Purinergic modulation of spontaneous activity and of responses to high potassium and acetylcholine in rat ileal smooth muscle

0742-8413/93 S6.00 + 0.00 0 1993 Pergamon Press Lid

Camp. Eiochem. Physiol.Vol. 106C, No. 1, pp. 79-85, 1993 Printed in Great Britain

PURINERGIC MODULATION OF SPONTANEOUS ACTIVITY AND OF RESPONSES TO HIGH POTASSIUM AND ACETYLCHOLINE IN RAT ILEAL SMOOTH MUSCLE S. M. MAHMOD* and H. HUDDART~ Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, U.K. (Tel. 0524 65201; Fax 0524 843854) (Received 10 February 1993; accepted for publication 30 April 1993)

Abstract-l. In rat ileal smooth muscle both adenosine and ATP at 10V4M significantly enhanced spontaneous mechanical activity. The excitatory actions of adenosine were blocked by the P, receptor antagonist 8-phenyltheophylline and the excitatory effects of ATP were significantly reduced by the Pr receptor antagonist quinidine. 2. The P, receptor desensitizer a,/?-methylene-ATP was without effect on ACh responses nor did the stable analogue B,y-methylene-ATP exert any effect on spontaneous mechanical activity. 3. Pretreatment with adenosine caused a dose-dependent enhancement of K-induced contractures in the ileum. Low adenosine concentrations slightly inhibited and high concentrations slightly enhanced ACh-induced contractures in the ileum. 4. ATP potentiated the phasic component of the ileal K-induced contractme but strongly inhibited tonic force at high concentrations. This agent slightly inhibited the phasic component of the ACh-induced contracture while strongly inhibiting ACh-induced tonic force. 5. a,/?-methylene-ATP inhibited ileal muscle ACh induced contractures while it potentiated both phasic and tonic K-induced contractures. /?,y-methylene ATP inhibited ACh-induced contractures but it enhanced K-induced phasic contractures while inhibiting K-induced tonic force. 6. The results of this study suggest that rat ileum may contain the A, subtype of the P, receptor but the evidence for a PZ receptor subtype is conflicting despite the inhibition of ATP actions by quinidine. 7. The inhibition of K- and ACh-induced tonic force suggests that adenosine and ATP interactions with ileal smooth muscle may inactivate slow voltage-dependent calcium channels leading to EC uncoupling.

responding to adenosine and a P, type responding to ATP. Numerous subtypes of these broad classifications such as A, and AZ type of P, receptor and the Pz,, P,,, Ph and P, have now been delineated (see Williams and Cusack, 1990). The rat ileum is strongly responsive to adenosine compounds and this study was undertaken in an attempt to unravel the type of possible purinoreceptor which may be present in the ileal longitudinal smooth muscle.

INTRODUCTION Ptttine compounds have been known for many years to have potent actions on excitable tissues (Drury and Szent-Gyorgi, 1929; Green and Stoner, 1950). These actions are not confined to mammals since purinergic

modulation of flounder heart and gut has recently been demonstrated (Lennard and Huddart, 1989a, b) and adenosine compounds and purinoreceptors have been found in a number of invertebrates (Hoyle and Greenberg, 1988; Hoyle et al., 1989; Knight et al., 1992a, b). The mounting evidence of non-cholinergic, nonadrenergic transmission in the gastrointestinal tract, the urinary bladder and the vascular system led to the formulation of the concept of purinergic transmission (Bumstock, 1975, 1981) and this has now been extended to mammalian vas deferens (Stone, 1981; Sneddon and Bumstock, 1984; Lynch and Huddart, 1991). The identification of membrane receptors to adenosine compounds (Stone, 1985) showed a P, type

MATERIALS

Male Wistar strain albino rats (200-300 g) were used throughout this study. Animals were killed by a blow to the head followed by cervical dislocation. The ileum was dissected out and placed in Krebs saline containing (in mM) NaCl: 120.7, KCl: 5.9, CaCl,: 2.5, MgCl,: 1.2, NaH,PO,: 1.2, NaHCO,: 15.5 and glucose: 11.5. The saline was adjusted to pH 7.3, aerated and maintained at 37°C. Preparations were ligated with monofilament nylon and suspended in jacketed organ baths maintained at 37”C, between the bath hook and Grass PT.03 force displacement transducers connected to a 4 channel Grass 79D

*Present address: Biology Department, College of Education II, IBn Al-Haitham, Baghdad University, Aadhamiya, Baghdad, Iraq. tTo whom correspondence should be addressed. cap(C)

106,1--F

AND METHODS

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S. M. MAHMOD

80

and H.

Adenosine

Ig lmin

I I

Fig. I. The effect of (a) 10m4M adenosine and (b) 10m4M ATP on spontaneous mechanical activity of rat ileal longitudinal smooth muscle. Tension and time scales apply to both traces.

Polygraph to display tension generated by the ileal longitudinal smooth muscle. RESULTS

Effects on spontaneous activity

Both adenosine and ATP, at a concentration of 10m4M, were found to induce a significant enhance-

HUDDART

ment of spontaneous mechanical activity of rat ileal longitudinal smooth muscle (Fig. 1). In an attempt to delineate the type of receptor that may be involved in these actions we examined the effect of 8-phenyltheophylline and quinidine. 8-phenyltheophylline has been shown to be a potent antagonist of the A, subtype of the P, receptor (Burns, 1980; Griffith et af., 1981) and while it was without effect on ATP-induced activity in the ileum, at lo-’ M it strongly inhibited the excitatory actions of adenosine on ileal smooth muscle (Fig. 2b). The lack of a precise antagonist for the P, type of receptor responding to ATP has raised some difficulty, but recently quinidine has been shown to possess antagonistic actions on ATP responses (Shuba and Vladimirova, 1980; Burnstock and Kennedy, 1985). Quinidine, at 10e4 M, was found to have a marked effect on the excitatory actions of ATP, reducing this by about 50% (Fig. 2d). It has been reported that the stable nonhydrolysable ATP analogue, x,/I-methylene-ATP, will desensitize P, type purinoceptors in urinary bladder smooth muscle (Karakasov and Burnstock, 1982) in vas deferens (Meldrum and Burnstock, 1983) and in flounder gut (Lennard and Huddart, 1989b). In rat ileum, ATP induced a contracture at 10m3M, but after three applications of 10e4 M o$methylene-ATP at 5min intervals, the ileal muscle response to ATP was largely unaffected (Fig. 3). Furthermore, the effect of another ATP derivative

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Fig. 2. (a) Control response of ileum to 10m4M adenosine, (b) inhibition by a 4 min pretreatment with low5 M 8-phenyltheophylline (8-Pt) of the response to 10m4M adenosine. (c) Control response of ileum to 10m4M ATP, (d) inhibition of this response by a 3 min pretreatment with 10m4M quinidine. Calibrations in (a) also apply to (b), calibrations in (c) apply to (d).

Purine actions on rat ileum

(c)

81

I$ I(-methylene ATP ‘I

Ig1 min Fig. 3. (a) Control ileal muscle contracture response to 10-j M ATP, (b) response to IO-’ M ATP after three 5 min applications of 10e4 M a$-methylene ATP. Note the lack of inhibition of the ATP response. (c) Lack of effect of 10e4M /?,y-methylene ATP on spontaneous activity of ileal smooth muscle. Calibrations apply to all traces.

with P, receptor activity, fi,y-methylene-ATP was examined and was found to be without activity on ileal smooth muscle, even at 10e4 M (Fig. 3). Efects on agonist-induced responses

If adenosine and ATP are effective neuromodulators of excitation-contraction coupling in the ileum they may be expected to modify agonist-induced responses. To investigate this possibility, their effects on K-induced and ACh-induced contractures were examined. Salines with 80 mM K and 5 x 10e5 M ACh gave strong but sub-maximal contractures and these agents were used as a screen for adenosine and ATP actions.

80mMK I

Preparations were pretreated with adenosine for 3 min before agonist exposure. Adenosine was found to induce a dose-dependent enhancement of subsequent K-induced contractures (Fig. 4). With ACh contractures the results were more enigmatic since low adenosine concentrations caused a slight inhibition while higher concentrations caused a slight enhancement (Fig. 4). The overall effects are shown graphically in Fig. 5. ATP was seen to potentiate the phasic component of K-induced contractures but it had a remarkably inhibitory effect on the tonic component at high concentrations (Fig. 6). With ACh responses, ATP slightly inhibited the phasic component but strongly

80mMK 1

80mMK I

lmin ACh (e)

ACh 1

ACh (f)

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Jlmin Fig. 4. The effect of a 3 min pretreatment with adenosine on the 80 mM K- and 5 x 10e5 M ACh contractures of ileal smooth muscle. (a) Control K-contracture, (b) K-contracture with lo-* M adenosine, (c) K-contracture with IO-’ M adenosine. (d) Control ACh contracture, (e) ACh contracture with lo-’ M adenosine, (f) ACh contracture with IO-‘M adenosine. Calibrations in (a) also apply to (b) and (c), calibrations in (d) also apply to (e) and ( f).

S. M. MAHMODand H. HUDDART

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Fig. 7. The effect of ATP on peak 80mM K-contracture tension (filled squares) and peak 5 x 10m5M ACh contracture tension (filled circles) of rat ileum. Each point represents the mean + S.E. (N = 8).

Fig. 5. The effect of adenosine concentration upon peak height of 80mM Kcontractures (filled squares) and ACh contractures (filled circles) of ileal smooth muscle. Each point represents the mean f S. E. (N = 8).

both phasic and tonic components of K-induced contractures (Fig. 8). The derivative /?,y-methyleneATP inhibited both components of ACh contractures but with K-induced responses it enhanced the phasic component while inhibiting the tonic

inhibited the tonic component (Fig. 6). These overall effects are shown graphically in Fig. 7. The ATP derivative a,/?-methylene-ATP was found to inhibit both phasic and tonic components of ACh contractures but in contrast it potentiated

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Fig. 6. The effect of ATP on phasic and tonic components of 80 mM K- and 5 x IO-’ M ACh contractures of ileum. (a) Control 80 mM K-contracture, (b) Contracture with 10e6 M ATP, (c) contracture with 10msM ATP. (d) Control 5 x IO-’ M ACh contracture, (e) ACh contracture with lo-’ M ATP, ( f) ACh contracture with 10e5 M ATP. Calibrations apply to all traces.

83

Purine actions on rat ileum

(a) ACh

(b)

ACh (c) ACb

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(d)

80mM K

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(f) 8OmMK 1

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Fig. 8. The effect of a,j3-methylene ATP upon 5 x 10m5M ACh and 80 mM K-contractures of ileal smooth muscle. (a) Control ACh response., (b) ACh response with lo-’ M a,/?-methylene ATP, (c) ACh response with 10m6M a,@-methylene ATP. (d) Control 80 mM Kcontracture, (e) Kcontracture with lo-’ M a$-methylene ATP, ( f) K-contracture with 10e6 M a,/?-methylene ATP. Calibration in (a) applies also to (b) and (c), calibration in (d) also applies to (e) and (f).

component. Fig. 9.

These overall actions are summarized in

DISCUSSION

The results here show that both adenosine and ATP have excitatory effects on normal spontaneous (4

220 -

activity of the ileum. Excitatory actions are seen at fairly high concentrations (10e4 M), and could possibly be mediated unspecifically such as complexing with magnesium at the plasma membrane and favouring calcium entry (see Daniel and Irwin, 1965). However non-cholinergic, non-adrenergic excitatory nerves have heen demonstrated in the supply to the

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Fig. 9. (a) The effect of a,/?-methylene ATP upon 80 mM K responses (filled squares) and 5 x 10e5 M ACh responses (filled circles) of rat ileal smooth muscle. (b) The effect of B-y methylene ATP upon 80 mM K phasic contractures (filled squares) and 5 x 10e5 M ACh phasic contractures (filled circles). Each point is the mean f SE. (N = 8).

84

S. M. MAHMODand H. HIJDDART

small intestine of rabbit (Day and Warren, 1968), guinea-pig (Fumess, 1971) and cat (Day and Warren, 1968). To assess whether the ileal responses to adenosine and ATP were indirect or mediated via purinoceptors we used 8-phenyltheophylline which is

more potent than theophylline itself as an antagonist of P, receptors (Smellie et al., 1979; Burns, 1980). The antagonistic effect of this agent on the adenosine excitation of the rat ileal preparations strongly suggests the presence of P, type purinoceptors in this muscle. Whether a P, type receptor is present in the ileum is more problematic. Neither of the non-hydrolysable analogues of ATP had any effect on the contractions induced by ATP or on natural spontaneous activity. It is possible that any PZ receptors present in this muscle may simply not be specifically antagonized by these agents. Alternatively it is necessary to consider the interrelationship between ATP itself and adenosine. Adenosine, both in vivo and in vitro can be formed by hydrolysis of ATP and ATP can be formed from adenosine by energy-dependent phosphorylation. This means that some of the actions ascribed to in vitro application of ATP may actually be mediated by the P, receptor after breakdown to adenosine, making it difficult to separate the two possible actions in a tissue like ileal smooth muscle where both adenosine and ATP possess excitatory actions. Despite the lack of action of P, acting analogues of ATP on ileal smooth muscle, quinidine was found to reduce the excitatory effect of ATP. While quinidine is not an exceptionally specific competitive antagonist of the P2 receptor it does antagonize ATP actions in many tissues and is as good a P, antagonist as anything available so far. Its actions in ileum do point to the probability that ileal smooth muscle may contain a population of P, receptors. Caution needs to be exercised here since in one isolated case it has been reported that quinidine may antagonize the action of adenosine in isolated guinea-pig and cat uterine smooth muscle (Arulappu, 1967). The actions of adenosine and ATP on K- and ACh-induced contractures were examined to see if these agents modified the excitation-contraction coupling mechanism. Both adenosine and ATP potentiated the phasic component of the K-induced contracture and both strongly inhibited the tonic components of these contractures. While adenosine actions on ACh contractures were rather variable, ATP strongly inhibited ACh-induced tonic force. The most important underlying feature of both compounds is the strong inhibition of tonic force. In smooth muscle, tonic force is generated by the entry of calcium through slow voltage dependent noninactivating calcium channels (see Sperelakis, 1984; Langton and Huddart, 1988). The membrane depolarization induced by high K salines and the secondary phosphorylation of such calcium channels by ACh action opens such channels to permit calcium

entry, the essential link in excitation-contraction coupling generating sustained tonic force. The results here seem to suggest that adenosine and ATP interactions with ileal muscle receptors may lead to inactivation of these slow voltage-dependent calcium channels leading to excitation-contraction uncoupling and inhibition of tonic force. Acknowledgement-This study was carried out while Dr Mahmod was in receipt of a postgraduate research fellowship from the Ministry of Education of the Republic of Iraq. REFERENCES Arulappu R. G. S. (1967) The action of nucleic acid derivatives on smooth muscle. Fellowship thesis, Pharmaceutical Society of Victoria, Melbourne. Burns R. F. (1980) Adenosine receptor activation in human fibroblast: nucleoside agonists and antagonists. Can. J. Physiol.

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Stone T. W. (1981) The effect of 4-aminopyrridine on isolated vas deferens and its effect on inhibitory properties of adenosine, morphine, noradrenaline and I-aminobutyric acid. Br. J. Pharmac. 73, 791-796. Stone T. W. (1985) Classification of adenosine receptors in the nervous system. In Methodr in Pharmacology (Edited by Paton D. M.). Plenum, New York. Williams M. and Cusack N. J. (1990) Neuromodulatory roles of purine nucleosides and nucleotides: their receptors and ligands. Neurotransmissions 6, l-6.