Investigation of the role of adrenergic and non-nitrergic, non-adrenergic neurotransmission in the sheep isolated internal anal sphincter

T. Eames1, A. Acheson1, S. Rayment2, J. Scholefield1 and V. G. Wilson2 Centre for Integrated Systems Biology and Medicine, Department of Surgery1 & School of Biomedical Sciences2, The University of Nottingham Medical School, Queen’s Medical Centre, Clifton Boulevard, Nottingham, NG7 2UH, UK.

Nitric oxide is the principal neurotransmitter involved in the control of anal sphincter tone in a number of species, including sheep (Mundey et al., 2000) and man (Burleigh, 1992) but the possibility of a second inhibitory transmitter has been suggested. The role of noradrenaline in the control of sphincter tone has not been well characterised, although oral administration of α-adrenoceptor antagonists reduces anal sphincter pressure in man (Pitt et al., 2001). We have investigated the contribution of adrenergic transmitters to neurogenic relaxations, and evaluated the possible role of a second inhibitory non-nitrergic, non-adrenergic transmitter in the sheep internal anal sphincter.

The magnitude and duration of neurogenic responses were examined by measuring responses to electrical field stimulation (10Hz or 30Hz; 30 seconds) in segments of sheep internal anal sphincter that had developed spontaneous myogenic tone under tension. The effect of inhibitors and antagonists on these responses was assessed in a minimum of 6 preparations from different animals.

Electrical field stimulation (30Hz) induced transient neurogenic relaxations that, in approx. 70% of preparations, were coupled to a contraction. Bretylium (adrenergic neurone blocker; 30μM) significantly increased myogenic relaxations at 30Hz (2.66 ± 0.43g, mean ± standard error of mean, n=10) compared to control tissues (2.00 ± 0.49g, n=10; p<0.05, paired Student t-test). Similarly, responses in the presence of prazosin (α 1-adrenoceptor antagonist, 0.1μM) also showed significantly greater relaxations at 30Hz (1.59 ± 0.28g, n=8) than in control tissues (1.07 ± 0.39g, n=8; p<0.05, paired Student t-test) as well as abolishing neurogenic contractions. RX8211059 (α 2-adrenoceptor antagonist, 0.1μM) also significantly affected the response (-0.17 ± 1.15 g wt) vs control ( -1.79 ± 0.26 g wt, n=8; p<0.05, paired Student t-test). In the presence of both L-NAME (nitric oxide synthase inhibitor, 100μM) and bretylium, approximately 60% of preparations stimulated at 10Hz for 30 seconds produced neurogenic relaxations >0.5g, which were abolished by the addition of apamin (potassium channel blocker, 0.1μM). PPADS and suramin (non-selective P2 receptor antagonists, 10μM and 100μM respectively), α.β-methylene ATP (used to desensitise P2X receptors, 10μM) and vasoactive intestinal polypeptide (VIP; 0.3μM) failed to affect this response, although MRS2179 (P2Y1 selective antagonist, 10μM) significantly reduced the relaxations observed from 1.21 ± 0.25g to 0.78 ± 0.14g (n=14; p<0.05, paired Student t-test).

Taken together, these results suggest that noradrenaline has a significant role in high frequency neurogenic responses in sheep internal anal sphincter. These responses are likely to be mediated through α1- adrenoceptors since responses in the presence of prazosin were similar to those observed with bretylium. There was also evidence for a non-nitrergic, non-adrenergic apamin-sensitive relaxation, which was partially inhibited by MRS2179, suggesting a contribution of adenine nucleotides in this response.

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Pitt J. et al., (2001) Colorectal Disease 3:165-168