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This study evaluated whether improved insulin-stimulated glucose uptake in recovery from acute exercise coincides with reduced malonyl-CoA (MCoA) content in human muscle. Furthermore, we investigated whether a high-fat diet [65 energy-% (Fat)] would alter the content of MCoA and insulin action compared with a high-carbohydrate diet [65 energy-% (CHO)]. After 4 days of isocaloric diet on two occasions (Fat/CHO), 12 male subjects performed 1 h of one-legged knee extensor exercise (~80% peak workload). Four hours after exercise, insulin-stimulated glucose uptake was determined in both legs during a euglycemic-hyperinsulinemic clamp. Muscle biopsies were obtained in both legs before and after the clamp. Four hours after exercise, insulin-stimulated glucose uptake was improved (~70%, P < 0.001) independent of diet composition and despite normal insulin-stimulated regulation of insulin receptor substrate-l-associated phosphatidylinositol 3-kinase, Akt, GSK-3, and glycogen synthase. Interestingly, exercise resulted in a sustained reduction (~20%, P < 0.05) in MCoA content 4 h after exercise that correlated (r = 0.65, P < 0.001) with improved insulin-stimulated glucose uptake. Four days of Fat diet resulted in an increased content of intramyocellular triacylglycerol (P < 0.01) but did not influence muscle MCoA content or whole body insulin-stimulated glucose uptake. However, at the muscular level proximal insulin signaling and insulin-stimulated glucose uptake appeared to be compromised, although to a minor extent, by the Fat diet. Collectively, this study indicates that reduced muscle MCoA content in recovery from exercise may be part of the adaptive response leading to improved insulin action on glucose uptake after exercise in human muscle. AMP-activated protein kinase; acetyl coenzyme A carboxylase; intramuscular triacylglycerol; Akt; diet

Protein kinase B (PKB) is known to mediate a number of biological responses to insulin and growth factors, its role in glucose uptake being one of the most extensively studied. In this work, we have employed a recently described allosteric inhibitor of PKB, Akti, to clarify the role of PKB in lipid metabolism in adipocytes--a subject that has received less attention. Pretreatment of primary rat and 3T3L1 adipocytes with Akti resulted in dose-dependent inhibition of PKB phosphorylation and activation in response to insulin, without affecting upstream insulin signaling [insulin receptor (IR), insulin receptor substrate ORS)] or the insulin-induced phosphoinositide 3-kinase (PI3K)-dependent activation of the ERK/p90 ribosomal kinase (RSK) pathway. PKB activity was required for the insulin-induced activation of phosphodiesterase 3B (PDE3B) and for the antilipolytic action of insulin. Moreover, inhibition of PKB activity resulted in a reduction in de novo lipid synthesis and in the ability of insulin to stimulate this process. The regulation of the rate-limiting lipogenic enzyme acetyl-CoA carboxylase (ACC) by insulin through dephosphorylation of S79, which is a target for AMP-activated protein kinase (AMPK), was dependent on the presence of active PKB. Finally, AMPK was shown to be phosphorylated by PKB on S485 in response to insulin, and this was associated with a reduction in AMPK activity. In summary, we propose that PKB is required for the positive effects of insulin on lipid storage and that regulation of PDE3B and AMPK by PKB is important for these effects. Akt; phosphodiesterase 3B; acetyl-coenzyme A carboxylase; adenosine 5'-monophosphate-activated protein kinase; lipogenesis

The sarcolemmal [Na.sup.+]-[K.sup.+] pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to [epsilon]-protein kinase C ([epsilon]PKC), previously implicated in ANG II-induced [Na.sup.+]-[K.sup.+] pump inhibition. A role for [epsilon]PKC was also supported by an ANG H-induced increase in coimmunoprecipitation of [epsilon]PKC with the receptor for the activated kinase and with the cytosolic [p47.sup.phox] subunit of NADPH oxidase. ANG II decreased electrogenic [Na.sup.+]-[K.sup.+] pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by [epsilon]PKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the [Na.sup.+]-[K.sup.+] pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The [[alpha].sub.1]-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated [p22.sup.phox] and cytosolic [p47.sup.phox] NADPH oxidase subunits at baseline. ANG II had no effect on [[alpha].sub.1]/caveolin 3 or [[alpha].sub.1]/[p22.sup.phox] interaction, but it increased [[alpha].sub.1]/[p47.sup.phox] coimmunoprecipitation. We conclude that ANG II inhibits the [Na.sup.+]-[K.sup.+] pump via PKC-dependent NADPH oxidase activation. reduced nicotinamide adenine dinucleotide phosphatase oxidase; oxidant signaling; glutathionylation; protein kinase C; [Na.sup.+]-[K.sup.+]-ATPase

Acidic luminal pH and low [HC[O.sup.-.sub.3]] maintain sperm quiescent during maturation in the epididymis. The vacuolar [H.sup.+]-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HC[O.sup.-.sub.3], induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and nonhuman primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by [N.sup.6]-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [[sup.32]P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. vas deferens; male reproductive tract; metabolism; soluble adenylyl cyclase; acid secretion

Skeletal muscle fibers differ considerably in their metabolic and physiological properties. Skeletal muscle displays a high degree of metabolic flexibility, which allows the myofibers to adapt to various physiological demands by shifting energy substrate utilization. Transcriptional events play a pivotal role in the metabolic adaptations of skeletal muscle. The expression of genes essential for skeletal muscle glucose and lipid metabolism is tightly coordinated in support of a shift in substrate utilization. AMP-activated protein kinase (AMPK) and calcineurin (a calcium-regulated serine/threonine protein phosphatase) regulate skeletal muscle metabolic gene expression programs in response to changes in the energy status and levels of neuronal input, respectively. AMPK and calcineurin activate transcriptional regulators such as peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] and myocyte enhancer factor as well as increase skeletal muscle oxidative capacity and mitochondrial gene expression. Activation of either the AMPK or calcineurin pathway can also enhance the glycogen storage capacity and insulin sensitivity in skeletal muscle. Characterization of pathways governing skeletal muscle metabolism offers insight into physiological and pharmacological strategies to prevent or ameliorate peripheral insulin resistance associated with metabolic disorders such as type 2 diabetes. adenosine 5'-monophosphate-activated protein kinase

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-[delta] and PKC-[epsilon] reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-[delta] and -[epsilon], reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-[delta] and -[epsilon] isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-[epsilon] redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-[epsilon] overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-[delta] and -[epsilon] isoforms translocate to specific acinar cell compartments and modulate zymogen activation. translocation; PKC activator phorbol ester; PKC inhibitor

Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial [Ca.sup.2+] reabsorption in kidney. Many hormones alter renal [Ca.sup.2+] reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal [Ca.sup.2+] reabsorption by PTH. dynamin; caveolin; parathyroid hormone; phospholipase C; Madin-Darby canine kidney cells

Obese conditions increase the expression of adipocytokine monocyte chemoattractant protein-1 (MCP-1) in adipose tissue as well as MCP-1 plasma levels. To investigate the mechanism behind increased MCP-1, we used a model in which 3T3-L1 adipocytes were artificially hypertrophied by preloading with palmitate in vitro. As observed in obesity, under our model conditions, palmitate-preloaded ceils showed significantly increased oxidative stress and increased MCP-1 expression relative to control cells. This increased MCP-1 expression was enhanced by adding exogenous tumor necrosis factor-[alpha] (TNF-[alpha]; 17.8-fold vs. control cells, P < 0.01 ) rather than interleukin-1[beta] (IL-1[beta]; 2.6-fold vs. control cells, P < 0.01). However, endogenous TNF-[alpha] and IL-1[beta] release was not affected in hypertrophied cells, suggesting that these endogenous cytokines do not mediate hypertrophy-induced increase in MCP-1. MCP-1 secretion from hypertrophied cells was significantly decreased by treatment with antioxidant N-acetyl-cysteine, JNK inhibitors SP600125 and JIP-1 peptide, and I[kappa]B phosphorylation inhibitors BAY 11-7085 and BMS-345541 (P < 0.01). MCP-1 secretion was not affected by peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) antagonists assayed. Adiponectin, another adipocytokine studied in parallel, also showed increased release in hypertrophy relative to control cells. But in contrast to MCP-1, adiponectin release was significantly suppressed by both exogenous TNF-[alpha] and IL-1[beta] as well as by PPAR[gamma] antagonists bisphenol A diglycidyl ether and T0070907 (P < 0.01). JNK inhibitors and I[kappa]B phosphorylation inhibitors showed no significant effect on adiponectin. We conclude that adipocyte hypertrophy through palmitate loading causes oxidative stress, which in turn increases MCP-1 expression and secretion through JNK and I[kappa]B signaling. In contrast, the parallel increase in adiponectin expression appears to be related to the PPAR[gamma] ligand properties of palmitate. c-Jun N[H.sub.2]-terminal kinase; monocyte chemoattractant protein-1; tumor necrosis factor-[alpha]; interleukin-1[beta]

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum long us (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of [H.sub.2][O.sub.2], EDL muscles from rats were preincubated with the [H.sub.2][O.sub.2] scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that [H.sub.2][O.sub.2] is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct [H.sub.2][O.sub.2] also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 [micro]mol/l [H.sub.2][O.sub.2]. [H.sub.2][O.sub.2]-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor [N.sup.G]-monomethyl-L-arginine. [H.sub.2][O.sub.2] stimulated the phosphorylation of Akt [Set.sup.473] (7-fold) and [Thr.sup.308] (2-fold) in isolated EDL muscles. [H.sub.2][O.sub.2] at 600 [micro]mol/l had no effect on ATP concentrations and did not increase the activities of either the [alpha]1 or [alpha]2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism. hydrogen peroxide; Akt phosphorylation; adenosine monophosphate-activated protein kinase

Lipopolysaccharide (LPS) endotoxin is a causative agent of sepsis. The aim of this study was to examine LPS effects on intestinal fructose absorption and to decipher mechanisms. Sepsis was induced by intravenous injection of LPS in rabbits. The ultrastructural study and DNA fragmentation patterns were identical in the intestine of LPS and sham animals. LPS treatment reduced fructose absorption altering both mucosal-to-serosal transepithelial fluxes and uptake into brush border membrane vesicles (BBMVs). Cytochalasin B was ineffective on fructose uptake, indicating that GLUT5, but not GLUT2, transport activity was targeted. GLUT5 protein levels in BBMvs were lower in LPS than in sham-injected rabbits. Thus lower fructose transport resulted from lower levels of GLUT5 protein. LPS treatment decreased GLUT5 levels by proteasome-dependent degradation. Specific inhibitors of PKC, PKA, and MAP kinases (p38MAPK, JNK, MEK 1/2) protected fructose uptake from adverse LPS effect. Moreover, a TNF-[alpha] antagonist blocked LPS action on fructose uptake. We conclude that intestinal fructose transport inhibition by LPS is associated with diminished GLUT5 numbers in the brush border membrane of enterocytes triggered by activation of several interrelated signaling cascades and proteasome degradation. GLUT5; small intestine; LPS; intracellular signals; rabbit

Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK), which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied overnight-fasted adult male rats by use of an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU x [min.sup.-1] x [kg.sup.-1]) was begun either 10 min before ischemia ([Insuli.sub.BI]), 5 min before reperfusion ([Insulin.sub.BR]), or 30 min after the onset of reperfusion ([Insulin.sub.AR]), and continued until the end of the study. Compared with saline control, insulin decreased the infarct size in both [Insuli.sub.BI] (P < 0.001) and [Insulin.sub.BR] (P < 0.02) rats hut not in [Insulin.sub.AR] rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK compared with the nonischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg iv 10 min before ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in [Insulin.sub.BI] and [Insulin.sub.BR] rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB-239063, a potent and specific inhibitor of p38 MAPK, 10 rain before reperfusion enabled insulin-mediated myocardial protection in [Insulin.sub.AR] rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion, and activation of p38 MAPK abolishes insulin's cardioprotective effect. insulin; myocardial infarction; anisomycin

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular [Ca.sup.2+] handling in hydrogen sulfide ([H.sub.2]S)-preconditioned cardiomyocytes and its consequent effects on iscbemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an [H.sub.2]S donor, [10.sup.-4] M) preconditioning (SP, 30 min). Intracellular [Ca.sup.2+] was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKC[alpha], PKC[epsilon], and PKC[delta] to membrane fraction but only translocation of PKC[epsilon] and PKC[delta] was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [[Ca.sup.2+]] transients, which were reversed by a selective PKC inhibitor cbelerythrine. These data suggest that SP facilitated [Ca.sup.2+] removal via both accelerating uptake of [Ca.sup.2+] into sarcoplasmic reticulum and enhancing [Ca.sup.2+] extrusion through [Na.sup.+]/[Ca.sup.2+] exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against [Ca.sup.2+] overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKC[alpha], PKC[epsilon], and PKC[delta] in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular [Ca.sup.2+] handling. protein kinase C isoforms; ischemia and reperfusion; cardioprotection; ATP-sensitive potassium channel

The vacuolar [H.sup.+]-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland ceils energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of [V.sub.0] and [V.sub.1] subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of [V.sub.1] components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced [V.sub.1] translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA. vacuolar [H.sup.+]-adenosine 5'-triphosphatase; adenosine 3',5'-cyclic monophosphate; exchange protein directly activated by adenosine 3',5'-cyclic monophosphate; insect

Activation of the p38 mitogen-activated protein kinase (MAPK) pathway induces inflammation, apoptosis, and fibrosis. However, little is known of the contribution of the upstream kinases, MMK3 and MKK6, to activation of the p38 kinase in the kidney and consequent renal injury. This study investigated the contribution of MKK3 to p38 MAPK activation and renal injury in the obstructed kidney. Groups of eight wild-type (WT) or Mkk3-/- mice underwent unilateral ureteric obstruction (UUO) and were killed 3 or 7 days later. Western blotting showed a marked increase in phospho-p38 (p-p38) MAPK in UUO WT kidney. The same trend of increased p-p38 MAPK was seen in the UUO Mkk3-/- kidney, although the actual level of p-p38 MAPK was significantly reduced compared with WT, and this could not be entirely compensated for by the increase in MKK6 expression in the Mkk3-/- kidney. Apoptosis of tubular and interstitial cells in WT UUO mice was reduced by 50% in Mkk3-/- UUO mice. Furthermore, cultured Mkk3-/- tubular epithelial cells showed resistance to [H.sub.2][O.sub.2]-induced apoptosis, suggesting a direct role for MKK3-p38 signaling in tubular apoptosis. Upregulation of MCP-1 mRNA levels and macrophage infiltration seen on day 3 in WT UUO mice was significantly reduced in Mkk3-/- mice, but this difference was not evident by day 7. The development of renal fibrosis in Mkk3-/-UUO mice was not different from that seen in WT UUO mice. In conclusion, these studies identify discrete roles for MKK3-p38 signaling in renal cell apoptosis and the early inflammatory response in the obstructed kidney. MKK6; macrophage; MCP-1, fibrosis

The androgen-secreting Leydig cells produce cGMP, but the pathways responsible for generation and actions of this intracellular messenger have been incompletely characterized in these cells. Here, we show the presence of mRNA transcripts for the membrane-bound and soluble guanylyl cyclases (sGC), the cGMP-specific phosphodiesterase 5, and the cGMP-dependent protein kinase I (PKG I) and PKG II in purified rat Leydig cells from adult animals. Stimulation of both guanylyl cyclases and inhibition of phosphodiesterase 5 in vitro were accompanied by elevations in cGMP and androgen production, whereas inhibition of sGC and PKG led to a decrease in steroidogenesis. The stimulatory action of cGMP on steroidogenesis was preserved in cells with inhibited cAMP-dependent protein kinases. Experiments with exogenously added substrates revealed the dependence of cGMP-induced progesterone and androgen synthesis on cholesterol but not on 22-OH cholesterol, pregnenolone, progesterone, and [[DELTA].sup.4]-androstenedione. Treatment with nitric oxide donor increased phosphorylation of the steroidogenic acute regulatory protein (STAR). In contrast, inhibition of sGC and PKG, but not protein kinase A, significantly reduced StAR phosphorylation. These results suggest that cGMP contributes to the control of basal steroidogenesis in Leydig cells through the PKG-dependent modification of the StAR protein. cGMP; steroidogenic acute regulatory protein

5'-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pymvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKcd activity in [[alpha].sup.2]KO mice (90%, P < 0.01) and increased AMPK[alpha]2 activity in wild-type (WT) mice (110%, P < 0.05), leading to increased AMPK[alpha] [Thr.sup.172] (WT: 40%, [[alpha].sup.2]KO: 100%, P < 0.01) and ACC[beta] [Ser.sup.227] phosphorylation (WT: 70%, [[alpha].sub.2]KO: 210%, P < 0.01). Compared with rest, exercise significantly induced PDH-[E.sub.1] [alpha] site 1 (WT: 20%, [[alpha].sub.2]KO: 62%, P < 0.01) and site 2 (only [[alpha].sub.2]KO: 83%, P < 0.01) dephosphorylation and [PDH.sub.a] [~200% in both genotypes (P < 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the [[alpha].sub.2]KO mice both at rest and during exercise. The increased [PDH.sub.a] activity during exercise was associated with elevated glycolytic flux, and muscles from the [[alpha].sub.2]KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of [[alpha].sub.2]KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-[E.sub.1] [alpha] phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPK[alpha]2 expression. During exercise, [[alpha].sub.2]KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and [PDH.sub.a] activity. This may be linked to decreased mitochondrial oxidative capacity. adenosine 5'-monophosphate-activated protein kinase; 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside; treadmill running; glucose metabolism

Airway goblet cell mucin secretion is controlled by agonist activation of [P2.sub.2] purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (I[P.sub.3]), diacylglycerol, [Ca.sup.2+] and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC[alpha], nPKC[delta], nPKC[epsilon], and nPKC[eta]; of these, only nPKC[delta] translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC[alpha], nPK[delta], and nPKC[eta] had the same levels of ATP[gamma]S- and phorbol-l,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC[epsilon] (14.6 and 23.5%, for ATP[gamma]S and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC[epsilon] exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATP[gamma]S-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in [P2Y.sub.2]-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC[delta] knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC[epsilon] KO mice relative to its WT littermates. We conclude that nPKC[epsilon] is the effector isoform downstream of [P2Y.sub.2]-R activation in the goblet cell secretory response. The translocation of nPKC[delta] observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung

To determine whether ceramide mediates regulation of the renal epithelial sodium channel (ENaC) by tumor necrosis factor-[alpha] (TNF-[alpha]), confocal microscopy and patch-clamp experiments were performed in A6 distal nephron cells. We found that TNF-[alpha] (100 ng/ml) had no effect on ENaC activity and ceramide level when the cells were grown in the presence of aldosterone, but significantly inhibited ENaC and induced ceramide production after the cells were pretreated with LY 294002, an inhibitor of phosphatidylinositol 3-kinase, for 24 h. The inhibition of ENaC induced by TNF-[alpha] was mimicked by exogenous sphingomyelinase (0.1 U/ml) and [C.sub.2]-ceramide (50 [micro]M), but neither [C.sub.2]-dihydroceramide, a membrane-impermeable analog of [C.sub.2]-ceramide, nor choline, and abolished by pretreatment with GF109203X, a protein kinase C (PKC) inhibitor. [C.sub.2]-ceramide failed to affect ENaC in the cells pretreated with GF109203X, but not in the cells pretreated with PD-98059, a mitogen-activated protein kinase kinase inhibitor. [C.sub.2]-ceramide induced the externalization of phosphatidylserine (PS) in control A6 cells, but not in the cells pretreated with GFI09203X. Together with our previous finding that cytosolic PS maintains ENaC activity in A6 cells, these data suggest that ceramide mediates TNF-[alpha] inhibition of the renal ENaC via a pathway associated with PKC-dependent externalization of PS. A6 cells; single-channel recordings; confocal microscopy; sphingomyelinase; [C.sub.2]-ceramide; phosphatidylserine

[PGE.sub.2] plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and [PGE.sub.2] synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that [PGE.sub.2] has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of [PGE.sub.2] on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by [PGE.sub.2]. In addition, [PGE.sub.2] is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of [PGE.sub.2] on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer. colon cancer; prostaglandin [E.sub.2]; Akt; protein kinase A; phosphatidylinositol 3-kinase

Secretin stimulates ductal secretion by activation of cAMP [right arrow] PKA [right arrow] CFTR [right arrow] [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger in cholangiocytes. We evaluated the expression of [[alpha].sub.2A-], [[alpha].sub.2B-], and [[alpha].sub.2C-]-adrenergic receptors in cholangiocytes and the effects of the selective [[alpha].sub.2]-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the [Na.sup.+]/[H.sup.+] exchanger isoform NHE3; and 3) cAMP levels, PKA activity, [Cl.sup.-] efflux, and [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger activity in purified cholangiocytes. [[alpha].sub.2]-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, [Cl.sup.-] efflux, and [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. [[alpha].sub.2]-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases. bicarbonate secretion; chloride efflux; gastrointestinal hormones; intrahepatic biliary epithelium: protein kinase A

5'AMP-activated protein kinase (AMPK) signaling initiates adaptive changes in skeletal muscle fibers that restore homeostatic energy balance. The purpose of this investigation was to examine, in rats, the fiber-type protein expression patterns of the [alpha]-catalytic subunit isoforms in various skeletal muscles, and changes in their respective contents within the tibialis anterior (TA) after chronic low-frequency electrical stimulation (CLFS; 10 Hz, 10 h daily), applied for 4 [+ or -] 1.2 or 25 [+ or -] 4.8 days. Immunocytochemical staining of soleus (SOL) and medial gastrocnemius (MG) showed that 86 [+ or -] 4.1 to 97 [+ or -] 1.4% of type IIA fibers stained for both the [alpha]1- and [alpha]2-isoforms progressively decreased to 63 [+ or -] 12.2% of type IID/X and 9 [+ or -] 2.4% of IIB fibers. 39 [+ or -] 11.4% of IID/X and 83 [+ or -] 7.9% of IIB fibers expressed only the [alpha]2 isoform in the MG, much of which was localized within nuclei, [alpha]1 and [alpha]2 contents, assessed by immunoblot, were lowest in the white gastrocnemius [WG; 80% myosin heavy chain (MHC) IIb; 20% MHCIId/x]. Compared with the WG, [alpha]1 content was 1.6 [+ or -] 0.08 (P < 0.001) and 1.8 [+ or -] 0.04 (P < 0.0001)-fold greater in the red gastrocnemius (RG: 13%, MHCIIa) and SOL (21%, MHCIIa), respectively, and increased in proportion to MHCIIa content. Similarly, [alpha]2 content was 1.4 [+ or -] 0.10 (P < 0.02) and 1.5 [+ or -] 0.07 (P < 0.001)-fold greater in RG and SOL compared with WG. CLFS induced 1.43 [+ or -] 0.13 (P < 0.007) and 1.33 [+ or -] 0.08 (P < 0.009)-fold increases in the [alpha]1 and [alpha]2 contents of the TA and coincided with the transition of faster type IIB and IID/X fibers toward IIA fibers. These findings indicate that fiber types differ with regard to their capacity for AMPK signaling and that this potential is increased by CLFS. myosin heavy chain; fast-twitch muscle; slow-twitch muscle

Arterial smooth muscle constriction in response to pressure, i.e., myogenic tone, may involve calcium-dependent and calcium-sensitization mechanisms. Calcium sensitization in vascular smooth muscle is regulated by kinases such as PKC and Rho kinase, and activity of these kinases is known to be altered in cardiovascular disorders. In the present study, we evaluated the relative contribution of PKC and Rho kinase to myogenic tone in cerebral arteries in hypertension. Myogenic tone and arterial wall calcium in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were measured simultaneously, and the effect of PKC and Rho kinase inhibitors on myogenic tone was evaluated. SHR arteries showed significantly greater myogenic tone than WKY arteries. Pressure/wall tension-arterial wall calcium curves showed a hyperbolic relation in WKY rats, but the curves for SHR arteries were parabolic. Myogenic tone was decreased by the Rho kinase inhibitors Y-27632 and HA-1077, with a significantly greater effect in SHR than in WKY arteries. Reduction in myogenic tone produced by the PKC inhibitor bisindolylmaleimide I in WKY and SHR arteries was significantly less than that produced by Rho kinase inhibition. The pressure-dependent increase in myogenic tone was significantly decreased by Y-27632, and the decrease was markedly greater than that produced by bisindolylmaleimide I in SHR arteries. In WKY arteries, the pressure-dependent increase in myogenic tone was decreased to a similar extent by Y-27632 and bisindolylmaleimide I. These results suggest greater myogenic tone with increased calcium sensitization in SHR arteries, largely because of Rho kinase activation, with a minor contribution of PKC activation. spontaneously hypertensive rats; cerebral arteries; myogenic tone

The extrusion process of Ca2+ ions in human T-lymphocytes is analyzed by observing the fluorescence due to fura 2 decay in the peripheral T-lymphocytes. Kinetic studies of this phenomenon indicate the influence of Ca2+, Na+, calmodulin and temperature on this transport mechanism. Cytosolic Ca2+ homeostasis is affected by small amounts of protein kinase C, adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. Ca2+ uptake is determined by the Mn2+-mediated quenching of intracellular fura 2 contained in the human peripheral T-lymphocytes.

Astudy examined the assumption that the stimulation of phospholipase C (PLC) and G protein is linked to the myogenic tone in the cerebral circulation. The influence of U-73122, a new putative inhibitor of PLC (28), on the continued pressure-stimulated constriction of rat cerebral arteries was studied. Through pathways superimposed on basal myogenic tone, G proteins modulate mechanotransduction. A total loss of pressure stimulated constriction is caused by the pharmacological inhibition of PLC. In arteries with myogenic tone, PLC is stimulated.

Guanosine-3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PK) is characterized by its ability to activate large-conductance calcium-activated potassium channels (Kca channels) in reversed patches of smooth muscles of rabbit cerebral arteries, which necessitates the presence of cGMP and adenosine triphosphate (ATP). The pathway of cGMP-PK involves phosphorylation of Kca channel or an associated protein. The activation of Kca is considered responsible for vasodilating activity of nitrovasodilators such as nitric oxide (NO).

Inositol efflux in C6 glioma cells of rat, resulting from chronic acclimatization of C6 cells to hypertonic media, follows a Na(plus or minus) independent transport mechanism, characterized by its partial resistance to inositol. The inositol efflux is hindered by quinidine, quinine, numerous anion transport blockers and Cis-unsaturated fatty acids. The role of lipoxygenase metabolites in swelling-stimulated activation of inositol efflux is indicated by the inhibition of basal and swelling-activated inositol efflux. This inhibition is attributed to the action of ketoconazole, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate and gossypol, which are specific inactivators of lipoxygenase enzymes.

Arginine vasopressin (AVP) possesses the natriuretic property of enhancing Na+ reabsorption in epithelial cells and acts as an intermediary in the adenosine 3,'5'-cyclic monophosphate (cAMP)-dependent pathway associated with protein kinase A (PKA) activation. The PKA activation is targeted at the G protein pathway, and is responsible for regulating apical epithelial Na+ channel conductance.

An assay of the relationship between protein kinase A (PKA) stimulated activation of the 9-Ps apical Na+ channel and the actin filament arrangement, by utilizing patch-clamp techniques, illustrates the functional role of the actin filament organization in the PKA activation of epithelial Na+ channels. AVP also stimulates transepithelial water reabsorption through the incorporation of water-channel-containing vesicles into the apical plasma membrane in sensitive epithelia.