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

Abstract

The physiological actions of somatostatin-14 (SRIF: somatotrophin release inhibitory factor) receptor subtypes (sst(1)-sst(5)), which are endogenously expressed in growth cells (GC cells), have not yet been elucidated, although there is evidence that sst(2) receptors are negatively coupled to cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and adenosine 3,5'-cyclic monophosphate (cAMP) accumulation. In addition, both sst(1) and sst(2) 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 sst(2) and sst(5) receptors are present at different relative densities, while the presence of sst(3) and sst(4) receptors appears to be negligible. The absence of sst(1) receptor binding was unexpected in view of sst(1) receptor functional effects on GH secretion. This suggests very efficient receptor-effector coupling of a low-density population of sst(1) receptors. Functionally, only sst(2) receptors are coupled to the inhibition of [Ca(2+)](i) and cAMP accumulation and the selective activation of sst(5) receptors facilitates the stimulation of adenylyl cyclase activity through G(i/o) proteins. This effect was not observed when sst(2) and sst(5) receptors were simultaneously activated, suggesting that there is a functional interaction between sst(2) and sst(5) receptors. In addition, sst(1), sst(2) and sst(5) receptor activation inhibits GH release, further indicating that SRIF can modulate GH secretion in GC cells through mechanisms both dependent and independent on [Ca(2+)](i) and cAMP-dependent pathways. The present data suggest SRIF-mediated functional effects in GC cells to be very diverse and provides compelling arguments to propose that multiple native SRIF receptors expressed in the same cells are not simply redundant, but contribute to marked signalling diversity.

The β isoenzyme of Ca2+/calmodulin-dependent kinase type II as possible mediator of somatostatin functions in pituitary tumour cells

“…These evidences are confirmed by previous studies performed in rat anterior pituitary GC cells, an adenoma cell line which exhibits pacemaker activity and concomitant spontaneous oscillations of [Ca 2+ ] i and GH secretion (Cervia et al 2002b). In GC cells, sst 2 activation is responsible for the [Ca 2+ ] i decrease and the block of Ca 2+ transients which, in turn, results in the sst 2 -mediated decrease of GH secretion (Petrucci et al 2000;Cervia et al 2002aCervia et al ,b, 2003. Ca 2+ /calmodulin-dependent kinase type II (CaMKII), a multifunctional serine/threonine protein kinase, is highly expressed in neuronal tissues (Takeuchi et al 2000;Griffith 2004;Schulman 2004;Ishida et al 2005;Yamauchi 2005;Kamata et al 2006;Wayman et al 2008).…”

“…1A, we detected the amplified product corresponding to CaMKIIα, CaMKIIβ, CaMKIIγ and CaMKIIδ mRNAs. In another set of experiments, we found that 30 min SRIF application at 100 nM, a concentration giving maximal receptor occupancy in our system (Petrucci et al 2000;Cervia et al 2003), decreased the expression of the band corresponding to CaMKIIβ mRNA while the bands corresponding to CaMKIIα, CaMKIIγ and CaMKIIδ mRNAs were not affected (Fig. 1B,C).…”

“…Using published protocols (Petrucci et al 2000;Cervia et al 2002aCervia et al , 2003Cervia et al , 2005Cervia et al , 2006 6 cells, as determined with a standard hemocytometer chamber and trypan blue exclusion. During cell treatment (i.e., 100 nM SRIF, 10/100 μM KN-62), the fluorescence intensity of fura-2/AM was quantified with a LS-50B Luminescence Spectrometer (Perkin-Elmer, Norwalk, CT, USA) with a double excitation wavelength set at 340 nm and 380 nm, respectively, and an emission wavelength monitored at 510 nm.…”

“…Using published protocols (Cervia et al 2002b(Cervia et al , 2003, GH was detected and quantified by means of a commercially available EIA kit (SPI Bio, Montigny Le Bretonneux, France) utilizing the double-antibody sandwich technique, according to the manufacturer's recommended procedure. Briefly, GC cells (transfected or untransfected) were detached from 100-mm dishes, and seeded in new dishes at a concentration of 10 7 cells.…”

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

“…Calcium and activated G proteins are known to regulate the exocytosis of different factors, i.e., chemokines, synergistically from the activated macrophages, although a Ca 2ϩ -independent component may also be involved [76]. SRIF receptor coupling to Ca 2ϩ homeostasis is well established [2] as well as the fact that SRIF receptors may regulate exocytosis through mechanisms dependent and independent on Ca 2ϩ [2,35,51]. Whether the SRIF system couples to Ca 2ϩ homeostasis in human macrophages remains to be established.…”