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

Abstract.

Total [³H]phosphoinositide (IP_x) 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 µM) stimulated total [³H]-IP_x production 200% and 1070% over basal levels, and increased intracellular Ca²⁺ ([Ca²⁺]_i) 1600% and 2790%, in cells expressing hsst₃ and hsst₅ receptors, respectively. The SRIF₁₄-stimulated IP_x production was partly blocked by 100 ng/ml pertussis toxin (PTX) (30% and 15% inhibition, respectively). At hsst₁, hsst₂, and hsst₄ receptors, only weak or no stimulation of PLC activity was found (E _max=114%, 122%, and 102%, respectively). Consequently, hsst₃ and hsst₅ receptors were subjected to more detailed studies to establish pharmacological profiles of PLC stimulation.

At hsst₃ receptors, the relative efficacies of most ligands were in the same range (maximum response E _max=218–267%). At hsst₅ 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 SRIF₁₄-induced total [³H]-IP_x accumulation with a pK _B (negative logarithm of antagonist binding constant) of 6.74 at hsst₃ receptors, and of 6.94 at hsst₅ receptors. The putative cycloantagonist SA showed weak antagonist activity on SRIF₁₄-induced total [³H]-IP_x levels at hsst₃ (pK _B=5.85), but not at hsst₅ receptors. The [³H]-IP_x accumulation profiles at sst₃/sst₅ receptors were compared to their respective radioligand binding ([¹²⁵I]LTT-SRIF₂₈, [¹²⁵I][Tyr¹⁰]CST₁₄, [¹²⁵I]CGP 23996, [¹²⁵I][Tyr³]octreotide binding), to [³⁵S]GTPγS 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 (sst₃: r=0.90–0.97; sst₅: r=0.80–0.87). However, [³⁵S]GTPγS binding correlated only minimally with stimulation of [³H]-IP_x levels at sst₅ receptors (r=0.59), but rather well at sst₃ receptors (r=0.80). A moderate correlation was also observed between inhibition of FSAC activity and stimulation of PLC activity for hsst₃ and hsst₅ receptors with correlation coefficients of 0.85 and 0.70, respectively.

In summary, most SRIF analogues behave as full agonists at hsst₃ receptors and agonist-induced phosphoinositide turnover correlates well with radioligand binding, [³⁵S]GTPγS 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, [³⁵S]GTPγS binding or inhibition of adenylate cyclase activity, all measured in the same cell line. Agonist-induced phosphoinositide turnover, [³⁵S]GTPγS binding and inhibition of adenylate cyclase activity, show differences both in the rank orders of potency and relative efficacy at hsst₃ and markedly at hsst₅ receptors, suggesting either that PLC activity is functionally irrelevant or, more probably, that agonist-dependent receptor trafficking is taking place in CCL39 cells.