Cyclic AMP-dependent expression of the steroidogenic acute regulatory (StAR) protein is thought to be the controlling step for steroid production, but the mechanisms through which external signals are translated into increased transcription of the StAR gene are unknown. We demonstrate that cyclic AMP-induced steroid synthesis is dependent upon the phosphorylation and activation of ERKs and that ERK activation results in enhanced phosphorylation of SF-1 and increased steroid production through increased transcription of the StAR gene. Adenylate cyclase activation with forskolin (FSK) caused a time-dependent increase in ERK activity and translocation from cytoplasm to nucleus, which correlated with an increase in StAR mRNA levels, StAR protein accumulation, and steroidogenesis. Similarly, ERK inhibition led to a reduction in the levels of FSKstimulated StAR mRNA, StAR protein, and steroid secretion. These effects were attributed to the finding that ERK activity is required for SF-1 phosphorylation, a transcription factor required for the regulation of StAR gene transcription. This conclusion was supported by our demonstration of an ERK-dependent increase in the binding of SF-1 from FSK-treated Y1 nuclei to three consensus double-stranded DNA sequences from the StAR promoter region. These observations suggest that the activation of ERK2/1 by increasing cAMP is an obligatory and regulated stage in the stimulation of steroid synthesis by cyclic AMP-generating stimuli.

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Aims/hypothesis: This study aimed to identify the expression of angiotensin II receptors in isolated human islets and beta cells and to examine the functional consequences of their activation. Materials and methods: Singlecell RT-PCR was used to identify whether human islet cells express mRNA for type 1 angiotensin II receptors (AT 1 ), and western blotting was used to determine AT 1 protein expression by human islets and MIN6 beta cells. We measured changes in intracellular calcium by microfluorimetry using Fura 2-loaded MIN6 cells and human islet cells. Dynamic insulin secretory responses were determined by RIA following perifusion of human islets and MIN6 cells. Results: Human islets expressed mRNAs for both the angiotensin precursor, angiotensinogen, and for angiotensin-converting enzyme. In addition, human and mouse beta cells expressed AT 1 . These were functionally coupled to increases in intracellular calcium, which occurred at least in part through phospholipase-C-sensitive mechanisms and calcium influx through voltage-operated calcium channels.Short-term exposure of human islets and MIN6 cells to angiotensin II caused a rapid, short-lived initiation of insulin secretion at 2 mmol/l glucose and potentiation of insulin secretion induced by glucose (at 8 and 16.7 mmol/l). Conclusions/interpretation: These data demonstrate that the AT 1 is expressed by beta cells and that angiotensin II effects a short-lived and direct stimulation of human and mouse beta cells to promote insulin secretion, most probably through elevations in intracellular calcium. Locally produced angiotensin II may be important in regulating a coordinated insulin secretory response from beta cells.

A technique for the perfusion of the rat adrenal cortex is described. With tissue culture Medium 199 the preparation was responsive in terms of steroid production of both ACTH and K+ ions. Production of corticosterone and 18-hydroxydeoxycorticosterone (18-hydroxy-DOC) was stimulated by ACTH when it was administered at rates between 5 uu.'/min and 5 mu./min. Increasing the K+ ion concentration of the perfusate from 3.6 to 5.4 and 8.9 mmol/l stimulated the production of aldosterone, 18-hydroxycorticosterone and deoxycorticosterone, although not of corticosterone or 18-hydroxy-DOC. This preparation has been used to study further the mechanism of secretion of corticosterone and 18-hydroxy-DOC. Thus, production of these two steroids was measured at different perfusion flows, varying between 0.1 and 0.6ml/min, with different levels of ACTH stimulation. Corticosterone production was significantly (P less than 0.001) increased by increasing flows both under control conditions and with ACTH was administered at constant rates of 50 uu./min or 1 mu./min. Production of 18-hydroxy-DOC was not affected by flow either under control conditions or with 50 uu. ACTH/min. However, when ACTH was administered at 1 mu./min. 18-hydroxy-DOC production was also significantly (P less than 0.001) increased by flow. The results are consistent with those obtained in previous in-vitro studies and have been interpreted as suggesting that the main mechanism of corticosterone secretion is simple diffusion. In contrast, 18-hydroxy-DOC secretion, at least at sub-maximal levels of stimulation, appears to require a more complex process.

Mast cells were identified in the rat adrenal gland, located in the walls of arterioles at the point at which they penetrate the connective tissue capsule. The mast cell products, histamine and serotonin, both caused dose-dependent increases in rates of perfusion medium flow and steroid secretion in the isolated, perfused rat adrenal gland in situ. Compound 48-80, a mast cell degranulator, caused a significant increase in perfusion medium flow rate and steroid secretion by the in-situ perfused rat adrenal. Administration of disodium cromoglycate, a mast cell stabilizer, before administration of ACTH(1-24) virtually abolished the normal flow rate increment and significantly attenuated the corticosterone secretory response to ACTH(1-24). These observations strongly suggest that adrenal mast cells modulate both vascular and secretory responses in the intact adrenal gland of the rat.