Biological activity of somatostatin receptors in GC rat tumour somatotrophs: evidence with sst1-sst5 receptor-selective nonpeptidyl agonists

The physiological actions of somatostatin-14 (SRIF) receptor subtypes (sst1-sst5), which are endogenously expressed in GC cells, have not yet been elucidated, although there is evidence that sst2 receptors are negatively coupled to cytosolic free Ca2+ concentration ([Ca2+]i) and cAMP accumulation. In addition, both sst1 and sst2 receptors are negatively coupled to growth hormone (GH) secretion in GC cells. Here we report on studies concerning the expression, the pharmacology and the functional role of native SRIF receptors in GC cells with the use of five nonpeptidyl agonists, highly selective for each of the SRIF receptors. Radioligand binding studies show that sst2 and sst5 receptors are present at different relative densities, while the presence of sst3 and sst4 receptors appears to be negligible. The absence of sst1 receptor binding was unexpected in view of sst1 receptor functional effects on GH secretion. This suggests very efficient receptor-effector coupling of a low density population of sst1 receptors. Functionally, only sst2 receptors are coupled to the inhibition of [Ca2+]i and cAMP accumulation and the selective activation of sst5 receptors facilitates the stimulation of adenylyl cyclase activity through Gi/o proteins. This effect was not observed when sst2 and sst5 receptors were simultaneously activated, suggesting that there is a functional interaction between sst2 and sst5 receptors. In addition, sst1, sst2 and sst5 receptor activation inhibits GH release, further indicating that SRIF can modulate GH secretion in GC cells through mechanisms both dependent and independent on [Ca2+]i and cAMP-dependent pathways. The present data suggest SRIF-mediated functional effects in GC cells to be very diverse and provide compelling arguments to propose that multiple native SRIF receptors expressed in the same cells are not simply redundant, but contribute to marked signalling diversity.L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com

The dual orexin receptor antagonist almorexant induces sleep and decreases orexin-induced locomotion by blocking orexin 2 receptors

Orexin peptides regulate locomotor activity and sleep-wake balance by activating orexin 1 and orexin 2 receptors (OX1R and OX2R). Dual OX1R and OX2R antagonists reduce activity and promote sleep in multiple species, including man. We tested the effects of orexin A and almorexant, a dual OX1R/OX2R antagonist, in C57BL/6J mice and in mice lacking OX1Rs, OX2Rs or both to investigate the roles of the two receptors in orexin-induced locomotion and in sleep/wake regulation. Orexin A induced locomotion primarily by activating OX2Rs as locomotion was increased following intracerebroventricular orexin in OX1R-/- mice but not in OX2R-/- or OX1R-/-/OX2R-/- mice. Almorexant attenuated the orexin A-induced locomotion, demonstrating in vivo that almorexant specifically inhibits the actions of orexin. As in other species, almorexant dose-dependently increased rapid eye movement (REM) and non-REM (NREM) sleep in C57BL/6J mice. Sleep promotion by almorexant was mediated by inhibition of the known OXRs as almorexant was ineffective in OX1R-/-/OX2R-/- mice. Almorexant induced sleep in OX1R-/- mice but not in OX2R-/- mice, demonstrating that antagonism of OX2Rs is sufficient for sleep induction. When almorexant was incubated with the receptors for short periods of time, it behaved as a dual antagonist against orexin A-induced Ca2+ responses. However, with increasing incubation times, almorexant acquired OX2R selectivity confirming the findings from binding assays and further suggesting it may behave as an OX2R antagonist in vivo. Thus, OX2R activation mediates the stimulatory effects of orexin A on locomotion and antagonism of OX2R is sufficient to promote sleep in mice

SRA880, in vitro characterization of the first non-peptide somatostatin sst(1) receptor antagonist.

This report describes the in vitro features of the first somatostatin sst(1) receptor selective non-peptide antagonist, SRA880 ([3R,4aR,10aR]-1,2,3,4,4a,5,10,10a-Octahydro-6-methoxy-1-methyl-benz[g] quinoline-3-carboxylic-acid-4-(4-nitro-phenyl)-piperazine-amide, hydrogen malonate). SRA was evaluated in a number of in vitro systems of various species, both at native and recombinant receptors, using radioligand binding and second messenger/transduction studies. SRA880 has high affinity for native rat, mouse, monkey and human cerebral cortex somatostatin sst(1) receptors (pK(d) = 7.8-8.6) and for human recombinant sst(1) receptors (pK(d) = 8.0-8.1). SRA880 displayed significantly lower affinity for the other human recombinant somatostatin receptors ( pK(d) < or = 6.0) or a wide range of neurotransmitter receptors, except for the human dopamine D4 receptors. SRA880 was characterized in various transduction assays: somatotropin release inhibiting factor (SRIF) induced inhibition of forskolin-stimulated cAMP accumulation, SRIF stimulated-GTPgammaS binding, and SRIF stimulated luciferase gene expression; in all tests, SRA880 was devoid of intrinsic activity and acted as an apparently surmountable antagonist with pK(B) values of 7.5-7.7. Combined with the data from binding studies, these results suggest that SRA880 acts as a competitive antagonist. Thus, SRA880 is the first non-peptide somatostatin sst(1) receptor antagonist to be reported; SRA880 will be a useful tool for the characterization of somatostatin sst(1) receptor-mediated effects both in vitro and in vivo