Characterisation of α1- and α2-adrenoceptor subtypes in isolated sheep internal anal sphincter

Sarah Rayment1, Michael Dashwood2, Thomas Eames1, Yvette Henry3, Hans-Jurgen Gruss3, Austin Acheson1, John Scholefield1, Vincent Wilson1

1University of Nottingham, Nottingham, UK, 2Royal Free Hospital and University College medical school, London, UK, 3Norgine International Ltd, Harefield, UK

Anal sphincter pressure is important in maintaining faecal continence and studies in man indicate that α-adrenoceptors are involved in modulating sphincter tone (Guttierrez et al., 1975). Recent work on the pig internal anal sphincter (IAS) suggests that noradrenaline-induced contractions involve α1-adrenoceptors, with a minor contribution by α2-adrenoceptors (Mills et al., 2008). In light of this observation, we have characterised the density, distribution and subtypes of α1- and α2-adrenoceptors in sheep IAS (as a model for man) to assess the potential utility of agonists in the treatment of faecal incontinence.

Saturation binding with 3H-prazosin and 3H-RX821002 was used to confirm the presence and density (Bmax values) of α1- and α2-adrenoceptors in sheep IAS, whilst competition binding with subtype selective agents was used to identify α-adrenoceptor subtype. A combination of in vitro receptor autoradiography and immunohistochemistry was used to study the regional distribution of binding sites.

Saturation binding confirmed the presence of both α1- and α2-adrenoceptors with Bmax values of 222±4 fmol/mg protein (n=3) and 72±7 fmol/mg protein (n=3), respectively. Immunohistochemical analysis revealed that α1-adrenoceptors were localised to areas corresponding to smooth muscle fibres. Determination of α1-adrenoceptor subtype involved 3 agents: RS100329 (an α1A-adrenoceptor selective antagonist), 5-methyl-urapidil (discriminates α1A- from α1B/α1D-adrenoceptors) and BMY7378 (α1D-adrenoceptor selective antagonist). With the exception of RS100329, all of the Hill slope values were close to unity (>0.8) and a comparison of pKi values produced a rank order of affinity of 5-methyl-urapidil (9.52±0.03; n=3) ≥ RS100329 (9.14±0.11; n=3) >> BMY7378 (6.61±0.04; n=3), suggesting the presence of the α1A-adrenoceptor subtype. Four agents were used to identify the α2-adrenoceptor subtype present; (i) BRL44408 (a selective α2A/D-adrenoceptor antagonist), (ii) prazosin, which discriminates between α2C- and α2A/D-adrenoceptors, plus (iii) phentolamine and rauwolscine – the relative affinity discriminates between α2A- and α2D-adrenoceptors. The pKi values from this series of experiments (n=3) indicates that the rank order of affinity for these agents is BRL44408 (8.85±0.13) > phentolamine (8.66±0.13) > rauwolscine (7.91±0.12) > prazosin (5.32±0.13), which is consistent with the presence of the α2D-adrenoceptor subtype.

This study has confirmed that α1- and α2-adrenoceptors are expressed in the sheep IAS with the former associated with smooth muscle bundles. Competition binding data indicates that sheep IAS expresses α1A/L-adrenoceptors as previously noted in pig IAS (Mills et al., 2008). Evidence of both α-adrenoceptor subtypes in IAS tissue provides a sound basis for considering receptor agonist in the treatment of faecal incontinence.

Guttierrez, J. G. et al. (1975). In Trappen G, ed. Vth International Symposium on Gastrointestinal Motility. Leuven: Typoff Press. 363-373

Mills, K et al. (2008), Br. J. Pharmacol. 155(1) 110-117