Characterisation of the fish sst3 receptor, a member of the SRIF1 receptor family:: atypical pharmacological features

The first cloned non-mammalian somatostatin (somatostatin release-inhibiting factor = SRIF) receptor previously obtained from the teleost fish Apteronotus albifrons and generically named somatostatin receptor 3 (fsst(3)), was stably expressed and characterised in Chinese hamster lung fibroblast (CCL39) cells. Radioligand binding studies were performed with four radioligands selective for SRIF receptors in CCL39 cells expressing the fsst(3) receptors; [I-125]LTT-SRIF28 ([Leu(8), D-Trp(22), I-125-Tyr(25)]-SRIF28), [I-125]Tyr(10)-cortistatin, [I-125]CGP 23996, and [I-125]Tyr(3)-octreotide labelled the fsst(3) receptor with high affinity (pK(d) values: 10.47, 10.87, 9.59 and 9.57) and in a saturable manner, but defined different B-max values; 4500, 4000, 3400 and 1500 fmol/mg, respectively The affinities of SRIF peptides and analogues determined for fsst(3), receptors displayed the following rank order of potency: seglitide = SRIF25 > SRIF14 = SRIF28 > cortistatin 14 > BIM 23014 > RC160 = L361,301 = octreotide greater than or equal to BIM 23052 greater than or equal to L362,855 > CGP23996 > BIM 23056 > BIM 23030 = cycloantagonist > SRIF22. The pharmacological profiles determined with [I-125]LTT-SRIF28 [I-125]CGP 23996 and [I-125]Tyr(10)-cortistatin correlated highly significantly (r=0.96-0.99), whereas [I-125]Tyr(3)-octreotide binding was rather divergent (r = 0.78-0.81). Further, [I-125]Tyr(3)-octreotide- and [I-125]CGP 23996-Iabelled sites showed higher affinity for the various peptides than [I-125]LTT-SRIF28, and [I-125]Tyr(10)-cortistatin-labelled sites, although there were exceptions. [I-125]LTT-SRIF28-binding to fsst(3) receptors and human sst(1-5) receptors was compared; the fsst(3) binding profile correlated better with the hsst(5)- than with the hsst(3) receptor profile. SRIF inhibited potently forskolin-stimulated adenylate cyclase activity in fsst(3) transfected CCL39 cells; this effect was blocked by pertussis toxin, suggesting coupling of the fast, receptor to G(i alpha) and/or G(o alpha). [I-125]LTT-SRIF28 binding was detected in fish brain, liver, heart, spleen, and stomach, but not in gut. The pharmacological profile of [I-125]LTT-SRIF28-labelled sites in brain, but not in liver, correlated significantly with the recombinant fsst(3) receptor, in agreement with expression of the fsst(3), receptor gene found by RT-PCR in the brain. However, biphasic binding curves obtained with two SRIF-analogues in brain, as well as the distinct pharmacological profile of the liver SRIF receptor, suggest the existence of several yet to be defined SRIF receptor subtypes in fish. The present data demonstrate that the recombinantly expressed fsst(3) receptor has a pharmacological profile compatible with that of a SRIF1 receptor, although the rank order of affinity of fsst(3) is closer to that of hsst(5) than hsst(3) receptors, as may be found when comparing very distantly related species. The fsst(3) receptor expressed in CCL39 cells, is negatively coupled to adenylate cyclase activity via pertussis toxin-sensitive G-proteins, like mammalian set, receptors. Radioligand binding performed with fish tissue suggests the presence of a native sst, receptor in brain as well as other yet to be defined SRIF receptor subtypes. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.