Neuropeptides in sea urchins

265 NEUROPEPTIDES IN SEA URCHINS M.R. ELPHICK*, K. PARKER and M.C. THORNDYKE Biology Department, RHBNC, University of London, Egham, TW20 0EX. *Present address: Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton, BN1 9QG. HPLC of whole-body extracts of the sea urchin Echinus esculentus resolves five peaks that are immunoreactive with antisera to the starfish SALMFamide neuropeptides (peaks A, B, C, D1 and I)2; Elphick et al., 1991, Reg. Peptides, 35:235). D2 has been purified and sequenced and is an amidated nonapeptide, FPVGRVHRFamide. Experiments directed towards obtaining the full sequences of A, B, C and D1 are underway. Immunocytochemistry using the same antisera employed for HPLC isolation reveals a widespread distribution of SALMFamide-like immunoreactivity throughout the gut of E. esculentus. In particular, the anterior segment of the gut (pharynx-oesophagus) displays extensive tracts of immunoreactivity associated with the basi-epithelial nerve plexus that runs between the secretory epithelium and the underlying layers of connective tissue and muscle. SALMFamide-like immunoreactivity was also detected in association with the outer sub-coelomic epithelial nerve plexus which runs between the outer connective tissue layer and the coelomic epithelium. In contrast to our findings with the starfish Astertas rubens, no immunoreactive epithelial endocrine or neuroendocrine cells were observed. It seems likely that the antibodies used for immunocytochemistry are detecting all of the peptides associated with the five peaks observed in our HPLC studies and the sequenced pepfide (FPVGRVHRFamide) is likely to be present in only a sub-set of the immunoreactive nerve fibres. We are presently raising specific antibodies to FPVGRVHRFamide with a view to establishing a more specific map of its distribution. In addition, we also plan to use immunological and molecular methods to analyse neuropeptide expression during sea urchin development.

ACTIVITY OF VASOACTIVE INTESTINAL POLYPEPTIDE AT SECRETIN-PREFERRING RECEPTORS ON ISOLATED CARDIOMYOCYTES D Bell and B J McDermott, Department of Therapeutics and Pharmacology, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland In contrast to the hearts of other mammalian species which possess VIP-preferring receptors, it has been proposed that the rat heart is unique in that both secretin and VIP may bind to a "relativelynon-selective receptor".(1). This early study measured adenylate cyclase activity in crude preparations of ventricular membranes. Such suspensions have the inherent disadvantage that they are derived from a number of ventricular cell types. The present investigation was designed to more dearly define the receptor(s) for secretin and VIP on the rat ventricular cardiomyocyte using a pure suspension of intact cardiomyoeytes. We demonstrate a direct stimdatory effect of both secretin and VIP on the accumulation of cAMP which is mediated by a selective receptor population. In the presence of adenosine deaminase (5 u/ml) and the phosphodiesterase in~bitor, isobutylmethylxanthine (1 mM), both secretin and VIP increased intracellular levels of cAMP maximally after 5 minutes and in a concentration-dependant manner: EC50 values were 8 nM and 58 nM respectively. At maximally effective concentrations, secretin (1 uM) increased intracellular levels of cAMP 3.4 fold above basal levels, whereas a 1.3 fold elevation was observed with VIP (10 uM). The selective antagonist for VIP-preferring receptors, p-chloro DPhe-6 Leu-17 VIP (< 10 uM), failed to block the action of VIP (50 aM). In the presence of the selective antagonist at receptors for secretin, secretin 7-27 (1-100 uM), the concentration-respouse curve for secretin exhibited a rightward parallel shift: the pA2 value for secretin 7-27 was 4.89. Secretin 7-27 also induced a rightward parallel shift of the VIP concentrationresponse curve. VIP (10 uM) was additive with low concentrations of secretin (< 10 uM) but antagonised cAMP production at higher concentrations of secretin (> 10 aM). It is concluded that rat ventricular cardiomyocytes are devoid of VIP-preferring receptors but do possess secretin-preferring receptors at which VIP behaves as a partial agonist to induce the observed response. Chatelain, Pet al (1980). Pflugers Arch, 389: 21-27.