Characterisation of human recombinant somatostatin receptors. 4. Modulation of phospholipase C activity

Total [H-3]phosphoinositide (IPx) accumulation, a measure of phospholipase C (PLC) activity, induced by somatostatin (somatotropin release-inhibiting factor, SRIF) and cortistatin (CST) analogues was studied at human somatostatin receptor subtypes 1-5 (hsst(1-5)) recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. SRIF14 (10 mu M) stimulated total [H-3]-IPx production 200% and 1070% over basal levels, and increased intracellular Ca2+ ([Ca2+](i)) 1600% and 2790%, in cells ex pressing hsst(3) and hsst(5) receptors, respectively. The SRIF14-stimulated IPx production was partly blocked by 100 ng/ml pertussis toxin (PTX) (30% and 15% inhibition, respectively). At hsst(1), hsst(2), and hsst(4) receptors, only weak or no stimulation of PLC activity was found (E-max=114%, 122%, and 102%, respectively). Consequently, hsst(3) and hsst(5) receptors were subjected to more detailed studies to establish pharmacological profiles of PLC stimulation. At hsst(3) receptors, the relative efficacies of most ligands were in the same range (maximum response E-max=218-267%). At hsst(5) receptors E-max varied over a broad range, seglitide, CST17, SRIF28 displaying almost full agonism compared to SRIF14, whereas octreotide and BIM 23052 showed very low partial agonism. BIM 23056 behaved as an antagonist on SRLF14-induced total [H-3]-IPx accumulation with a pK(B) (negative logarithm of antagonist binding constant) of 6.74 at hsst(3) receptors, and of 6.94 at hsst(5) receptors. The putative cycloantagonist SA showed weak antagonist activity on SRIF14-induced total [H-3]-IPx levels at hsst(3) (pK(B)=5.85), but not at hsst(5) receptors. The [H-3]-IPx accumulation profiles at sst(3)/sst(5) receptors were compared to their respective radioligand binding ([I-125]LTT-SRIF28, [I-125[Tyr(10)]CST14, [I-125]CGP 23996, [125I][Tyr(3)]octreotide binding), to [S-35]GTP gamma S binding, and to forskolin-stimulated adenylate cyclase (FSAC) inhibition profiles determined previously in CCL39 cells. The different affinity profiles correlated relatively well at both receptor subtypes with PLC activation (sst(3): r=0.90-0.97; sst(5): r=0.80-0.87). However, [S-35]GTP gamma S binding correlated only minimally with stimulation of [H-3]-IPx levels at sst(5) receptors (r=0.59), but rather well at sst(3) receptors (r=0.80). A moderate correlation was also observed between inhibition of FSAC activity and stimulation of PLC activity for hsst(3) and hsst(5) receptors with correlation coefficients of 0.85 and 0.70, respectively. In summary, most SRIF analogues behave as full agonists at hsst(3) receptors and agonist-induced phosphoinositide turnover correlates well with radioligand binding, [S-35]GTP gamma S binding and inhibition of adenylate cyclase activity, all measured in CCL39 cells. By contrast, at hsst5 receptors, most SRIF analogues behave as intermediate or very low partial agonists (although receptor levels are comparatively high, 7000 vs. 400 fmol/mg), and the agonist-induced phosphoinositide turnover correlates rather poorly with radioligand binding, [S-35]GTP gamma S binding or inhibition of adenylate cyclase activity, all measured in the same cell line. Agonist-induced phosphoinositide turnover, [S-35]GTP gamma S binding and inhibition of adenylate cyclase activity show differences both in the rank orders of potency and relative efficacy at hsst(3) and markedly at hsst(5) receptors, suggesting either that PLC activity is functionally irrelevant or, more probably, that agonist-dependent receptor trafficking is taking place in CCL39 cells.