Journal of the Autonomic Nervous System, 40 (1992) 57-62 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00

57

JANS 01299

Alpha-adrenoreceptor modulation of neurally evoked circular muscle responses of the guinea pig stomach K. H i l l s l e y ~, M . S c h e m a n n b a n d D . G r u n d y

a

a Department of Biomedical Science, The University of Sheffield, Sheffield, UK, and b Institute of Zoophysiology, University of Hohenheim, Stuttgart, FRG (Received 12 February 1992) (Revision received 21 April 1992) (Accepted 25 April 1992)

Key words: A l p h a - a d r e n o r e c e p t o r ; Stomach; T r a n s m u r a l s t i m u l a t i o n Abstract The effects of alpha-adrenergic agonists on transmural-evoked motor responses were investigated in guinea pig gastric corpus in vitro, using preparations stripped of mucosa and orientated to record changes in circular muscle tension. Three tetrodotoxin-sensi-

tive components to a 10 s burst of transmural stimulation could be distinguished: an initial 'on' contraction, an 'off' contraction and a transient relaxation. The 'on' response was blocked by atropine (0.1/zM), while the 'off' response and relaxation were unaffected at this dose. A submaximal dose of acetylcholine was used to assess the sensitivity of the preparation. The alpha I agonist L-phenylephrine decreased the amplitude of the 'off' response while simultaneously increasing both the 'on' response and the relaxation, although the response to acetylcholine was unchanged. These effects were dose-dependent and reversed by pretreatment with prazosin. In marked contrast, the alpha 2 agonist clonidine inhibited the 'on' response in a dose-dependent manner without affecting the 'off' response, the relaxation or the response to acetylcholine. Yohimbine reversed the effect of clonidine. We conclude that the inhibitory action of alpha-agonists involves both cholinergic and non-cholinergic pathways, with alpha I and alpha 2 adrenoreceptors modulating different circuits within the enteric nervous system.

Introduction G a s t r i c m o t o r f u n c t i o n is u n d e r the i n f l u e n c e of b o t h intrinsic a n d extrinsic n e u r a l controls. Local control is achieved by the e n t e r i c n e r v o u s system which c a n o p e r a t e i n d e p e n d e n t l y of the c e n t r a l n e r v o u s system [16]. However, extrinsic i n p u t s from p a r a s y m p a t h e t i c nerves a n d sympathetic nerves have a m o d u l a t i n g i n f l u e n c e over these local controls. N o r a d r e n a l i n e , r e l e a s e d from

Correspondence to: D. Grundy, Department of Biomedical Science, The University of Sheffield, Sheffield S10 2TN, UK.

sympathetic nerve endings, has a p r e d o m i n a n t l y inhibitory effect o n gastric m o t o r f u n c t i o n by a n action m a i n l y at the level of the m y e n t e r i c plexus. Thus, few sympathetic nerve t e r m i n a l s i n n e r v a t e the muscle layers directly [3,8] a n d f u n c t i o n a l studies suggest that sympathetic i n h i b i t i o n of gastric t o n e a n d motility is m e d i a t e d by i n h i b i t i o n of cholinergic pathways [1,6], a l t h o u g h a direct action o n gastric muscle c a n n o t be r u l e d out [2]. I n the small i n t e s t i n e it has b e e n shown that n o r a d r e n a l i n e acts via a l p h a 2 receptors, both pre-synaptically to inhibit cholinergic transmission t h r o u g h the e n t e r i c n e r v o u s system, a n d p o s t - s y n p a t i c a l l y to g e n e r a t e I P S P s [ 5 , 1 0 -

58 12,14,17]. Thus, noradrenaline acts to suppress transmission through the enteric network in the small intestine. Recently, it has been demonstrated that the sympathetic innervation of the stomach is functionally distinct from that in the small intestine. A pre-synaptic action of noradrenaline on cholinergic transmission mediated via alpha 2 receptors was similar to that observed in the small intestine. However, a post-synpatic depolarisation mimicked by L-phenylephrine and blocked by prazosin was observed in 40% of gastric myenteric neurones [14]. In the stomach it appears, therefore, that alpha l and alpha 2 adrenoreceptors have markedly different effects on transmission through enteric circuits. Alpha 2 adrenoreceptors act on cholinergic transmission while alpha~ adrenoreceptors activate a subpopulation of myenteric neurones, whose function in terms of effector response is not known. The aim of the present study was therefore to characterise the effects of alpha-adrenergic agonists on transmural evoked motor responses of the guinea pig stomach.

Materials and Methods

Technique Albino guinea pigs (Duncan-Hartley strain, female, 500-600 g), which had been allowed free access to food and water, were stunned and Ned. Following incision of the abdomen, the whole stomach was removed and opened by cutting in a longitudinal direction along the lesser curvature. After washing the contents of the stomach out with cold Krebs solution, the corpus was dissected out and pinned under cold Krebs solution to a petri dish filled with Sylgard 184 Elastomer. The tissue was stretched to its approximate in situ size, serosal side down, and the mucosal layer removed. Circular muscle strips (15-20 mm long and 10 mm wide) were isolated, together with the longitudinal layer, and positioned in a Palmer 50 ml organ bath containing Krebs solution gassed with 95% oxygen, 5% carbon dioxide at 37°C. The composition of the Krebs solution was as follows: sodium chloride 125 mM, potassium chloride 4.5 mM, sodium dihydrogen phosphate 1

mM, calcium chloride 2.5 raM, magnesium sulphate 1 mM, glucose 5.6 raM, sodium bicarbonate 25 mM. Both ends of the tissue strips were connected by surgical ligatures, one end was fixed at the bottom of the chamber and the other end to a force transducer (Ormed Engineering- UF1) for continuous recording of isometric tension. The strips were placed at a resting tension of 1 g and allowed to equilibrate with the experimental environment for 40 mins. Two platinum electrodes (0.5 mm diameter) were placed on either side of the tissue, and transmural electrical stimulation was applied from a Digitimer S100 stimulator, triggered by a Digitimer pulse generator. Stimulation parameters were set at 1 ms pulses with 70 V intensity at 10 Hz for 10 s, which produced reproducible responses. Contractions to acetylcholine were matched with those to electrical stimulation.

Protocol The following drugs were used: acetylcholine, atropine, L-phenylephrine, clonidine, yohimbine and tetrodotoxin (Sigma) and prazosin (Pfizer). Prazosin and yohimbine were dissolved in 70% ethanol to a stock solution of 10 mM; all other drugs were dissolved in distilled water. Before any drugs were added, control responses to transmural electrical stimulation and acetylcholine (matched by varying concentrations of acetylcholine between 10 nM and 10/zM, but typically 0.10 ~ M or 0.5/~M) were elicited. Statistics The mean and S.E.M. are shown in all figures and tables, with n = number of muscle strips. Using paired t-tests the data was compared between the controls and the relevant antagonists a n d / o r agonists. For all tests a P value < 0.05 was considered as statistically significant.

Results

Three tetrodotoxin-sensitive components to a 10 s burst of transmural electrical stimulation could be distinguished, although the relative mag-

59 1 rain

and blocked by atropine (0.1 tzM), while the 'off' contraction and relaxation were unaffected at this dose. The agonists and antagonists used in this study have been shown to differentiate between alpha 1 and alpha 2 responses in gastric myenteric neurones.

19

Effect of L-phenylephrine

Fig. 1. This representative trace shows a typical response to transmural stimulation. T h e dot marks a 10 s period of transmural stimulation. The initial response to the stimulation, 1, is the 'on' contraction. The 'off' response, 2, follows, either directly, or with an intermittent relaxation as shown. The relaxation, 3, is the decline in the tone below the baseline, and was quantified as the m a x i m u m fall, whether this occurred prior to or following the 'off' contraction.

nitude of each was variable, and not all were present in every preparation, hence the variable n values quoted in the text and tables. A representative response to transmural stimulation is shown in Fig. 1. An 'on' contraction immediately followed the onset of stimulation and was usually maintained for the duration of the stimulation period. The m e a n amplitude of the 'on' contraction was 1.36 + 0.2 g (n = 44). On cessation of stimulation the tension fell rapidly to a level below the pre-stimulation baseline upon which was superimposed one or more phasic 'off' contractions. The mean maximum amplitude of the 'off' contractions was 1.99 + 0.29 g (n = 26) and was comparable to the 'on' contraction. Following the 'off' contraction there was a transient relaxation. The maximum relaxation seen in response to a single stimulus, irrespective of whether it was prior to or after the 'off' response, was recorded; the m e a n maximum amplitude of the transient relaxation was 0.88 + 0.12 g (n = 44). The 'on' response was cholinergically mediated

L-phenylephrine gave a dose-dependent relaxation which lasted for 20-30 s before returning to baseline. The maximum relaxation seen with the highest dose of phenylephrine (5/zM) was around 1 g. This effect was tetrodotoxin-resistant. A desensitisation of the relaxatory response to L-phenylephrine was observed which was quickly reversed upon washout. In addition to this direct effect of the alpha 1 agonist on muscle tone, Lphenylephrine also modified the response to transmural stimulation (Fig. 2) at a time when muscle tone had been re-established. The most prominent effect of L-phenylephrine at 5 / ~ M was the inhibition of the 'off' contraction (Fig. 3). In contrast, both the 'on' contraction and the relaxation were potentiated at a time when the response to exogenous acetylcholine was unchanged. These effects were dose-dependent (Fig. 3) and reversed by pre-treatment with prazosin (1 /~M), which alone had no significant effect on any I-'-1 Phenylephrine 22Z Phenylephdne x. Prazosin (1)JM)

120-

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-80Fig. 2. The effect of L-phenylephrine (5/zM) on the responses to transmural stimulation. Application of L-phenylephrine significantly potentiated the 'on' response ( P < 0.02), the relaxation ( P < 0.005), and significantly inhibited the ' o f f response ( P < 0.005). Pretreatment with prazosin significantly reversed the effects on the 'off' response and relaxation ( P < 0.01): prazosin also reduced the potentiation of the 'on' response, although this was not significant.

60 TABLE I The effects of 5 tx M L-phenylephrine alone and in the presence of 1 # M prazosin, on the magnitude (g + S.E.) of the three components of the response to transmural stimulation. The significance values shown relate to the relative controls, indicating L-phenylephrine's effects were blocked by prazosin

On

Off

Relaxation

Control

1.62 + 0.33 (n = 151

2.56 _+0.47 (n = 121

1.24 ± 0.19 (n = 161

i.-phenylephrine

2.35 ± 0.4 P < 0.02

1.26 + 0.41 P < 0.005

t.72 ± (/.26 P < 0.005

Control

1.06 ± 0.45 (n = 8)

3.42 _+0.82 (n = 8)

0.9 ± 0.16 (n = 12)

~.-phenylephrine and prazosin

1.39 ± 0.65 NS

3.71 + 0.9 NS

0.81 ± 0.13 NS

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Fig. 3. Application of L-phenylephrine caused a significant dose-dependent inhibition of the 'off' response, which was significantly reversed by prazosin.

Fig. 4. Application of clonidine significantly inhibited the 'on' response (P < 0.002), whilst having no effect on either the "off' response or the relaxation.

q u i t e v a r i a b l e . In five o f 15 p r e p a r a t i o n s , 1 / z M c a u s e d a l m o s t 1 0 0 % i n h i b i t i o n w h i l e in t h e rem a i n d e r t h e e f f e c t was c o n s i d e r a b l y less a n d d o s e s

c o m p o n e n t o f t h e r e s p o n s e to t r a n s m u r a l s t i m u lation. T h e e f f e c t s o f L - p h e n y l e p h r i n e a n d p r a zosin on transmural stimulation are summarised in T a b l e I. Effect of clonidine A l o n e , t h e a l p h a 2 a g o n i s t c l o n i d i n e slightly increased the basal tone of the preparation. C l o n i d i n e also h a d a n e f f e c t o n t h e m o t o r response to t r a n s m u r a l s t i m u l a t i o n (Fig. 4), m a r k e d l y d i f f e r e n t to t h a t p r o d u c e d by L-phenyle p h r i n e . A t d o s e s o f 0 . 0 1 - 1 / z M c l o n i d i n e h a d no e f f e c t o n e i t h e r t h e ' o f f ' c o n t r a c t i o n o r t h e relaxation but dose-dependently inhibited the magnit u d e o f t h e ' o n ' c o n t r a c t i o n (Fig. 5) w i t h n o e f f e c t o n t h e r e s p o n s e to e x o g e n o u s a c e t y l c h o l i n e . T h e e f f e c t o f c l o n i d i n e o n i n d i v i d u a l p r e p a r a t i o n s was

• - - ~ Clordalne 0--0 Cloflldlneand Yohimblnll(1)JM) p

Alpha-adrenoreceptor modulation of neurally evoked circular muscle responses of the guinea pig stomach.

The effects of alpha-adrenergic agonists on transmural-evoked motor responses were investigated in guinea pig gastric corpus in vitro, using preparati...
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