Greater importance of Ca(2+)-calmodulin in maintenance of ang II- and K(+)-mediated aldosterone secretion: lesser role of protein kinase C.

In this study we have investigated various components of the stimulus-secretion coupling process leading to aldosterone secretion from the calf adrenal glomerulosa cells as evoked by angiotensin II (AII) and potassium (K+). The roles of Ca2+, calmodulin and protein kinase C in the sustained phase rather than initiation of aldosterone secretion were of special interest. Our investigations revealed that the reduction of extracellular Ca2+ by EGTA or interruption of Ca2+ influx by nitrendipine at various time points after stimulation with either AII or K+ markedly compromised aldosterone secretion. Calmodulin inhibitors, calmidazolium and W-7 reduced aldosterone secretion profoundly. Agonists of protein kinase C, phorbol ester or diacylglycerol analogues failed to stimulate aldosterone secretion while the protein kinase C inhibitor, H-7, only partially inhibited aldosterone secretion at a concentration which completely inhibited protein kinase C activity. Calmodulin inhibitors produced significantly greater inhibition of aldosterone secretion than inhibitors of protein kinase C.     

p38 MAP kinase is involved in the signalling of sphingosine in osteoblasts: sphingosine inhibits prostaglandin F(2alpha)-induced phosphoinositide hydrolysis

We previously showed that sphingosine inhibits prostaglandin F(2alpha) (PGF(2alpha))-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine on phospholipase C-catalyzing phosphoinositide hydrolysis induced by PGF(2alpha) in these cells. Sphingosine inhibited the inositol phosphates formation by PGF(2alpha) or NaF, a GTP-binding protein activator. Sphingosine induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase but did not affect the phosphorylation of p42/p44 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, rescued the inhibitory effect of sphingosine on the formation of inositol phosphates by PGF(2alpha) or NaF. These results indicate that sphingosine inhibits PGF(2alpha)-induced phosphoinositide hydrolysis by phospholipase C via p38 MAP kinase in osteoblasts.     

Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway

Sphingosine, a metabolite of membrane sphingolipids, is generally considered to be cytotoxic for a variety of cell types. However, we have found that sphingosine at low concentrations stimulates DNA synthesis and acts synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, had no mitogenic effects, suggesting that sphingosine did not induce nonspecific membrane perturbations. Sphingosine, which has been proposed to be a physiological inhibitor of protein kinase C, also markedly potentiates the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Sphingosine still stimulates DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester. At mitogenic concentrations, sphingosine does not bind to protein kinase C as shown by its lack of effect on phorbol dibutyrate binding. Only at higher concentrations, in the cytotoxic range, was there a displacement of phorbol dibutyrate from its cellular-binding sites. In contrast to sphingosine, H-7, a known inhibitor of protein kinase C, inhibited the mitogenic response to TPA and the TPA-induced phosphorylation of the 80 kDa cellular substrate of protein kinase C. Our results suggest that sphingosine may play an important role as a positive regulator of cell growth acting in a fundamentally different, protein kinase C-independent pathway.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.     

Involvement of protein kinase C in angiotensin II-mediated release of 12-hydroxyeicosatetraenoic acid in bovine adrenal glomerulosa cells.

The present study was conducted to determine whether protein kinase C was involved in angiotensin II-mediated release of 12-hydroxyeicosatetraenoic acid (12-HETE) from bovine adrenal glomerulosa cells. Activators of protein kinase C, 12-O-tetradecanoylphorbol 4-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG), significantly increased release of 12-HETE. The effect of OAG was potentiated by BAYK8644, a stimulator of calcium entry. Sphingosine, H-7 and staurosporine, which inhibited the activity of protein kinase C in vitro, almost completely blocked 12-HETE release induced by TPA. These agents also significantly reduced angiotensin II-mediated 12-HETE release. When time course of the liberation of 12-HETE was measured, angiotensin II elicited sustained release of 12-HETE, which was inhibited by staurosporine. These results indicate that angiotensin II induces sustained release of 12-HETE, a feed forward regulator of aldosterone secretion, and that protein kinase C may be involved in this process.     

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.     

IL-5-induced IgA synthesis by LPS-stimulated mouse B cells is prevented by protein kinase C inhibitors.

We have previously demonstrated that activation of protein kinase C (PKC) by phorbol esters induces selectively IgA synthesis by mouse B cells. In this study, we investigated the effects of a number of protein kinase inhibitors on IgA secretion induced by a recombinant murine IL-5 in LPS-stimulated mouse B cells. The results show that PKC inhibitors, such as sphingosine (SPH), staurosporine (STP) and H-7, blocked IL-5-induced IgA synthesis; the protein kinase A inhibitor HA-1004 and the inhibitor of calcium/calmodulin dependent protein kinase W-7 had no effect on IgA secretion induced by IL-5. The proliferation of the IL-5 sensitive B13 cell line in response to IL-5 was also inhibited by addition of SPH or STP or H-7. The data suggest an involvement of the PKC pathway in IL-5-induced B cell differentiation into IgA secreting cells.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

The role of chloride ions in the regulation of steroidogenesis in rat Leydig cells and adrenal cells

The role of chloride ions in the regulation of steroidogenesis in rat Leydig cells and adrenal cells has been investigated. It was found that the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) inhibited LH but not dibutyryl cAMP (dbcAMP)-stimulated steroidogenesis in the Leydig cells. This was found to be via an inhibition of cAMP production, because both LH- and forskolin-stimulated cAMP productions were inhibited by DIDS. The exclusion of chloride ions enhanced steroidogenesis during incubation of Leydig cells and adrenal cells with dbcAMP. The adrenal cells were found to be more sensitive to dbcAMP than Leydig cells and the enhancing effects of chloride removal were higher. In the presence of chloride ions, near maximum steroidogenesis was achieved with approximately 60 μM and 1 mM dbcAMP in the adrenal and Leydig cells, respectively. In the absence of chloride ions the concentrations required decreased approximately 50-fold and 10-fold, respectively. It is concluded that although LH may regulate DIDS sensitive chloride channels, the enhanced stimulation of cAMP-mediated steroidogenesis by chloride exclusion is not mediated via these channels. We propose a model based on the present and previous studies [1] with Leydig tumour (MA10) cells i.e. that intracellular chloride ion depletion enhances the action of cAMP on protein synthesis which results in increased synthesis of the Steroidogenic Acute Regulator (StAR) protein and consequently increased steroidogenesis.     

Atrial natriuretic factor inhibits angiotensin-induced aldosterone secretion: not through cGMP or interference with phospholipase C

ANF did not prevent the formation of [3H] inositol trisphosphate in response to AII but inhibited aldosterone secretion in calf adrenal glomerulosa cells. 8-bromo cGMP did not affect either inositol phosphate formation or aldosterone secretion. Changes in cytosolic Ca++ concentration induced by AII, as measured by Quin 2 fluorescence, were also unaffected by ANF. No difference in adrenal cell protein phosphorylation with AII or AII + ANF was observed. The results suggest that ANF may inhibit aldosterone secretion through a non-guanyl cyclase linked receptor system not involving the formation of phosphoinositide-derived second messengers. Interference with protein kinase C activity cannot be ruled out.     

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.     

Influence of agents that affect intracellular calcium regulation on progesterone secretion in small and large luteal cells of the sheep

Enriched fractions of small and large luteal cells were incubated for 2 h with 1 or 10 microM calcium ionophore, A23187: unstimulated secretion of progesterone and viability in small cells were not affected but these measures were decreased (P less than 0.01) for unstimulated large cells and were significantly correlated (P less than 0.05). This effect in large cells was independent of extracellular calcium. Therefore, incubations of the two cell types were made in the presence of increasing concentrations of a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA). Secretion of progesterone and viability were not augmented in unstimulated small cells, but TPA prevented (P less than 0.05) the full stimulation of secretion of progesterone by LH. Secretion of progesterone in unstimulated large cells was inhibited (P less than 0.01) by TPA (100 nM and 10 microM), although viability was unaffected. The non-tumour promoting phorbol ester, 4 alpha-phorbol didecanoate, had no effect on large cells. Extracellular calcium was not required for the observed effect of TPA. Sphingosine, an agent inhibitory to protein kinase C activity, inhibited (P less than 0.01) secretion of progesterone in small and large cells, and also reduced (P less than 0.01) cell viability. These values were significantly correlated (P less than 0.05) in both cell types. The above observations suggest that protein kinase C may invoke negative regulation on progesterone production in unstimulated large and hormone-stimulated small luteal cells of sheep. Since sphingosine significantly reduced viability in small and large cells and ionophore selectively inhibited viability in large cells, the ability of these agents to influence calcium-mediated intracellular regulation of steroidogenesis is still uncertain.     

Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.     

Effects of angiotensin, prostaglandin E2 and indomethacin on the early and late steps of aldosterone biosynthesis in isolated adrenal cells

The involvement of prostaglandins in the regulation of aldosterone biosynthesis was investigated in isolated adrenal glomerulosa cells. Cells were treated with cyanoketone to inhibit the 3 beta-hydroxy-steroid dehydrogenase and isolate the early step of aldosterone synthesis and the late step. Angiotensin II and PGE2 stimulated the synthesis of aldosterone in a concentration-related manner. The stimulation by both compounds was inhibited by indomethacin, a prostaglandin synthesis inhibitor. Indomethacin also inhibited arachidonic acid-stimulation of 6-keto PGF1 alpha synthesis, whereas cyanoketone was without effect. Both angiotensin II and PGE2 stimulated the synthesis of pregnenolone (the early step) in a concentration-related manner. At higher concentrations, angiotensin II also stimulated the conversion of [3H]corticosterone to [3H]aldosterone (the late step). PGE2 did not alter the late step significantly. Indomethacin had no effect on either biosynthetic step when added alone. However, it inhibited the angiotensin- and PGE2-stimulated pregnenolone synthesis by 41 and 59%, respectively (P less than 0.05). Indomethacin did not alter angiotensin stimulation of the conversion of corticosterone to aldosterone. These findings indicate that PGE2 increases the synthesis of aldosterone by stimulating the conversion of cholesterol to pregnenolone. Indomethacin inhibits angiotensin II- and PGE2-induced steroidogenesis at the same early biosynthetic step. These findings suggest that indomethacin may act by a mechanism other than a reduction in the concentration of prostaglandins.     

Synthesis and evaluation of sphingoid analogs as inhibitors of sphingosine kinases

Sphingosine 1-phosphate (S1P), a product of sphingosine kinases (SphK), mediates diverse biological processes such as cell differentiation, proliferation, motility, and apoptosis. In an effort to search and identify specific inhibitors of human SphK, the inhibitory effects of synthetic sphingoid analogs on kinase activity were examined. Among the analogs tested, we found two, SG12 and SG14, that have specific inhibitory effects on hSphK2. N,N-Dimethylsphingosine (DMS), a well-known SphK inhibitor, displayed inhibitory effects for both SphK1 and SphK2, as well as protein kinase C. In contrast, SG12 and SG14 exhibited selective inhibitory effects on hSphK2. Furthermore, SG14 did not affect PKC. In isolated platelets, SG14 blocked the conversion of sphingosine into sphingosine 1-phosphate significantly. This is the first report on the identification of a hSphK2-specific inhibitor, which may provide a useful tool for studying the biological functions of hSphK2.     

Inhibition of hCG- and cAMP-stimulated progesterone production in MA-10 mouse Leydig tumor cells by ketoconazole

In this study we attempted to examine the effects of ketoconazole on steroid biosynthesis and to determine which steps in the steroidogenic pathway were blocked using MA-10 Mouse Leydig tumor cells. This cloned cell line produces progesterone as the major steroid following stimulation by hCG or dbcAMP. At a concentration of 1 microM ketoconazole completely inhibited the hCG- and dbcAMP-stimulated progesterone synthesis in MA-10 Leydig cells. The conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone was also suppressed by this drug. The presence of ketoconazole inhibited mitochondrial steroid synthesis but required high concentrations of the drug as compared to inhibition in intact cells. No accumulation of pregnenolone was observed in the presence of ketoconazole indicating that the activity of 3 beta-hydroxysteroid dehydrogenase was not affected. We conclude that ketoconazole directly inhibits the activity of cholesterol side-chain cleavage enzyme (CSCC), a rate-determining enzymatic step in steroidogenesis, by interacting with cytochrome P-450scc.     

Dantrolene inhibits adrenal steroidogenesis by a mechanism independent of effects on stored calcium release

The muscle relaxant dantrolene has been widely used in signal transduction studies as an inhibitor of intracellular calcium release. However, in vivo studies have shown that the drug may inhibit steroidogenesis by a mechanism which is distinct from its effects on calcium mobilization. Using freshly isolated cells and mitochondria from the outermost regions of bovine adrenal cortex we have shown that dantrolene (0.2 mM) significantly inhibits steroid synthesis stimulated by either angiotensin II (AII) or by addition of various precursors. Our results suggest that dantrolene inhibits the rate-limiting steps of adrenocortical steroidogenesis, i.e. the intramitochondrial conversion of cholesterol to pregnenolone (for both aldosterone and cortisol) and the conversion of corticosterone to aldosterone (for aldosterone), by a mechanism independent from its known effects on calcium release. A possible alternative mechanism may involve direct inhibition of cytochrome P450-dependent hydroxylation reactions.     

Angiotensin stimulation of adrenal fasciculata cells

In this paper we provide evidence to show that the pathways by which adrenocorticotropic hormone (ACTH) and angiotensin II (AII) stimulate steroidogenesis in bovine fasciculata cells are only partially independent. Both hormones have the same intrinsic activity but a 500-fold higher dose of AII is required to achieve 50% stimulation of steroidogenesis. Whereas ACTH acts by way of cAMP, AII appears to operate through protein kinase C. The phorbol ester, 12-O-tetradecanoylphorbol-13 acetate (TPA), and the calcium ionophore, A23187, each stimulate steroidogenesis and, when added together, act synergistically. To test the relationship between the ACTH and AII pathways, we added the two hormones simultaneously and measured steroid production. When the hormones were present at submaximal concentrations, their effects were additive. At maximal doses, steroid production was 40% above that elicited by either hormone alone. In contrast to the action of AII in the glomerulosa cell where it inhibits ACTH-stimulated cAMP formation, AII causes no inhibition in the fasciculata. Cycloheximide inhibits steroidogenesis stimulated by AII or a mixture of TPA and A23187. Scatchard analysis of the binding of 125I-AII to particulates from adrenal cortical fasciculata indicates the presence of a single class of binding sites (Kd = 0.6 X 10(-8) M). Binding is not inhibited by ACTH. Biotin-containing AII analogs that bind specifically to the particulates have been evaluated as potential tools for avidin-biotin affinity chromatography of the receptor. One of these, [N epsilon-6-(biotinylamido)hexyllys1, Val5] AII, is a promising candidate for receptor isolation.     

Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid capable of regulating critical physiological and pathological functions. Here, we report for the first time that S1P stimulates aldosterone secretion in cells of the zona glomerulosa of the adrenal gland. Regulation of aldosterone secretion is important because this hormone controls electrolyte and fluid balance and is implicated in cardiovascular homeostasis. S1P-stimulated aldosterone secretion was dependent upon the protein kinase C (PKC) isoforms alpha and delta and extracellular Ca2+, and it was inhibited by pertussis toxin (PTX). S1P activated phospholipase D (PLD) through a PTX-sensitive mechanism, also involving PKC alpha and delta and extracellular Ca2+. Primary alcohols, which attenuate the formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD activity, blocked S1P-stimulated aldosterone secretion. Furthermore, propranolol, chlorpromazine, and sphingosine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked aldosterone secretion. These data suggest that the PLD/PAP pathway plays a crucial role in the regulation of aldosterone secretion by S1P and that Gi protein-coupled receptors, extracellular Ca2+, and the PKC isoforms alpha and delta are all important components in the cascade of events controlling this process.     

Sphingosine induces apoptosis in hippocampal neurons and astrocytes by activating caspase-3/-9 via a mitochondrial pathway linked to SDK/14-3-3 protein/Bax/cytochrome c

The present study examined sphingosine-induced apoptosis in cultured rat hippocampal neurons and astrocytes. Sphingosine induced apoptosis in a concentration (1-100 μM)-dependent manner, that is inhibited by the PKC-δ inhibitor rottlerin, and a similar effect was obtained with the sphingosine kinase inhibitors, to raise intracellular sphingosine concentrations. Sphingosine increased presence of sphingosine-dependent protein kinase (SDK), and the effect was suppressed by rottlerin. Sphingosine increased phosphorylated 14-3-3 protein, thereby transforming the protein from a dimeric structure into a monomeric structure. Sphingosine accumulated Bax in the mitochondria and stimulated cytochrome c release into the cytosol, and those effects were inhibited by rottlerin. Sphingosine disrupted mitochondrial membrane potentials, that was abolished by silencing the PKC-δ-targeted gene. Moreover, sphingosine activated caspase-9 and the effector caspase-3 in a PKC-δ-dependent manner. Taken together, the results of the present study indicate that sphingosine activates SDK, produced through proteolytic processing of an active form of PKC-δ, to phosphorylate 14-3-3 protein and transform into a monomeric structure, causing Bax dissociation from 14-3-3 protein and accumulation in the mitochondria, which perturbs mitochondrial membrane potentials allowing cytochrome c release into the cytosol, to activate caspase-9 and the effector caspase-3, responsible for apoptosis in hippocampal neurons and astrocytes.