Mouse spermatozoa require extracellular Na+ for both capacitation and acrosomal exocytosis, but the minimum concentrations differ widely: > 1 < or = 25 mmol Na+ l-1 will support capacitation, but > 125 mmol Na+ l-1 is needed for acrosomal exocytosis in capacitated cells. Our conclusions are based on evidence obtained from sperm cells preincubated in iso-osmotic media with differing concentrations of Na+ and then analysed for occurrence of the acrosome reaction, capacitation-related changes in chlortetracycline (CTC) fluorescence and in vitro fertilization. The modified Tyrode's medium used as the control medium in these experiments contained 150 mmol Na+ l-1 and supported full sperm function. At least some of the Na+ needs to be internalized to promote the functional changes, as evidenced by the ability of the monovalent cation ionophore monensin to accelerate capacitation and trigger acrosomal exocytosis in control medium. However, in low Na+ (25 mmol l-1) medium, monensin could only modulate the transition to the capacitated state, assessed with CTC, indicating that higher concentrations of extracellular Na+ are required for initiation of acrosomal exocytosis. We suggest that changes in the composition of the female reproductive tract fluids serve to control expression of sperm functional potential. Before ovulation in the mouse, sufficient Na+ and Ca2+ are present to promote capacitation. However, the Na+ concentration is marginal for support of acrosomal exocytosis and the relatively high K+ reinforces an inhibition of exocytosis. At ovulation, the release of follicular fluid would increase the Na+ and decrease the K+ concentrations, thereby permitting full expression of fertilizing potential. Possible mechanisms that might be involved in the Na(+)-related responses, including a Na(+)-Ca2+ exchanger, a Na(+)-K+ ATPase and a Na(+)-H+ exchanger, were also investigated. If a Na(+)-Ca2+ exchanger has a role to play, it is not during capacitation per se. Incubation of sperm cells in high Na+, low Ca2+ (90 mumol CaCl2 l-1) medium that supports capacitation, followed by introduction of monensin, which would have promoted an influx of Na+ and could have, in turn, activated a Na+ out, Ca2+ in response, did not accelerate transition to the capacitated state (B pattern of CTC fluorescence). In contrast, it is possible that a Na(+)-K+ ATPase may play a role during capacitation. Incubation of suspensions in control medium plus ouabain, which would inhibit the ATPase, significantly accelerated the transition from the incapacitated to the capacitated state, although it did not trigger acrosomal exocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

In addition to the well-documented role of protein kinases in the regulation of steroid production, phosphoprotein phosphatase (PP) activity is required for steroidogenesis. In the present study, we have used the mouse Y1 adrenocortical cell line to identify the site of action of PPs on steroid production by measuring the effects of PP inhibition on the expression of the steroidogenic acute regulatory (StAR) protein and on steroid production. Forskolin-induced activation of cyclic AMPdependent protein kinase (PKA) enhanced steroidogenesis and this was accompanied by an increased expression of StAR protein. Both steroidogenesis and StAR protein expression were inhibited by two structurally dissimilar inhibitors of PP1 and PP2A activities, okadaic acid and calyculin A. These results suggest that inhibition of PP1 and PP2A inhibits steroid production by preventing the expression of the StAR protein, implicating PP1/2A dephosphorylation reactions as important regulators of stimulus-dependent StAR protein expression, and thus of steroidogenesis.

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.

We have recently demonstrated that the protein phosphatases PP1, PP2A and PP2B are expressed in the rat adrenal cortex and that hctionally active PP 1 and/or PP2A are required for the maintenance of steroidogenic responses [ 11. Thus, inhibition of PP1/2A activity with calyculin A and okadaic acid blocked the response to cyclic AMP-mobilizing stimuli, and this effect was localized to a site occurring after the activation of protein kinase A but before the delivery of cholesterol to the mitochondrion. The conversion of cholesterol to pregnenolone is the rate-limiting step in steroidogenesis and known to be controlled by the steroidogenic acute regulatory (StAR) protein [2]. In order to determine whether the effect of protein phosphatase inhibitors might be occurring at the level of StAR protein expression, we have now examined the effects of calyculin A, an inhibitor of PP1/2A activity, in Y1 mouse adrenocortical tumor cells. Y1 cells (150,000 celM200 pl) were cultured in DMEM medium in the presence or absence of forskolin and calyculin A for 6 hours. Steroid production was measured by RIA. In parallel experiments, mitochondria were isolated from Y1 cells that had been exposed to DMEM alone, DMEM + CA, DMEM + FSK, or DMEM + FSK + CA. Protein extracts (1 50-200 pg of protein) were separated by SDS-PAGE, and probed with antibody recognising the 30 kDa form of StAR [2]. StAR protein expression was quantified by scanning densitometry of Western 413 Copyright C 1998 by Marcel Dekker. Inc w w w .dekker.com Endocr Res Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.

Background: Salt used in the household is the most common way for iodine fortification to prevent the public health concerns of iodine deficiency disorders. The objective of the study was to assess household coverage with adequately iodized salt in Telangana State, India. Methods: Secondary data published in the District Level Household and Facility Survey-4 is analyzed. Results: Overall, 55.7 percent of the households were using cooking salt which was iodized at the recommended level of 15 ppm or more. Around 60.8 percent of households in urban areas used salt with 15 ppm or more iodine content compared with 51.9 percent of households in rural areas though the difference was not significant. Conclusions:The coverage of adequately iodized salt in the state was below the national average. To further expand the coverage of adequately iodized salt, researchers and policy makers should take action.

Protein kinase C (PKC) consists of a large gene family of isoenzymes that differ in their structure, cofactor requirements, kinetic properties, substrate specificity and tissue distribution (reviewed in [ 11). Molecular cloning has revealed the existence of at least 10 isoforms which can be broadly divided into two groups: the calcium-dependent or conventional PKCs (a, 01, 011 and y isoforms) and the novel, calcium-independent isoforms (6, E , (, 7 and 0). There is now substantial evidence to support the involvement of PKC in both the initiation and termination of cellular signals elicited by receptor-operating platelet agonists and at least three isoforms of PKC (a, B and fl have been identified in human platelets [2,3]. Evidence for PKC involvement in platelet function has been drawn principally from studies employing kinase inhibitors, including H7 and staurosporine [4]. These agents inhibit kinase activity by competing with ATP and are known to inhibit a wide range of serine-threonine kinases in addition to PKC. More recently, a potent PKC inhibitor (Ro 31-8220) has been developed [5] which offers a greater degree of selectivity for PKC versus other kinases but which can still exert non-selective effects, particularly at high concentrations [6]. Pseudosubstrate or autoinhibitory sequences have been proposed to function as endogenous regulators of several protein kinases and peptides spanning this region of PKC are highly selective inhibitors of the enzyme both in vitro and in permeabilised cell preparations [7], including saponin-permeabilised platelets [8]. Recent studies have shown that N-terminal myristoylation of a nonapeptide PKC inhibitor based upon the pseudosubstrate sequence of PKC-a and -B renders the peptide cell permeant and allows its use as an inhibitor of the enzyme in intact cells [9]. In the present study we have examined the effects of the myristoylated peptide (myr-PKC,,,) and a non-myristoylated peptide with the same sequence (PKC,,,) on platelet PKC activity in vitro, and on protein phosphorylation and secretion in intact human platelets. Measurements of protein phosphorylation and dense-granule secretion were carried out, respectively, in ["PIP,-and 5-hydr~xy['~C]tryptamine-labelled washed human platelets using previously described methodologies [ 101. PKC activity in vitro was measured essentially as described by Wheeler-Jones ef al.[ll]. Fig.1 depicts the effects of incubation with either the myristoylated peptide or PKC,,, on PKC activity partially purified from human platelets. Myr-PKC,,, potently attenuated PKC activity with maximum effects observed at 100 pM. Enzyme activity was also inhibited in a dose-dependent manner by PKC,,, with maximum inhibitory effects achieved at approximately 1 mM.Stimulation of platelets with phorbol esters or with receptor-operating agonists which lead to PKC activation results in the characteristic phosphorylation of several protein substrates, most notably pleckstrin (approx. 45 kDa) and myosin light chain (20 kDa). In [3ZP]Pi-labelled platelets m...

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