The involvement of protein kinases in the signal transduction pathways controlling adrenal steroidogenesis is well established, and the phosphorylation of substrates by cAMP-dependent protein kinase is a major mechanism in ACTH action. However, the possibility that protein phosphatases (PPs) might also be involved in this process has not been investigated. The aim of this study was, therefore, to measure the function, expression and enzymic activity of PPs in zona glomerulosa (ZG) and zona fasciculata/reticularis (ZFR) tissue from the rat adrenal cortex. Immunoblot analysis using specific antisera demonstrated the presence in whole adrenals and capsules of PP type 1 (PP1) migrating with an apparent molecular mass of 37 kDa, and PP type 2A (PP2A) migrating with apparent molecular masses of 38 and 31 kDa. The PP inhibitors, okadaic acid (OA), calyculin A (CA), tautomycin and microcystin RR, caused a reduction in PP activity in vitro, at doses between 1 nM and 1 microM. In addition, treatment of ZG cells with the adenylate cyclase stimulator, forskolin (10 microM) resulted in a significant reduction in PP activity. The effects of CA and OA on steroid secretion by ZG and ZFR cells were also investigated. Neither CA nor OA had any effect on basal steroid secretion or on yields of steroid obtained from 22R-hydroxycholesterol at doses between 1 and 100 nM. However, both OA and CA (10 and 100 nM respectively) significantly reduced ACTH-stimulated aldosterone and corticosterone production by ZG and ZFR cells. CA and OA (10 and 100 nM respectively) also reduced steroid secretion by cells stimulated by forskolin (10 microM) or dibutyryl cAMP (200 microM). These results suggest that PPs may be involved in the intracellular mechanisms through which adrenocortical steroidogenesis is regulated, acting at a point after cAMP generation and action, but proximal to the side-chain cleavage of cholesterol.
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SB Sayed, BJ Whitehouse, and PM Jones
SJ Persaud, H Al-Majed, A Raman, and PM Jones
To determine whether extracts of Gymnema sylvestre may have therapeutic potential for the treatment of non-insulin-dependent diabetes mellitus (NIDDM), we examined the effects of an alcoholic extract of G. sylvestre (GS4) on insulin secretion from rat islets of Langerhans and several pancreatic beta-cell lines. GS4 stimulated insulin release from HIT-T15, MIN6 and RINm5F beta-cells and from islets in the absence of any other stimulus, and GS4-stimulated insulin secretion was inhibited in the presence of 1 mM EGTA. Blockade of voltage-operated Ca(2+) channels with 10 microM isradipine did not significantly affect GS4-induced secretion, and insulin release in response to GS4 was independent of incubation temperature. Examination of islet and beta-cell integrity after exposure to GS4, by trypan blue exclusion, indicated that concentrations of GS4 that stimulated insulin secretion also caused increased uptake of dye. Two gymnemic acid-enriched fractions of GS4, obtained by size exclusion and silica gel chromatography, also caused increases in insulin secretion concomitant with increased trypan blue uptake. These results confirm the stimulatory effects of G. sylvestre on insulin release, but indicate that GS4 acts by increasing cell permeability, rather than by stimulating exocytosis by regulated pathways. Thus the suitability of GS4 as a potential novel treatment for NIDDM can not be assessed by direct measurements of beta-cell function in vitro.
HT Al-Majed, PM Jones, SJ Persaud, D Sugden, GC Huang, S Amiel, and BJ Whitehouse
It has previously been suggested that ACTH and ACTH-related peptides may act as paracrine modulators of insulin secretion in the islets of Langerhans. We have, therefore, examined the expression and function of the ACTH receptor (the melanocortin 2 receptor, MC2-R) in human and mouse primary islet tIssue and in the MIN6 mouse insulinoma cell line. Mouse MC2-R mRNA was detected in both MIN6 cells and mouse islet tIssue by PCR amplification of cDNA. In perifusion experiments with MIN6 pseudo-islets, a small, transient increase in insulin secretion was obtained when ACTH(1-24) (1 nM) was added to medium containing 2 mM glucose (control) but not when the medium glucose content was increased to 8 mM. Further investigations were performed using static incubations of MIN6 cell monolayers; ACTH(1-24) (1 pM-10 nM) provoked a concentration-dependent increase in insulin secretion from MIN6 monolayer cells that achieved statistical significance at concentrations of 1 and 10 nM (150 +/- 13.6% basal secretion; 187 +/- 14.9% basal secretion, P<0.01). Similar responses were obtained with ACTH(1-39). The phosphodiesterase inhibitor IBMX (100 microM) potentiated the responses to sub-maximal doses of ACTH(1-24). Two inhibitors of the protein kinase A (PKA) signaling pathway, Rp-cAMPS (500 microM) and H-89 (10 microM), abolished the insulin secretory response to ACTH(1-24) (0.5-10 nM). Treatment with 1 nM ACTH(1-24) caused a small, statistically significant increase in intracellular cAMP levels. Secretory responses of MIN6 cells to ACTH(1-24) were also influenced by changes in extracellular Ca2+ levels. Incubation in Ca2+-free buffer supplemented with 0.1 mM EGTA blocked the MIN6 cells' secretory response to 1 and 10 nM ACTH(1-24). Similar results were obtained when a Ca2+ channel blocker (nitrendipine, 10 microM) was added to the Ca2+-containing buffer. ACTH(1-24) also evoked an insulin secretory response from primary tIssues. The addition of ACTH(1-24) (0.5 nM) to perifusions of mouse islets induced a transient increase in insulin secretion at 8 mM glucose. Perifused human primary islets also showed a secretory response to ACTH(1-24) at basal glucose concentration (2 mM) with a rapid initial spike in insulin secretion followed by a decline to basal levels. Overall the results demonstrate that the MC2-R is expressed in beta-cells and suggest that activation of the receptor by ACTH initiates insulin secretion through the activation of PKA in association with Ca2+ influx into beta-cells.
BJ Whitehouse, SL Gyles, PE Squires, SB Sayed, CJ Burns, SJ Persaud, and PM Jones
Y1 adrenocortical cells respond to activators of the cyclic AMP-dependent protein kinase (PKA) signalling pathway not only with increases in steroid secretion but also with a characteristic change in cell morphology from flat and adherent to round and loosely attached. This change of shape, which may facilitate cholesterol transport to the mitochondrion, requires tyrosine dephosphorylation of the focal adhesion protein, paxillin, and can be blocked by inhibitors of phosphotyrosine phosphatase (PTP) activity. In a previous study we demonstrated that inhibition of phosphoserine/threonine phosphatase 1 and 2A (PP1/2A) activities caused a similar morphological response to PKA activation whilst opposing the effects on steroid production. We have now investigated the responses to PKA activation and inhibition of PP1/2A and used PTP inhibitors to examine the relationship between the morphological changes and enhanced steroid production. Both forskolin (FSK) and the PP1/2A inhibitor, calyculin A (CA), caused rapid and extensive rounding of Y1 cells. FSK-induced cell rounding was reversible and accompanied by a reduction in the tyrosine phosphorylation of paxillin. Rounding was prevented by the PTP inhibitors pervanadate (PV) and calpeptin (CP) and was associated with the maintained tyrosine phosphorylation of paxillin. In contrast, CA-induced cell rounding was not reversible over a 2-h period and was not affected by the presence of PTP inhibitors, and CA had no effect on the tyrosine phosphorylation of paxillin. Although neither CA nor FSK produced any gross changes in cell viability as judged by Trypan Blue exclusion or mitochondrial activity, CA-treated cells showed a marked reduction in total protein synthesis assessed by (35)S-incorporation. The effects of FSK and the PTP inhibitors on cell rounding were reflected in their effects on steroid production since PV and CP also inhibited FSK-stimulated steroid production. These results suggest that the mechanism through which inhibition of PP1/2A activities induces morphological changes in Y1 cells is fundamentally different from that seen in response to activation of PKA. They are consistent with PKA-induced shape changes in adrenocortical cells being mediated through increased PTP activity and the dephosphorylation of paxillin, and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are intimately linked.