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Intracellular calcium ion responses to somatostatin in cells from human somatotroph adenomas
1Department of Medicine, University of Bristol, UK.
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 (Ca2+i) imaging.
Patients:
Adenoma specimens were obtained from 4 male and 11 female acromagalic 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 35S 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 Ca2+i 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.
Insights
Somatostatin (SRIH) analogues reduce intracellular calcium in most human somatotroph adenoma cells, inhibiting growth hormone release. However, some adenomas show paradoxical calcium increases or no response, explaining varied patient outcomes.
Area of Science:
- Endocrinology
- Molecular Cell Biology
- Oncology
Background:
- Acromegaly is characterized by excessive growth hormone (GH) secretion, often due to somatotroph adenomas.
- Somatostatin (SRIH) analogues are used to treat acromegaly, but their precise effects on intracellular calcium in adenoma cells are not fully understood.
- Understanding SRIH's impact on calcium homeostasis is crucial for explaining variable treatment responses.
Purpose of the Study:
- To investigate the effects of SRIH on intracellular calcium ion (Ca2+i) levels in human somatotroph adenoma cells.
- To elucidate the mechanisms underlying SRIH-induced calcium responses in these cells.
- To correlate cellular responses with clinical observations of SRIH analogue efficacy.
Main Methods:
- Human somatotroph adenoma tissues were obtained from 15 acromegalic patients.
- Cells were analyzed using computerized real-time Ca2+i imaging at a single-cell level.
- Somatostatin receptor subtype 2 (SSTR2) gene sequencing and immunocytochemistry were performed.
Main Results:
- SRIH significantly reduced basal Ca2+i and inhibited the GH-releasing hormone (GRH)-induced Ca2+i increase in cells from 11 of 15 adenomas.
- Pertussis toxin pretreatment abolished SRIH's inhibitory effects, indicating involvement of Gi/o proteins.
- A minority of adenomas showed paradoxical Ca2+i increases or no response to SRIH, irrespective of SSTR2 sequence (wild-type).
Conclusions:
- SRIH primarily reduces intracellular calcium and inhibits GRH-stimulated calcium influx in most human somatotroph adenomas.
- The observed heterogeneity in SRIH response at the cellular level supports the variability in clinical outcomes with SRIH analogues.
- No SSTR2 mutations were found, suggesting other mechanisms contribute to differential SRIH responsiveness.