Some characteristics of the muscularis mucosae of the cat lower esophageal sphincter

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Pergamon

0306-3623(94)E0011-A

Gen. Pharmac. Vol. 25, No. 4, pp. 639-643, 1994 Copyright © 1994 ElsevierScience Ltd Printed in Great Britain. All rights reserved 0306-3623/94 $7.00 + 0.00

Some Characteristics of the Muscularis Mucosae of the Cat Lower Esophageal Sphincter G . D O B R E V A * , Z. M I Z H O R K O V A , N. KORTEZOVA and M. PAPASOVA Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev St., bl.23, 1113 Sofia, Bulgaria [Fax (00359-2)71-91-091 (Received 3 November 1993)

Abstract--l.The contractile activity of muscularis

mucosae strips isolated from cat lower esophageal sphincter (LES) was studied. 2. LES muscularis mucosae strips were characterized by spontaneous phasic contractions. 3. Acetylcholine (ACh) at a threshold concentration of 10 -8 M dose-dependently increased the basal tone and decreased the amplitude of the phasic contractions. Noradrenaline (NA) also increased the basal tone but at a higher threshold concentration (10 -6 M). 4. Nicotine (N) at concentrations of 5 x 10-5-10 -4 M induced a relaxation of the LES muscularis mucosae. The N-induced relaxation was tetrodotoxin (TTX) (10 -6 M) sensitive and was insignificantly reduced after guanethidine (5 x 10-6 M) plus scopolamine (5 x 10-6 M) or propranolol (10 -6 M). The N-induced relaxation was strongly decreased by the blocker of nitric oxide (NO) synthesis N~'Nitro-L Arginine (L-NNA) (10 -4 M). The relaxation was restored after addition of L-arginine (10 -3 M). 5. These results suggest the involvement of NO in the N-induced relaxation of the muscularis mucosae. Key Words: Cat LES muscularis mucosae, acetylcholine, noradrenaline, nitric oxide

INTRODUCTION

gastrin and vasoactive intestinal peptide (VIP) are without effect (Christensen and Percy, 1984). The relaxation of the rat esophageal muscularis mucosae induced by electrical field stimulation (EFS) is sensitive to tetrodotoxin (TTX) (Akbarali et al., 1986), while the opossum esophageal muscularis mucosae responds to EFS with atropine-sensitive contractions (Domoto et aL, 1983). The biphasic response to EFS: contraction followed by relaxation is characteristic for the guinea-pig esophageal muscularis mucosae (Ohkawa, 1980). Angel et aL (1983) found that longitudinal strips from the canine gastric muscularis mucosae were spontaneously active and that acetylcholine (ACh) induced atropine-sensitive contractions, while adrenaline caused relaxation. Substance P produced contractions and VIP led to relaxation. The canine gastric muscularis mucosae relaxed in response to EFS which was accompanied by an increased amount of VIP in the effluent (measured radioimmunologically). All these findings and the specific responses of the cat lower esophageal sphincter (LES) to the classical

The muscularis mucosae is found throughout the length of the gastro-intestinal tract. The esophageal muscularis mucosae has a cholinergic and adrenergic innervation as the contraction is mediated via cholinergic and ct-adrenergic receptors and the relaxation via fl-adrenergic receptors (Ohkawa, 1980; Uchida et al., 1983). Many authors pointed out that the excitation of esophageal muscularis mucosae is realized mainly by cholinergic nerves, while nonadrenergic, noncholinergic innervation is not presented (Kamikawa and Shimo, 1979; Christensen and Percy, 1984; Percy, 1993). According to Kamikawa et al. (1982) the relaxation of the esophageal muscularis mucosae is mainly adrenergic. However, the responses of the esophageal muscularis mucosae to some peptides are different: substance P concentration-dependently increases the basal tone of the muscularis mucosae of cat, dog and opossum, while *To whom correspondence should be addressed. 639

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G. DOBREVAet al.

neurotransmitters ACh, noradrenaline (NA) and to some peptides, as well as the participation of nitric oxide (NO) in LES relaxation, stimulated our interest in studying the muscularis mucosae of the cat LES.

Data analysis

The means + SEM are presented. The data were assessed for statistical significance using Student's t-test for grouped data.

MATERIALS AND METHODS Methods

Male cats (2.5-3 kg) were anaesthetized with chloralose (100 mg/kg i.p.). The abdomen and the thorax were opened and the distal part of the esophagus together with the LES and the gastric fundus was removed cautiously without distension and placed in a Petri dish containing Krebs solution bubbled with 95% 02 and 5% CO2 at 37°C. The segment was opened along the line of the lesser curvature of the stomach and pinned out flat on the paraffin-covered bottom of the Petri dish. Two strips from the muscularis mucosae (2 x 20 mm) and two muscle strips from the outer layers of LES were cut along its circular axis. To be sure that the muscularis mucosae strips belonged to LES, we subjected the strips from the outer muscle layers to EFS (2 Hz, 0.2 ms, supramaximal current intensity, 20 sec duration). Only the muscularis mucosae strips belonging to these LES which responded to EFS with relaxation were used. It has been reported that for in vitro study of dog small intestinal muscularis mucosae the presence of mucosal epithelium on the outer aspect of the preparation causes it to deteriorate rapidly (King et al., 1947; Percy and Christensen, 1986). To avoid this, strips were stitched in the middle with two threads and folded the mucosal surfaces inward so that they were half their original length. The loop was mounted in a 10 ml organ bath with Krebs solution bubbled with 95% 02 and 5% CO2 at 37°C. One end of the loop was connected with a strain-gauge. The preparations were suspended under 1 g tension. There was a 60 min equilibration period before any measurements were made. During this period the nutrient solution was changed every 15 rain. Solutions and drugs

Modified Krebs solution containing (mM): NaCI 112.5; KCI 4.75; NaHCO3 25.00; KH2PO4 1.19; MgCI 2 1.2; CaCI2 2.4; glucose 11.5. Guanethidine (Sigma), ( - ) scopolamine (Sigma), nicotine hydrogen tartrate (Sigma), N'~-nitro-I.-arginine (I.-NNA) (Sigma), L-arginine hydrochloride (Sigma), acetylcholine chloride (Serva), L-noradrenaline-L-tartrate (Merck), phentolamine (Sigma), propranolol (Sigma), methionine-enkephalin (Serva), substance Pt Ji (Sigma), serotonin bimaleinate (Koch-Light Lab.), tetrodotoxin (Sigma).

RESULTS Strips of LES muscularis mucosae proved to be spontaneously active. Rhythmic, phasic contractions were observed in 78% of the strips (from 17 cats). The contraction frequency varied between 9 and 11 cpm. Acetylcholine (ACh) increased the tone and decreased the amplitude of the spontaneous phasic contractions. Occasionally, the increase of the tone led to a complete inhibition of the phasic contractions. The affect of ACh was concentration-dependent at a threshold concentration of 10 -8 M (Fig. 1). NA also increased the basal tone whereby the amplitude of the phasic contractions decreased. The increase of the tone was also dose-dependent, but at a higher threshold concentration of NA (10 -6 M). Neither propranolol (10-6M) nor phentolamine (10-6 M) affected the spontaneous activity of the LES muscularis mucosae, but the ~-adrenergic antagonist decreased the NA effect (Fig. 2). Substance P (10-12_ 10- 6 M), methionine-enkephaiin (10 -7 and 10 -6 M) and serotonin (10-7-10 -5 M) exerted no effect. EFS (2-50 Hz, 0.3 msec, 30 V, duration 15-60 sec) induced atropine-sensitive contractions in 4 out of 11 strips. Nicotine (N) at a threshold concentration of 5 x 10 -5 M relaxed the LES muscularis mucosae. The concentration of 10-4M was used throughout the experiments. No N-induced relaxation was observed in the presence of TTX (10 -6 M) suggesting its neurogenic nature. On the other hand, in strips pretreated for 90 min with guanethidine (5 x 10 -6 M) plus scopolamine (5 x 10 -6 M) the N-induced relaxation was only slightly reduced. This led us to believe that N A N C mechanisms are involved in the N-induced relaxation of the LES muscularis mucosae. Recent studies suggest the role of NO in the NANC-relaxation of the smooth muscles. Thus the strips were then treated with the blocker of NO synthesis L-NNA (10 -4 M) for 15 min and we found a great reduction of the N-induced relaxation (Fig. 3). The addition of L-arginine (10-aM) to the nutrient solution for 20 min restored at least partly the amplitude of the N-induced relaxation. The N-induced relaxation decreased after addition of the fl-adrenoceptor antagonist propranolol and remained decreased after addition of the fl-adrenoceptor antagonist phentolamine. However, the block-

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ade of N O synthesis by L-NNA significantly reduced the relaxation amplitude (from 1.73+0.28 to 0 . 1 2 + 0 . 0 5 m N ; P <0.01). The effect of N was restored in the L-arginine-pretreated strips (Fig. 4).

DISCUSSION The role of the muscularis mucosae in the different regions of the gastro-intestinal tract is still unclear.

The differences in the responses of esophageal, gastric, small and large intestinal muscularis mucosae to drugs and peptides suggest it to be a highly specialized tissue. The results of the present study indicate that the intrinsic innervation of the cat LES muscularis mucosae differs from that found by Christensen and Percy (1984) in the esophageal muscularis mucosae. According to Christensen and Percy (1984) the esophageal muscularis mucosae has a cholinergic

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innervation but no nonadrenergic, noncholinergic innervation. The LES muscularis mucosae is also characterized by cholinergic innervation. A C h increased the tone of the LES muscularis mucosae and this effect was antagonized by atropine. All strips of the LES muscularis mucosae responded to N A with contraction, while, also in experiments on cats, Christensen and Percy (1984) found that 40% of the esophageal strips did not respond to N A at all. These authors as well as Kamikawa and Shimo (1979) reported that the esophageal muscularis mucosae has no nonadrenergic, noncholinergic innervation and that the relaxation is mediated via fl-adrenergic receptors only.

The present study also showed a fl-adrenergic relaxation, though less pronounced, in the LES muscularis mucosae. The finding that the N-induced relaxation, sensitive to TTX, occurred in the presence of guanethidine and scopolamine suggests a nonadrenergic, noncholinergic innervation of the LES muscularis mucosae. The lack of effect of substance P and methionineenkephalin suggests that these peptides play no role in the LES muscularis mucosae. Recent data have indicated the participation of N O in the nonadrenergic, noncholinergic relaxation not only of the vascular muscles but also of those of the gastro-intestinal tract (for review see Rand, 1992; Sanders and Ward, 1992). There is also evidence for the participation of N O or nitrogen-releasing substances in the N A N C relaxation in the LES upon EFS (T~ttrup et al., 1991; De Man et al., 1991). In strips from cat LES, Kortezova et al. (1994) have found that N O is involved in N-induced relaxation, too. The fact that N-induced relaxation in the LES muscularis mucosae was antagonized by the blocker of N O synthesis L-NNA supports the suggestion about the involvement of N O in this relaxation. That only one-third of the LES muscularis mucosae strips responded to EFS could not be explained. Perhaps a large proportion of the neurons in these strips are mainly sensory in function. This argues in favour of the basic function of the LES, namely, to prevent the reflux of the gastric content or to relax allowing for its propulsion distally to the stomach.

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Fig. 4. Nicotine-induced relaxation of LES muscularis mucosae of cat Iq; the antagonistic effect of propranolol 10-6 M alone (15 min) I~; in the presence of propranolol and phentolamine 10-6 M (15 min) []; in the presence of propranolol and phentolamine and L-NNA 10 4 M (15 min) II. The N-induced relaxation was restored after addition of L-arginine 10-3 M (20 min pretreatment with L-arginine) I~. Values are means ___SEM of 5 experiments (**Significance of difference at P < 0.01; n.s. = no significance of difference).

Muscularis mucosae of cat LES Acknowledgement--This study was supported by Grant B-11 from the Ministry of Education and Sciences, Bulgaria.

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