Electrophysiological responses induced by human (h) growth hormone-releasing hormone (GHRH) were analyzed using the perforated whole cell clamp technique in human growth hormone (GH)-secreting adenoma cells. Application of hGHRH depolarized the membrane by increasing Na+ conductance. The reversal potential of the hGHRH-induced current was -20 to 0 mV. The channel was permeable to Na+, Li+ and K+ but not to TMA+. These properties were compatible with those of nonselective cation channels. Similar nonselective cation current was activated by 8-bromoadenosine 3',5'-cyclic monophosphate and forskolin, and the activation of the hGHRH-induced current was inhibited by protein kinase A (PKA) inhibitors, (R)-p-adenosine 3',5'-cyclic monophosphate and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoleinsulfonamide, and PKA inhibitor peptide PKI-(5-24), indicating that hGHRH-induced current was activated by PKA. Cholera toxin pretreatment eliminated the hGHRH-induced current, suggesting that Gs is involved in the activation of this current. This current became irreversible when the cells were pretreated with okadaic acid, suggesting that the recovery of the hGHRH-induced current was mediated by a serine/threonine protein phosphatase. GHRH-induced GH secretion was inhibited in Na+-free medium, suggesting the importance of the nonselective cation current on hGHRH-induced GH secretion. In human GH-secreting nonadenoma cells, hGHRH increased Na+ conductance, as was the case in GH-secreting adenoma cells.
Membrane capacitance ( C m) was measured as an index of exocytosis in human growth hormone-secreting adenoma cells using the perforated whole cell, patch-clamp technique; the effects of membrane depolarization, growth hormone-releasing hormone, and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) were examined. C m was increased by membrane depolarization to potentials beyond the threshold necessary to open voltage-gated Ca2+channels. These voltage-dependent changes in C m varied as a function of both depolarization amplitude and duration and were blocked in the presence of the Ca2+channel antagonist nitrendipine (10−6 M). When membrane potential was clamped at the holding potential (−78 mV), voltage-gated Ca2+ channels were closed, and neither application of growth hormone-releasing hormone nor 8-BrcAMP affected C m. However, when these agents were applied to depolarized cells, where the voltage-gated Ca2+ channels were open, the increases in C mwere augmented. From these data, it was concluded that elevation of intracellular cAMP, per se, did not stimulate exocytosis. Rather, Ca2+ influx through voltage-gated channels was a prerequisite for cAMP-induced exocytosis.
(1→4)- and (1→6)-linked disaccharides were epimerized at C-2 of the reducing terminal under mild conditions by calcium(II)–N-alkylated monoamine systems. Naturally rare (1→4)-linked heterodisaccharides having a D-mannose unit as a reducing terminal can be prepared in one step from disaccharides having a D-glucose unit which are abundunt in nature.
Ionic mechanisms play an important role in the regulation of hormone secretion. The GHRH-induced GH release by human GH-secreting cells is transmitted through protein kinase A (PKA), which activates nonselective cation current (NSCC) and induces membrane depolarization, intracellular Ca2+ increase, and GH secretion. To evaluate whether ionic mechanisms have pathophysiological significance in GH oversecretion of GH-secreting pituitary adenomas, we examined four adenomas with constitutively active Gs alpha mutation (gsp mutation) and compared with three gsp-negative adenomas. In primary-cultured cells of gsp-positive adenomas, GHRH did not increase the NSCC under voltage-clamp experiments. Detailed examination showed that NSCC was maximally activated at the basal level and application of GHRH did not increase the current in these adenomas. Furthermore, by using single-cell RT-PCR method, we demonstrated for the first time at the single cell level that gsp mutation is heterozygous in GH-secreting pituitary adenomas. These indicate that heterozygous gsp mutation fully activates NSCC at the basal level, which may account for the GH oversecretion in gsp-positive GH-secreting pituitary adenomas.
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