Intracellular calcium ion responses to somatostatin in cells from human somatotroph adenomas

OBJECTIVE Various GH secretory responses to long-acting somatostatin (SRIH) analogues have been observed during the treatment of acromegalic patients. The effects of SRIH on intracellular Ca2+ homeostasis in human somatotroph adenoma cells has not been examined in detail, and the underlying mechanisms therefore remain to be determined, Using isolated cells from human somatotroph adenomas, we have investigated the SRIH-induced intracellular Ca2+ responses at a single-cell level with computerized real time intracellular calcium ion (Ca-i(2+)) imaging. PATIENTS Adenoma specimens were obtained from 4 male and 11 female acromegalic patients (mean age 56, range 26-72 years) undergoing transsphenoidal hypophysectomy. METHODS The identity of the biopsy material obtained was confirmed by immunocytochemistry for hGH and in situ hybridization histochemistry using a S-35 end-labelled hGH oligodeoxynucleotide probe and probes complementary to proopiomelanocortin and prolactin. Genomic DNA coding for somatostatin receptor (SSTR2) from each adenoma was PCR amplified and sequenced. Cells cultured from these adenoma were subject to computerized real time intracellular Ca-i(2+) imaging at a single cell level. RESULTS In cells from 11 of the 15 adenomas, SRIH produced a reversible, dose-independent reduction in [Ca2+](i) from the mean of 167 +/- 11 to 43 +/- 3 nM within 51 +/- 1.8 s, and blocked the growth hormone releasing hormone (GRH)-induced increase in [Ca2+](i) as expected. In the same adenomas, withdrawal of SRIH after a 30 second exposure produced a small but significant increase in resting [Ca2+](i). Pretreatment with pertussis toxin abolished the SRIH-induced inhibition of [Ca2+](i) and prevented the SRIH-induced inhibition of the effect of GRH on [Ca2+](i). One of the remaining 4 adenomas was completely unresponsive to SRIH despite responding vigorously to other ligands and immunostaining strongly for GH. Surprisingly, cells from 3 adenomas showed a paradoxical increase in [Ca2+](i) in response to SRIH in some or, in one case, all of the cells examined. In all adenomas the sequence of SSTR2 corresponded to wild-type. CONCLUSIONS In the majority of cells derived from human somatotrophic adenomas, SRIH caused a reduction in baseline [Ca2+](i) and inhibition of GRH-induced [Ca2+](i) increase, as observed in somatotrophs of other species, In addition, SRIH was found either to induce a paradoxical increase in [Ca2+](i) or to have no effect on [Ca2+](i) in a small proportion of somatotroph adenomas examined. This finding corroborates the clinical observation that the response to SRIH analogues varies markedly between somatotroph adenoma patients, There was no evidence of SSTR2 mutations in any of the adenomas examined.