinergic agonist-receptor complexes by an allosteric effect (Mohr and Trankle, 1994), eliminated to a great extent the combined hyperpolarizing effect of Atr and CB (Fig. 3B,D, filled circles). When applied alone, Atr also hyperpolarized the RMP to − 68.9 mV (P B 0.001 versus control value), in the lowest concentration tested (1× 10 − 7 M; Fig. 3A). This hyperpolarization was not significantly (P \0.05) affected by W-84 (Fig. 3A, filled circle versus empty circles). The higher doses of Atr (1 and 5 ×10 − 6 M)

were without any substantial effect on the development of the early postdenervation depolarization (Fig. 3A, empty circles). To reveal the type of muscarinic receptor which is activated by CB to decrease the early postdenervation depolarization, the effects of several other muscarinic drugs on RMP were studied.

3.3. Pirenzepine Pirenzepine (Pir), a specific antagonist of M1 receptors, blocked the effect of Oxo on early postdenervation depolarization in a dose-dependent manner (Fig. 4). Pir also blocked the effects of CB. For example, 1×10 − 7 M Pir depolarized the RMP from − 72.2 mV in the presence of 1×10 − 8 M CB, to −65.3 mV (PB0.001; Table 1), when both drugs were applied together. The apparent inhibition constant Ki for Pir was 1×10 − 7 mM in the presence of 5 ×10 − 8 M CB. No concentration of Pir tested alone (1× 10 − 7, 1 × 10 − 6 and 5 × 10 − 6 M) influenced the development of early postdenervation depolarization (Fig. 4, empty triangles).

Fig. 3. Effect of several concentrations of atropine (in M: 0, 1× 10 − 7, 5×10 − 7, 1×10 − 6 and 5× 10 − 6) on the resting membrane potential (RMP) of 3-h denervated diaphragm fibres in the absence (A) and presence of 1 ×10 − 8 (B), 5 × 10 − 8 (C) and 1 ×10 − 7 M (D) carbachol (CB). Filled circles: 5 ×10 − 6 M hexamethylene-bis-[dimethyl-(3-phtalimidopropyl)ammonium] (W-84) present in the muscle bath together with atropine and carbachol for 3 h. Each point is the mean9 S.E.M. of 60 – 150 RMP values measured from three to five animals. Temperature: 20°C.

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3.5. 4 -DAMP 1,1-Dimethyl-4-diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors (Barlow and Shepherd, 1986) had no significant effect (PB0.05) on the CB-induced decrease of the postdenervation depolarization in concentrations of 1× 10 − 5 and 1 × 10 − 4 M. The mean RMPs in these experiments were −70.5 mV (PB 0.05) and −68.1 mV, respectively (Table 1, h,i). The pharmacological evidence therefore suggests that Oxo, CB and/or ACh protect the muscle membrane from early postdenervation depolarization through the M1 subtype of muscarinic cholinergic receptors. Fig. 4. Effect of different concentrations of pirenzepine on the resting membrane potential (RMP) of 3-h denervated diaphragm fibres in the absence (empty triangles) and presence of 1×10 − 8 M (filled triangles), 5× 10 − 8 M (open circles) and 1× 10 − 7 M (filled circles) oxotremorine. Each point is the mean 9S.E.M. of 50–150 RMP values measured from three to seven animals. Temperature: 20°C. Table 2 The effect of carbachol on the resting membrane potential (RMP, in mV) of muscles excised and incubated in vitro in the culture medium for 1, 2 and 3 daysa Days after excision

RMP in control RMP 1 h after perfusion medium with 1×10−8 M carbachol

1 day

−62.99 0.5 (100) −60.59 0.4 (100) −57.89 0.5 (100)

2 days 3 days

−65.69 0.4 (100) −64.690.6 (100) −64.690.5 (125)

a Results presented are the means 9S.E.M. The figures in parentheses indicate the number of fibres investigated from three to five diaphragms. Measurements were performed 23–25 h (1 day), 49–50 h (2 days) and 71–73 h (3 days) after muscle isolation. All data with carbachol significantly differ from respective controls (PB0.05).

3.4. Arecaidine esters The hyperpolarizing effect of the potent muscarinic agonist, arecaidine but-2-ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors (Barlow and Weston-Smith, 1985; Moser et al., 1989) on the denervated muscle membrane, was similar to the actions of CB and Oxo. The mean RMP in the fibres incubated with ABET (50 mM) was − 69.1 mV (P B 0.001; Table 1, f). However, the propargyle ester of arecaidine (APET), another muscarinic agonist with a slight preference for the M1 over the M2 receptor (Barlow and Weston-Smith, 1985; Moser et al., 1989), failed to decrease the depolarization of the denervated muscle membrane (Table 1, g).

3.6. Long-term cultured muscles To distinguish between the actions of CB on presynaptic nerve autoreceptors and/or on the postsynaptic muscle membrane, we used nerve-free muscles excised and incubated in vitro for 1, 2 or 3 days. It is known that the motor nerve terminals in these cultured muscles (‘in vitro denervated’) do not release any transmitter either quantal or non-quantal after 15–20 h (Zemkova´ et al., 1987; Nikolsky et al., 1996). The mean RMPs depolarized to −62.9, − 60.5, and − 57.8 mV on the 1st, 2nd, and 3rd day of incubation (Table 2). The membrane in these muscle fibres was significantly hyperpolarized by 5× 10 − 8 M CB (Table 2). It was not the result of an increase in the input resistance of the fibre membrane; the average input resistance of the muscle membrane incubated 24 h in the control medium was 0.74690.112 MV and it did not change (P\0.05) after 1-h exposure to 5× 10 − 8 M CB (0.7499 0.045 MV). Similar results have been obtained in muscle fibres denervated in vivo (data not given; for method see Bera´nek and Vyskocˇil, 1967). The denervation experiments show that CB can slow the development of denervation depolarization by direct action on the muscle membrane.

3.7. Effect of atropine on the time-course of muscle depolarization If muscarinic receptors are involved in the regulation of early postdenervation depolarization, then the incubation of in vitro denervated muscles with muscarinolytic drugs should facilitate the development of depolarization due to the protection of fibres from the action of ACh still being released for a period of several hours after the nerve section (Nikolsky et al., 1996). We incubated the muscle strips in the culture medium and measured the RMP every 30 min in the absence and presence of Atr. In control muscles, the RMP depolarized significantly by 2.5 mV after the first 30 min and this depolarization continued to increase for 3 h of

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muscle incubation. In the presence of both 1× 10 − 6 M Atr and 2×10 − 7 M Pir, the early postdenervation depolarization developed more rapidly (P B0.01; Table 3). This accelerating effect of Atr and Pir was highly significant (PB 0.001) during the first 30 and 60 min. After 3 h with either drug in the bath, the mean RMPs did not differ (P \ 0.05) from the values obtained in the control medium.

4. Discussion As we have shown recently, the CB action is not influenced by (+)tubocurarine or ouabain suggesting that neither nicotinic cholinergic receptors nor the sodium pump are involved in the prevention of early postdenervation depolarization by cholinergic agonists (Urazaev et al., 1997). On the other hand, the hyperpolarization is evidently mediated through muscarinic cholinergic receptors. In particular, the classical muscarinic antagonist atropine removed the CB-induced protection of RMP of denervated muscle membrane in a dose-dependent manner. In the presence of Atr, the early postdenervation depolarization developed more rapidly, probably due to elimination of the effect of exogenous ACh, which is still released during the first hours after denervation (Zemkova´ et al., 1987; Nikolsky et al., 1996). The greatest muscle membrane hyperpolarization occurred when CB was used in concentrations of 5× 10 − 8 and 1× 10 − 7 M (Fig. 1). CB appears to act similarly as ACh in this concentration range (Urazaev et al., 1997) and these concentrations are believed to be similar to the ACh concentration produced in the synaptic cleft by non-quantal release (Bray et al., 1982; Vyskocˇil et al., 1983; Nikolsky et al., 1994; Vyskocˇil et al., 1995). It is therefore reasonable to expect that the ‘trophic’ non-impulse control of the muscle fibre by motor nerve may be mediated by ACh. Using the dose-response curve for Atr-induced depolarization with 5×10 − 8 M CB (Fig. 2) the estimated Ki

for Atr was 7 × 10 − 8 M. At lower and higher concentrations of CB, the dose-response curve was interrupted by a hyperpolarization caused by 1× 10 − 8 M Atr (Fig. 3). Similar hyperpolarization was observed with Atr alone, but only at a lower concentration of 1×10 − 7 M. This observation could be ascribed to the known ability of some inhibitors to activate their particular receptors when used in low concentrations (for reviews see McDonald et al., 1994 and also Urazaev et al., 1998). Apparently, not only Atr alone, but also the presence of low doses of both CB and Atr may somehow interfere with the receptor and then have a hyperpolarizing effect. It is possible that Atr and CB, when applied together in such concentrations, cause a type of ‘unstable’ binding of Atr to its receptor which is then ineffective. When the allosteric binding of muscarinic antagonists to their receptor was stabilized by W-84 (Mohr and Trankle, 1994), this drug reversed the muscle fibre hyperpolarization to depolarization when applied together with Atr and CB. In other words, the inhibitory effect of 5×10 − 7 M Atr exists, but can be revealed only after stabilization of antagonist binding to the receptor. However, we have no explanation for the fact that W-84 was not able to protect the muscle membrane from the hyperpolarizing action of 1× 10 − 7 M Atr alone, when the RMP was hyperpolarized either with or without W-84. This might mean that 1×10 − 7 M Atr may activate the muscarinic receptor directly. There are at least five subtypes of muscarinic receptors. The M1, M3, and M5 subtypes can be classified as a group of receptors whose activation stimulates the phosphoinositide pathway, while M2 and M4 receptors initiate the adenylyl cyclase cascade (for review see Hosey, 1992). Recently, the expression of M1-muscarinic receptors coupled to phospholipase C and internal calcium stores has been convincingly demonstrated in cultured rat skeletal muscle by Reyes and Jaimovich (1996). They studied the influence of muscarinic and nicotinic stimulation on both phosphoinositide metabolism and intracellular calcium levels and found that the muscarinic effect was mimicked by ox-

Table 3 Effect of atropine and pirenzepine on the early postdenervation depolarizationa Period of incubation (min) 0 30 60 90 120 150 180

RMP in control medium

RMP in the medium containing 1 mM atropine

RMP in the medium containing 0.2 mM pirenzepine

−74.59 0.4 −72.19 0.4 −70.69 0.4 −69.19 0.3 −67.59 0.5 −66.89 0.3 −66.69 0.4

– −68.69 0.3 −68.39 0.4 −68.09 0.3 −65.990.3 −66.090.3 −66.590.3

– −68.59 0.4 −68.69 0.3 −68.29 0.4 −66.29 0.3 −65.99 0.4 −66.39 0.3

(75) (70) (70) (65) (39) (70) (100)

(42) (40) (40) (30) (40) (42)

(59) (49) (48) (45) (50) (50)

a Resting membrane potential (RMP) in mV. Results presented are the means9 S.E.M. The figures in parentheses represent the number of fibres investigated from three to five animals.

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otremorine (Oxo) and prevented by pirenzepine. Therefore, expression of M1-muscarinic receptors coupled to phospholipase C and to internal calcium stores in skeletal muscle was proposed. In agreement with this, we observed that pirenzepine, a specific antagonist of muscarinic M1 receptors, blocked the effect of CB and Oxo on RMP of incubated muscle fibres completely in a concentration of 5× 10 − 7 M. Thus, the effect of both agonists could be mediated through activation of M1 receptors. This idea was supported in particular by the experiments with Oxo, a highly specific muscarinic agonist, which has no nicotinic effects, in contrast to CB. Oxo hyperpolarized the denervated muscle membrane similarly to CB. It seemed to be even more potent than CB in several concentrations (Fig. 1) suggesting that muscle muscarinic receptors might be more sensitive to Oxo than to CB (Fig. 4). Arecaidine but-2-ynyl ester tosylate (ABET), a potent muscarinic agonist which is more selective to M2 receptors in the ileum versus M1 receptors in the atria (Barlow and Weston-Smith, 1985; Moser et al., 1989), had a significant hyperpolarizing effect on the denervated muscle membrane, similar to the actions of CB and Oxo. However, the propargyle ester of arecaidine (APET), another potent muscarinic agonist with a slight preference for the cardiac M1 over ileac M2 receptors (Barlow and Weston-Smith, 1985; Moser et al., 1989), failed to protect the denervated muscle membrane from depolarization. One can deduce that the protective action of cholinomimetics on the denervated muscle membrane might also be mediated at least partly by M2 receptors. On the other hand, the data obtained with Oxo and Pir, the known specific M1 binding compounds, strongly suggest that this particular subtype of muscarinic receptors is involved in the early postdenervation depolarization. The difference between the effects of ABET and APET also suggests that ABET probably acts as an M1 agonist in skeletal muscles rather than APET which apparently cannot bind to M1 muscle cholinergic receptors. In contrast to the drugs acting on M1 receptors, a specific antagonist of M3 cholinergic receptors, 4DAMP (Barlow and Shepherd, 1986), in concentrations of 1 ×10 − 5 and 1 × 10 − 4 M did not inhibit the protective action of 5× 10 − 8 M CB towards the early postdenervation depolarization. Thus, M3 receptors are not involved in this CB action. Our data therefore suggest that the hyperpolarizing action of cholinomimetics on the denervated muscle membrane is mediated through the M1 subtype of muscarinic cholinergic receptors whose pharmacological characteristics might, at the same time, be somewhat different from those of muscarinic receptors in smooth and cardiac muscles, as indicated by the ABET and APET effects. These receptors are considered to be localized in the skeletal muscle membrane; however, at

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the same time there exists the possibility that the muscarinic receptors which were activated by cholinergic agonists in our experiments and involved in the regulation of the muscle RMP, are localized in the membrane of presynaptic motor nerve terminals. There is evidence that muscarinic receptors are distributed not only in the skeletal muscle membrane (Magazanik and Vyskocˇil, 1969; Reyes and Jaimovich, 1996) but also in the motor nerve endings (Wessler 1989a,b; Vizi and Somogyi 1989; Nikolsky et al., 1991) where they may regulate both the quantal and non-quantal secretion of ACh (Zemkova´ et al., 1990; Nikolsky et al., 1994). These presynaptic receptors might be responsible for the trophic effects of ACh and could therefore be of physiological significance in maintaining the RMP in muscle fibres through modulation of non-quantal secretion of the ACh at the neuromuscular junction, as has already been suggested (Zemkova´ et al., 1990). We therefore used muscle fibres denervated for 1–3 days, where motor nerve terminals obviously release no transmitter, either quantally or non-quantally (Zemkova´ et al., 1987). After denervation, CB was still able to hyperpolarize the RMP even when the nerve terminal had degenerated. Denervation experiments therefore show that exogenous CB can slow the development of denervation depolarization directly, without stimulating the endogenous ACh release from the nerve terminals through muscarinic autoreceptors. The participation of nerve muscarinic autoreceptors also seems unlikely since the minimal effective concentration of CB (which can change the Ca2 + -dependent frequency of miniature end-plate potentials through presynaptic muscarinic receptors) was estimated to be rather high — 0.6 mM (Nikolsky et al., 1991). Apparently, this concentration cannot be attained non-quantally. It is also much higher than the doses of ACh and CB (5–10 × 10 − 8 M) which can maintain the RMP in denervated muscles through Ca2 + -regulated production of NO in our experiments (Urazaev et al., 1987a, 1997). Therefore, the hyperpolarizing effect of cholinomimetics on denervated muscles is mediated via postsynaptically localized M1-cholinergic receptors. We have recently proposed on the basis of our previous results (see also Grozdanovic and Baumgarten 1999 for review) that the resting non-quantal ACh (and probably glutanate as well) released from nerve endings might be a discrete trophic non-impulse message from motoneurones. It promotes Ca2 + entry into the sarcoplasm, Ca2 + -dependent activation of NO-synthase and production of NO (Urazaev et al., 1995; Urazaev et al., 1996; Urazaev et al., 1997; Urazaev et al., 1997), activation of the soluble guanylyl cyclase and cGMPdependent phosphorylation of Cl−-furosemide sensitive transporter, which remains inactive in the sarcolemma (Dulhunty, 1978; Urazaev et al., 1987a,b). After denervation, the secretion of transmitters disappears gradu-

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ally (Vyskocˇil et al., 1995) and the production of NO in the sarcoplasm is reduced. As a result, the Cl− influx and intracellular Cl− concentration increase. Because the equilibrium Cl− potential becomes more positive, the muscle membrane depolarizes.

Acknowledgements The authors thank Dr C. Edwards for extensive discussion and Dr P. Hnı´k for careful reading of the manuscript. This work was supported by grants VS 97099, EU ‘NESTING’, RFBR 98-04-48044, and grant A7011902 from the Grant Agency of the Czech Academy of Sciences.

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