Differences in the operational characteristics of the human recombinant somatostatin receptor types, sst1 and sst2, in mouse fibroblast (Ltk−) cells

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Abstract
1 The human recombinant somatostatin (SRIF) receptors, sst1 and sst2, have been stably expressed in mouse fibroblast (Ltk−) cells. Two stable clones, LSSR1/20 and LSSR11/13, expressing sst1 and sst2 receptors, respectively, have been used to characterize these receptor types using radioligand binding assays as well as measurements of changes in extracellular acidification rates using microphysiometry. 2 [125I]-[Tyr11]-SRIF bound to sst1 and sst2 receptors expressed in Ltk− cells with high affinity, Kd values being 1.52 nM and 0.23 nM, respectively. 3 In Ltk− cells expressing sst1 receptors, SRIF, SRIF-28, [D-Trp8]-SRIF and CGP 23996 all displaced [125I]-[Tyr11]-SRIF binding with high potency (IC50 values of 0.43-1.27 nM) whilst seglitide, BIM-23027, BIM-23056 and L-362855 were either weak inhibitors of binding or were ineffective. 4 In contrast MK-678 (seglitide) and BIM-23027 were the most potent inhibitors of [125I]-[Tyr11]-SRIF binding in Ltk− cells expressing sst2 receptors with IC50 values of 0.014 and 0.035 nM, respectively. 5 SRIF and a number of SRIF agonists, including seglitide and BIM-23027, caused concentration- dependent increases in extracellular acidification rates in Ltk− cells expressing sst2 receptors but not in Ltk− cells expressing sst1 receptors. The maximum increase in acidification rate produced by SRIF was 11.3 ± 0.7% above baseline (0.1-0.28 pH unit min−1). The relative potencies of the SRIF agonists examined in causing increases in extracellular acidification rates in Ltk− cells expressing sst2 receptors correlated well with their relative potencies in inhibiting [125I]-[Tyr11]-SRIF binding (r = 0.94). 6 The increase in extracellular acidification produced by SRIF was markedly inhibited by pretreatment of cells with pertussis toxin (100 ng ml−1) indicating the involvement of pertussis toxin-sensitive G proteins. 7 SRIF (1 μm) had no effect on basal cyclic AMP levels in Ltk− cells expressing sst1 or sst2 receptors nor did it inhibit forskolin stimulated increases in cyclic AMP levels in either cell type. 8 The results from the present study describe the operational characteristics of human sst2 receptors expressed in Ltk− cells where receptor activation causes increases in extracellular acidification rates. This receptor is coupled to a pertussis toxin-sensitive G protein. In contrast, activation of sst1 receptors, at a similar transfection density, did not cause increases in extracellular acidification rates.