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Although obesity is associated with multiple features of the metabolic syndrome (insulin resistance, leptin resistance, hepatic steatosis, chronic inflammation, etc.), the molecular changes that promote these conditions are not completely understood. Here, we tested the hypothesis that elevated ceramide biosynthesis contributes to the pathogenesis of obesity and the metabolic syndrome. Chronic treatment for 8 wk of genetically obese (ob/ob), and, high-fat diet-induced obese (DIO) mice with myriocin, an inhibitor of de novo ceramide synthesis, decreased circulating ceramides. Decreased ceramide was associated with reduced weight, enhanced metabolism and energy expenditure, decreased hepatic steatosis, and improved glucose hemostasis via enhancement of insulin signaling in the liver and muscle. Inhibition of de novo ceramide biosynthesis decreased adipose expression of suppressor of cytokine signaling-3 (SOCS-3) and induced adipose uncoupling protein-3 (UCP3). Moreover, ceramide directly induced SOCS-3 and inhibited UCP3 mRNA in cultured adipocytes suggesting a direct role for ceramide in regulation of metabolism and energy expenditure. Inhibition of de novo ceramide synthesis had no effect on adipose tumor necrosis factor-[alpha] (TNF-[alpha]) expression but dramatically reduced adipose plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattactant protein-1 (MCP-1). This study highlights a novel role for ceramide biosynthesis in body weight regulation, energy expenditure, and the metabolic syndrome. sphingolipids; insulin resistance; leptin resistance; insulin resistance; suppressor of cytokine signaling-3; tumor necrosis factor-[alpha]; plasminogen activator inhibitor-1; monocyte chemoattactant protein-1

In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a [gamma]-secretase inhibitor (compound E, 10 [micro]mol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract. Kruppel-like factor-4; notch signaling; gene expression; goblet cells

Myoglobin is an oxygen storage molecule that is selectively expressed in cardiac and slow-twitch skeletal muscles that have a high oxygen demand. Numerous studies have implicated hypoxia in the regulation of myoglobin expression as an adaptive response to hypoxic stress. However, the details of this relationship remain undefined. In the present study, adult mice exposed to 10% oxygen for periods up to 3 wk exhibited increased myoglobin expression only in the working heart, whereas myoglobin was either diminished or unchanged in skeletal muscle groups. In vitro and in vivo studies revealed that hypoxia in the presence or absence of exercise-induced stimuli reprograms calcium signaling and modulates myoglobin gene expression. Hypoxia alone significantly altered calcium influx in response to cell depolarization or depletion of endoplasmic reticulum calcium stores, which inhibited the expression of myoglobin. In contrast, our whole animal and transcriptional studies indicate that hypoxia in combination with exercise enhanced the release of calcium from the sarcoplasmic reticulum via the ryanodine receptors triggered by caffeine, which increased the translocation of nuclear factor of activated T-cells into the nucleus to transcriptionally activate myoglobin expression. The present study unveils a previously unrecognized mechanism where the hypoxia-mediated regulation of calcium transients from different intracellular pools modulates myoglobin gene expression. In addition, we observed that changes in myoglobin expression, in response to hypoxia, are not dependent on hypoxia-inducible factor-1 or changes in skeletal muscle fiber type. These studies enhance our understanding of hypoxia-mediated gene regulation and will have broad applications for the treatment of myopathic diseases. nuclear factor of activated T cells; calcineurin; skeletal muscle

Studies of cultured cells have indicated that the mammalian target of rapamycin complex 1 (mTORC1) mediates the development of insulin resistance. Because a role for mTORC1 in the development of skeletal muscle insulin resistance has not been established, we studied mTORC1 activity in skeletal muscles of ob/ob (OB) mice and wild-type (WT) mice. In vivo insulin action was assessed in muscles of mice 15 min following an intraperitoneal injection of insulin or an equivalent volume of saline. In the basal state, the phosphorylation of S6K on [Thr.sup.389], mTOR on [Ser.sup.2448], and PRAS40 on [Thr.sup.246] were increased significantly in muscles from OB mice compared with WT mice. The increase in basal mTORC1 signaling was associated with an increase in basal PKB phosphorylation on [Thr.sup.308] and [Ser.sup.473]. In the insulin-stimulated state, no differences existed in the phosphorylation of S6K on [Thr.sup.389], but PKB phosphorylation on [Thr.sup.308] and [Ser.sup.473] was significantly reduced in muscles of OB compared with WT mice. Despite elevated mTORC1 activity in OB mice, rapamycin treatment did not improve either glucose tolerance or insulin tolerance. These results indicate that the insulin resistance of OB mice is mediated, in part, by factors other than mTORC1. insulin resistance; signal transduction; skeletal muscle

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-[alpha] expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser l 5 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-[alpha], which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-[alpha] blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity. oxidative stress; mitochondrial death pathway

Glucagon receptor (GR) activity and expression are altered in several diseases, including Type 2 diabetes. Previously, we investigated the mechanism of GR desensitization and internalization. The present study focused on the fate of internalized GR. Using both hamster hepatocytes and human embryonic kidney (HEK)-293 cells, we showed that internalized GR recycled to the plasma membrane within 30-60 rain following stimulation of the cells with 100 nM glucagon. In HEK-293 cells and during recycling, GR colocalized with Rab4, Rabll, [beta]-arrestin1, [beta]-arrestin2, and actin filaments, in the cytosolic and/or perinuclear domains. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol. GR coimmunoprecipitated with [beta]-actin in both hepatocytes and HEK-293 cells. Downregulation of [beta]-arrestin 1 and [beta]-arrestin2 or disruption of the cytoskeleton inhibited recycling, but not internalization of GR. Deletion of the GR carboxyl-terminal 70 amino acids abolished internalization of GR in response to glucagon while deletion of the last 40 amino acids only did not affect GR internalization and recycling. After exposure of the cells to either high concentrations or prolonged duration of glucagon, GR colocalized with lysosomes. GR degradation was inhibited by lysosomal, but not proteosomal, inhibitors. In conclusion, GR recycles through Rab4- and Rab11-positive vesicles. The actin cytoskeleton, [beta]-arrestinl, [beta]-arrestin2, and the receptor's carboxyl terminus are involved in recycling. Prolonged stimulation with glucagon targets GR for degradation in lysosomes. Therefore, the present study provides a better understanding of the GR recycling mechanism, which could become useful in the treatment of certain diseases, including diabetes. actin; G protein-coupled receptor; lysosome; Rab4; Rabl 1

Polyuria, hypernatremia, and hypovolemia are the major clinical signs of inherited nephrogenic diabetes insipidus (NDI). Hypernatremia is commonly considered a secondary sign caused by the net loss of water due to insufficient insertion of aquaporin-2 water channels into the apical membrane of the collecting duct cells. In the present study, we employed transcriptome-wide expression analysis to study gene expression in V2 vasopressin receptor (Avpr2)-deficient mice, an animal model for X-linked NDI. Gene expression changes in NDI mice indicate increased proximal tubular sodium reabsorption. Expression of several key genes including [Na.sup.+]-[K.sup.+]-ATPase and carbonic anhydrases was increased at the mRNA levels and accompanied by enhanced enzyme activities. In addition, altered expression was also observed for components of the eicosanoid and thyroid hormone pathways, including cyclooxygenases and deiodinases, in both kidney and hypothalamus. These effects are likely to contribute to the clinical NDI phenotype. Finally, our data highlight the involvement of the renin-angiotensin-aldosterone system in NDI pathophysiology and provide clues to explain the effectiveness of diuretics and indomethacin in the treatment of NDI. vasopressin receptor; diabetes insipidus; G protein-coupled receptor; signal transduction; hypernatremia

Arginine vasopressin (AVP) and hypertonicity in the renal medulla play a major role in the urine concentration mechanism. Previously, we showed that rat vasopressin V2 receptor (rV2R) promoter activity was increased by vasopressin V2R stimulation and decreased by vasopressin Via receptor (V1aR) stimulation in a LLC-P[K.sub.1] cell line stably expressing rat V1aR (LLC-P[K.sub.1]/rV1aR). In the present study, we investigated the effects of hypertonicity on the rV2R promoter activity and on the suppression of rV2R promoter activity by VlaR stimulation in LLC-P[K.sub.1]/rV1aR cells, rV2R promoter activity was increased in NaCl- or mannitol-induced hypertonicity. The hypertonicity-responsive site in the rV2R promoter region was limited to 10 bp, including the Sp1 motif. The increase of V2R promoter activity by hypertonicity was significantly inhibited by a JNK inhibitor (SP600125) and PKA inhibitor (H89). In contrast, rV2R promoter activity was remarkably suppressed by V1aR stimulation in the hypertonic condition rather than in the isotonic condition. The AVP-stimulated intracellular [Ca.sup.2+] concentration was increased in the hypertonic condition, suggesting the functional activation of V1aR by hypertonicity. In conclusion, 1) V2R promoter activity is increased by hypertonicity via the JNK and PKA pathways, 2) suppression of V2R expression by the VlaR-[Ca.sup.2+] pathway is enhanced by hypertonicity, and 3) hypertonicity enhances the V1aR-[Ca.sup.2+] pathway. The counteractivity of V2R and V1aR could be required to maintain minimum urine volume in the dehydrated state. collecting ducts; transcription regulation; cell signaling

Postconditioning (POC), a novel strategy of cardioprotection against ischemia-reperfusion injury, is clinically attractive because of its therapeutic application at the predictable onset of reperfusion. POC activates several intracellular kinase signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt (RISK). The regulation of POC-induced survival kinase signaling, however, has not been fully characterized. JAK-STAT activation is integral to cardiac ischemic tolerance and may provide upstream regulation of RISK. We hypothesized that POC requires the activation of both JAK-STAT and RISK signaling. Langendorff-perfused mouse hearts were subjected to 30 min of global ischemia and 40 min of reperfusion, with or without POC immediately after ischemia. A separate group of POC hearts was treated with AG 490, a JAK2 inhibitor, Stattic, a specific STAT3 inhibitor, or LY-294002, a PI3K inhibitor, at the onset of reperfusion. Cardiomyocyte-specific STAT3 knockout (KO) hearts were also subjected to non-POC or POC protocols. Myocardial performance (+dP/ [dt.sub.max] mmHg/s) was assessed throughout each perfusion protocol. Phosphorylated (p-) STAT3 and Akt expression was analyzed by Western immunoblotting. POC enhanced myocardial functional recovery and increased expression of p-STAT3 and p-Akt. JAK-STAT inhibition abrogated POC-induced functional protection. STAT3 inhibition decreased expression of both p-STAT3 and p-Akt. PI3K inhibition also attenuated POC-induced cardioprotection and reduced p-Akt expression but had no effect on STAT3 phosphorylation. Interestingly, STAT3 KO hearts undergoing POC exhibited improved ischemic tolerance compared with KO non-POC hearts. POC induces myocardial functional protection by activating the RISK pathway. JAK-STAT signaling, however, is insufficient for effective POC without PI3K-Akt activation. phosphatidylinositol 3-kinase-Akt; STAT3; ischemia-reperfusion

Chronic allograft nephropathy (CAN), the most common cause of late kidney allograft failure, is not effectively prevented by immunosuppressive regimens. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) via MEK mediates actions of various growth factors, including transforming growth factor (TGF)-[beta]1, which plays a key role in CAN. Hence, we tested the therapeutic potential of MEK-ERK1/2 signaling disruption to prevent CAN. Kidneys from C57BL/6J ([H-2.sup.b]) mice were transplanted to bilaterally nephrectomized BALB/c ([H-2.sup.d]) mice. At 14 days after transplantation, the recipients were subjected to 28 days of treatment with the MEK inhibitor CI-1040. All six CI-1040-treated allografts survived, while two of seven grafts in the vehicle-treated group were lost. At the end of the experiment, the function and structure of grafts in the CI-1040-treated group were significantly preserved, as indicated by lower levels of serum creatinine or blood urea nitrogen than in the vehicle-treated group [30 [+ or -] 6 vs. 94 [+ or -] 39 [micro]M creatinine (P = 0.0015) and 22 [+ or -] 8 vs. 56 [+ or -] 25 mM BUN (P = 0.0054)] and reduced CAN in the CI-1040-treated group compared with vehicle controls (CAN score = 4.2 vs. 10.3, P = 0.0119). The beneficial effects induced by CI-1040 were associated with reduction of ERK1/2 phosphorylation and TGF[beta]1 levels in grafts. Also, CI-1040 potently suppressed not only TGF[beta] biosynthesis in kidney cell cultures but also antiallograft immune responses in vitro and in vivo. Our data suggest that interference of MEK-ERK1/2 signaling with a pharmacological agent (e.g., CI-1040) has therapeutic potential to prevent CAN in kidney transplantation. kidney transplantation; experimental therapy; transforming growth factor-[beta]1

Bladder suburothelial myofibroblasts may modulate both sensory responses from the bladder wall and spontaneous activity. This study aimed to characterize further these cells in their response to exogenous agents implicated in mediating the above activity. Detrusor strips, with or without mucosa, and isolated suburothelial myofibroblasts were prepared from guinea pig bladders. Isometric tension, intracellular [Ca.sup.2+], and membrane current were recorded. Cell pairs were formed by pushing two cells together. Tension, intracellular [Ca.sup.2+], and membrane potential were also recorded from bladder sheets using normal or spinal cord-transected (SCT) rats. Spontaneous contractions were greater in detrusor strips with an intact mucosa and were augmented by 10 [micro]M UTP. ATP, UTP, or reduced extracellular pH elicited [Ca.sup.2+] transients and inward currents ([E.sub.rev] -30 mV) in isolated cells. Capsaicin (5-30 [micro]M) reduced membrane current (37 [+ or -] 12% of control) with minor effects on [Ca.sup.2+] transients: sodium nitroprusside reduced membrane currents (40 [+ or -] 21% of control). Cell pair formation, without an increase in cell capacitance, augmented ATP and pH responses (180 [+ or -] 58% of control) and reduced the threshold to ATP and acidosis. Glivec (20-50 [micro]M) reversibly blocked the augmentation and also reduced spontaneous activity in bladder sheets from SCT, but not normal, rats. Glivec also disrupted the spread of [Ca.sup.2+] waves in SCT sheets, generating patterns similar to normal bladders. Suburothelial myofibroblasts respond to exogenous agents implicated in modulating bladder sensory responses; responses augmented by physical intercellular contact. The action of glivec and its selective suppression of spontaneous activity in SCT rats identifies a possible pathway to attenuate bladder overactivity. suburothelium; spontaneous activity; purinergic signaling

pH is one of the most important physiological parameters, with its changes affecting the function of vital organs like the heart. However, the effects of alkalosis on the regulation of cardiac myocyte function have not been extensively investigated. Therefore, we decided to study whether the mitogen-activated protein kinase (MAPK) signaling pathways [c-Jun [NH.sub.2]-terminal kinases (JNKs), extracellular signalregulated kinases (ERKs), and p38 MAPK] are activated by alkalosis induced with Tris-Tyrode buffer at two pH values, 8.5 and 9.5, in H9c2 rat cardiac myoblasts. These buffers also induced intracellular alkalinization comparable to that induced by 1 mM [NH.sub.4]Cl. The three MAPKs examined presented differential phosphorylation patterns that depended on the severity and the duration of the stimulus. Inhibition of [Na.sup.+]/[H.sup.+] exchanger (NHE)1 by its inhibitor HOE-642 prevented alkalinization and partially attenuated the alkalosis (pH 8.5)-induced activation of these kinases. The same stimulus also promoted c-Jun phosphorylation and enhanced the binding at oligonucleotides bearing the activator protein-1 (AP-1) consensus sequence, all in a JNK-dependent manner. Additionally, mitogen- and stress-activated kinase 1 (MSK1) was transiently phosphorylated by alkalosis (pH 8.5), and this was abolished by the selective inhibitors of either p38 MAPK or ERK pathways. JNKs also mediated Bc1-2 phosphorylation in response to incubation with the alkaline medium (pH 8.5), while selective inhibitors of the three MAPKs diminished cell viability under these conditions. All these data suggest that alkalosis activates MAPKs in H9c2 cells and these kinases, in turn, modify proteins that regulate gene transcription and cell survival. signal transduction; cardiomyoblast; cell death

We have previously reported the participation of mitogen-activated protein, Rho, and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study, we investigated the possible participation of phospholipase D (PLD) and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [[sup.3]H]phosphatidylbutanol and PA generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mammalian target of rapamycin (mTOR), by 80 and 72%, respectively, and partially blocked activation of extracellular signal-regulated kinase (ERK) by 30% but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1, and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by ~50%. Inhibition of 5-HT receptor 2A (R 2A) with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3-kinase inhibitor failed to block either activation of PLD by 5-HT or PA-dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT. pulmonary artery smooth muscle cell proliferation; serotonin; phospholipase D; phosphatidic acid; cell signal transduction

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3[beta] resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth. metabolism; cell signaling; S6 kinase; mammalian target of rapamycin; glycogen synthetase kinase; tuberous sclerosis complex

Mediators of angiogenesis such as VEGFs and angiopoietins may regulate pulmonary vascular permeability under normal and pathological conditions. Ephrin family receptor tyrosine kinases are expressed in the vasculature and also regulate angiogenesis under some circumstances, but whether they also modulate lung vascular permeability is unknown. We hypothesized that stimulation of lung endothelial EphA receptors with ephrin-a1 ligand would alter pulmonary vascular permeability and tested this idea in vivo and in vitro. We found that ephrin-a1 ligand and EphA2 receptors are expressed in distal normal lung vasculature and that their expression is increased in injured lung, suggesting a link to mechanisms of increased permeability. Intravenous injection of ephrin-a1 caused a large increase in the leakage of labeled albumin into the lungs of rats within 30 rain (293 [+ or -] 27 vs. 150 [+ or -] 6 ng/mg dry lung, P < 0.01), along with histological evidence of the formation of endothelial disruptions. In cultured lung vascular endothelial cells, stimulation with ephrin-a1 increased monolayer permeability by 44% (P < 0.01), a permeability change similar to that seen with VEGF stimulation of the same cells. Ephrin-a1 stimulation in vivo and in vitro was associated with histological evidence for disruptions of tight and adherens junctions. These observations describe a novel role for ephrin-a1 and EphA receptors in the regulation of vascular permeability in the lung. ephrin; EphA2; pulmonary edema

Glucagon counterregulation (GCR) is a key protection against hypoglycemia that is compromised in diabetes via an unknown mechanism. To test the hypothesis that [alpha]-cell-inhibiting signals that are switched off during hypoglycemia amplify GCR, we studied streptozotocin (STZ)-treated male Wistar rats and estimated the effect on GCR of intrapancreatic infusion and termination during hypoglycemia of saline, insulin, and somatostatin. Times 10 rain before and 45 min after the switch-off were analyzed. Insulin and somatostatin, but not saline, switch-off significantly increased the glucagon levels (P = 0.03), and the fold increases relative to baseline were significantly higher (P < 0.05) in the insulin and somatostatin groups vs. the saline group. The peak concentrations were also higher in the insulin (368 pg/ml) and somatostatin (228 pg/ml) groups vs. the saline (114 pg/ml) group (P < 0.05). GCR was pulsatile in most animals, indicating a feedback regulation. After the switch-off, the number of secretory events and the total pulsatile production were lower in the saline group vs. the insulin and somatostatin groups (P < 0.05), indicating enhancement of glucagon pulsatile activity by insulin and somatostatin compared with saline. Network modeling analysis demonstrates that reciprocal interactions between [alpha]- and [delta]-cells can explain the amplification by interpreting the GCR as a rebound response to the switch-off. The model justifies experimental designs to further study the intrapancreatic network in relation to the switch-off phenomenon. The results of this proof-of-concept interdisciplinary study support the hypothesis that GCR develops as a rebound pulsatile response of the intrapancreatic endocrine feedback network to switch-off of [alpha]-cell-inhibiting islet signals. pancreatic endocrine network; feedback control; insulin; somatostatin

We determined the effects of intravenous infusion of amino acids (AA) at serum insulin of 5, 30, 72, and 167 mU/l on anabolic signaling, expression of ubiquitin-proteasome components, and protein turnover in muscles of healthy young men. Tripling AA availability at 5 mU/l insulin doubled incorporation of [1-[sup.13]C]leucine [i.e., muscle protein synthesis (MPS), P < 0.01] without affecting the rate of leg protein breakdown (LPB; appearance of [d.sub.5]-phenylalanine). While keeping AA availability constant, increasing insulin to 30 mU/l halved LPB (P < 0.05) without further inhibition at higher doses, whereas rates of MPS were identical to that at 5 mU/l insulin. The phosphorylation of PKB [Ser.sup.473] and [p70.sup.s6k] [Thr.sup.389] increased concomitantly with insulin, but whereas raising insulin to 30 mU/l increased the phosphorylation of mTOR [Ser.sup.2448], 4E-BP1 [Thr.sup.37/46], or GSK3[beta] [Ser.sup.9] and decreased that of eEF2 [Thr.sup.56], higher insulin doses to 72 and 167 mU/l did not augment these latter responses. MAFbx and proteasome C2 subunit proteins declined as insulin increased, with MuRF-1 expression largely unchanged. Thus increasing AA and insulin availability causes changes in anabolic signaling and amounts of enzymes of the ubiquitin-proteasome pathway, which cannot be easily reconciled with observed effects on MPS or LPB. muscle protein synthesis; muscle protein breakdown

Neurokinin A (NKA) belongs to the tachykinin neuropeptide family. Its biological functions are primarily mediated by the neurokinin (NK)-2 receptor. NKA has been implicated in several inflammatory conditions. However, there are limited data about the mechanism of its pathogenetic action. Here, we investigated proinflammatory effects of NKA on peripheral immune cells using the mouse macrophage/ monocyte cell line RAW 264.7 and primary peritoneal macrophages. The signaling mechanistic pathways involved were also studied. In mouse macrophages with no detectable NK-2 receptors, NKA induces the upregulation of NK-1 but not NK-2 receptor expression. Furthermore, NKA engages this NK-1 receptor, resulting in inflammatory-like responses involving activation of the transcription factor nuclear factor (NF)-[kappa]B and induction of NF-[kappa]B-responsive proinflammatory chemokine expression. NKA activates NF-[kappa]B as evidenced by induced phosphorylation (leading to degradation) of its inhibitory protein I[kappa]B[alpha], increased cellular levels of the transactivation-active phospho([Ser.sup.276])-p65 and its nuclear translocation, as well as enhanced DNA-binding activity of NF-[kappa]B. These responses are specifically inhibited by selective NK-1 receptor antagonists but not NK-2 receptor antagonists, thereby excluding the role of NK-2 receptor. Further investigation on the upstream signaling mechanisms suggests that two NF-[kappa]B-activating pathways (extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/protein kinase B) are activated by NKA. Specific inhibitors of the two pathways block NF-[kappa]B-dependent chemokine expression. The inhibitory effects are mediated through regulation of nuclear translocation, DNA-binding activity, and/or transactivation activity of NF-[kappa]B. Together, we provide novel evidence that NKA engages NK-1 receptors on mouse macrophages to elicit NF-[kappa]B-dependent cellular responses. The findings reveal cellular mechanisms that may underlie NKA-mediated inflammatory and immunological conditions. neuroimmunomodulation; tachykinins; leukocytes; chemokines; signaling transduction pathways

We have investigated the regulation of translation during the period of rapid liver growth that occurs at the end of gestation in the rat. This work was based on our prior observation that fetal hepatocyte proliferation is resistant to the inhibitory effects of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a nutrient-sensing kinase that controls ribosome biogenesis and protein translation. We hypothesized that translation control in late-gestation fetal liver differs from that in adult liver. We first examined the ability of rapamycin to inhibit the translation of mRNAs encoding ribosomal proteins. Consistent with the effect of rapamycin on proliferation, the activation of adult liver 5'-terminal oligopyrimidine tracts (5'-TOP) translation that occurred during refeeding after food deprivation was sensitive to rapamycin. Fetal liver 5'-TOP translation was insensitive. We went on to examine the eukaryotic initiation factor (eIF) 4F cap-binding complex that controls global protein synthesis. The molecular weights of the multiple eIF4G1 isoforms present in fetal and adult liver eIF4F complexes differed. In addition, fetal liver expressed the eIF4A1 form of the eIF4A helicase, whereas adult liver contained eIF4AI and eIF4A2. Rapamycin administration before refeeding in adult rats inhibited formation of the preinitiation complex to a much greater degree than rapamycin administration to fetal rats in situ. We conclude that there are major structural and functional differences in translation control between late-gestation fetal and adult liver. These differences may confer differential sensitivity to the growth inhibitory effects of rapamycin. liver; hepatocyte; mammalian target of rapamycin; signal transduction; ribosome

Vasoconstrictors activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]), leading to calcium mobilization, protein kinase C activation, and contraction. Our aim was to investigate whether PLC-[[delta].sub.1], a PLC isoform implicated in [[alpha].sub.1]-adrenoreceptor signaling and the pathogenesis of hypertension, is involved in noradrenaline (NA) or endothelin (ET-1)-induced [PIP.sub.2] hydrolysis and contraction. Rat mesenteric small arteries were studied. Contractility was measured by pressure myography, phospholipids or inositol phosphates were measured by radiolabeling with [sup.33]pi or myo-[[sup.3]H]inositol, and caveolae/rafts were prepared by discontinuous sucrose density centrifugation. PLC-[[delta].sub.1] was localized by immunoblot analysis and neutralized by delivery of PLC-SI antibody. The PLC inhibitor U73122, but not the negative control U-73342, markedly inhibited NA and ET-1 contraction but had no effect on potassium or phorbol ester contraction, implicating PLC activity in receptor-mediated smooth muscle contraction. PLC-[[delta].sub.1] was present in caveolae/rafts, and NA, but not ET-1, stimulated a rapid twofold increase in PLC-[[delta].sub.1] levels in these domains. PLC-St is calcium dependent, and removal of extracellular calcium prevented its association with caveolae/rafts in response to NA, concomitantly reducing NA-induced [[sup.33]p][PIP.sub.2] hydrolysis and [3H]inositol phosphate formation but with no effect on ET-1-induced [[sup.33]p][PIP.sub.2] hydrolysis. Neutralization of PLC-[[delta].sub.1] by PLC-[[delta].sub.1] antibody prevented its caveolae/raft association and attenuated the sustained contractile response to NA compared with control antibodies. In contrast, ET-1-induced contraction was not affected by PLC-[[delta].sub.1] antibody. These results indicate the novel and selective role of caveolae/raft localized PLC-[[delta].sub.1] in NA-induced [PIP.sub.2] hydrolysis and sustained contraction in intact vascular tissue. signal transduction; vascular smooth muscle; phosphoinositide; caveolae; lipid rafts

Recently, we defined an [alpha]1A-adrenergic receptor-ERK ([alpha] 1A-AR-ERK) survival signaling pathway in adult cardiac myocytes. Previous studies in neonatal cardiac myocytes indicated that the cardiac-specific transcription factor GATA4 is a downstream mediator of [alpha]l-ERK signaling and that phosphorylation of GATA4 by ERK increases DNA binding and transcriptional activity. Therefore, we examined GATA4 as a potential downstream effector of [alpha]lA-ERK survival signaling in adult cardiac myocytes. We measured norepinephrine (NE)-induced cell death in cultured cardiac myocytes lacking a 1-ARs (cultured from [alpha]1A/B-AR double-knockout mice, [alpha]lABKO mice) that are susceptible to cell death induced by several proapoptotic stimuli, including NE. Our results show that overexpression of GATA4 is sufficient to protect [alpha]lABKO cardiac myocytes from NE-induced cell death. However, we found that the [alpha]1A-subtype did not induce phosphorylation or increase the activity of GATA4 in adult mouse cardiac myocytes in culture or in vivo. Furthermore, we examined the effect of siRNA-mediated knockdown of GATA4 on [alpha]1A-survival signaling. In [alpha]1B-knockout cardiac myocytes, which express only the a 1 A-subtype and are protected from NE-induced cell death, GATA4 knockdown did not reverse [alpha]1A-survival signaling in response to NE. In summary, we found that GATA4 acted as a survival factor by preventing cell death in [alpha]1ABKO cardiac myocytes, but GATA4 was not activated by [alpha]1-AR stimulation and was not required for [alpha]1A-survival signaling in adult cardiac myocytes. This also identifies an important mechanistic difference in a [alpha]1-signaling between adult and neonatal cardiac myocytes. alpha-1-adrenergic receptors; extracellular signal regulated kinase; norepinephrine

Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-hased signaling pathways that are associated with protection from lethal cell injury. cell signaling; protection; ischemia

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation. myoblast; skeletal muscle; differentiation; extracellular signal-regulated kinase; myogenin

It is well known that excessive intake of sodium chloride (sodium) is a risk factor for cardiovascular disease because it raises blood pressure. However, sodium loading reportedly promotes cardiovascular disease independently of its effect on blood pressure. To examine the mechanisms by which sodium loading promotes vascular inflammation independently of its effect on blood pressure, we examined the role of calcineurin in sodium loadinginduced vascular inflammation using a wire injury model of the rat femoral artery. Calcineurin mRNA expression in the wire-injured femoral artery was significantly higher in sodium-loaded normotensive rats, such as Wistar-Kyoto (WKY) rats, than that in control WKY rats. Neointimal formation was also significantly enhanced in sodium-loaded WKY rats compared with control WKY rats. Gene transfer of an adenovirus expressing a dominant negative mutant of calcineurin (AdCalAAC92Q) significantly suppressed neointimal formation in sodium-loaded WKY rats to a level similar to that observed in control WKY rats. Calcineurin expression and neointimal formation were more significantly enhanced in hypertensive rats, such as spontaneously hypertensive rats (SHRs), than those in control WKY rats. AdCalAAC92Q infection significantly suppressed neointimal formation in SHRs to a level similar to that observed in control WKY rats. These results suggest that sodium loading promotes neointimal formation, even in normotensive rats, and that hypertension further stimulates neointimal formation. These results also suggest that calcineurin plays a pivotal role in this process. signal transduction; vasculature; inflammation; hypertension

In this study, we have systematically evaluated the signaling mechanisms underlying stimulated nitric oxide (NO) synthesis by estrogen ([E.sub.2]) and other vasoactive agents at the level of a single endothelium-derived cell. To do so, we have characterized and contrasted rapid [E.sub.2]-evoked NO synthesis with that of ATP using single-cell microfluorimetry and patch-clamp recordings to monitor stimulated changes in cellular NO synthesis (via 4-amino-5-methylamino-2',7'-difluorofluorescein), [Ca.sup.2+] transients (via Fluo-3), and membrane hyperpolarization in cultured human EA.hy926 cells. Ez-evoked NO synthesis in single cells ([EC.sub.50] ~0.3 nM) was blocked by the E2 receptor antagonist ICI 182,780 and the NO synthase inhibitor [N.sup.[omega]]-nitro-L-arginine methyl ester. Although both [E.sub.2] and ATP stimulated comparable [Ca.sup.2+] transients, [E.sub.2]-induced NO synthesis was insensitive to intracellular BAPTA-AM or removal of external [Ca.sup.2+]. In contrast, ATP-evoked NO production was abolished by either one of these treatments. ATP-evoked hyperpolarizations (~20 mV) and NO production were both inhibited by the respective small-conductance and intermediate-conductance calciumactivated [K.sup.+] channel blockers apamin and charybdotoxin. [E.sub.2] minimally affected membrane potential, and stimulated NO synthesis was insensitive to calcium-activated [K.sup.+] channel blockers. Exposure to either the phosphatidylinositol 3-kinase inhibitor LY-294001 or the MAP kinase inhibitor PD-98059 abolished the NO response to [E.sub.2], but not that to ATP. Finally, the NO response evoked by a combined stimulus of [E.sub.2] plus ATP was similar to that of ATP alone. In conclusion, our data directly demonstrate that an individual human EA.hy926 cell contains at least two distinct mechanisms for stimulated NO synthesis that depend on either calcium or protein kinase signaling events. nitric oxide; endothelial function; calcium; signal transduction

Nitric oxide (NO) donors decrease intraocular pressure (IOP) by increasing aqueous outflow facility in the trabecular meshwork (TM) and/or Schlemm's canal. However, the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study, we investigated the effects of NO donors on outflow facility and NO-induced effects on TM cell volume. We tested the involvement of soluble guanylate cyclase (sGC), cGMP, PKG, and the large-conductance [Ca.sup.2+]-activated [K.sup.+] (B[K.sub.Ca]) channel using inhibitors and activators. Cell volume was measured using calcein AM fluorescent dye, detected by confocal microscopy, and quantified using NIH ImageJ software. An anterior segment organ perfusion system measured outflow facility. NO increased outflow facility in porcine eye anterior segments (0.4884-1.3956 [micro]l x [min.sup.-1] x mm[Hg.sup.-1]) over baseline (0.2373-0.5220 [micro]l x [min.sup.-1] x mm[Hg.sup.-1]) within 10 min of drug application. These NO-induced increases in outflow facility were inhibited by the the B[K.sub.Ca] channel inhibitor IBTX. Exposure of TM cells to NO resulted in a 10% decrease in cell volume, and these decreases were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and IBTX, suggesting the involvement of sGC and [K.sup.+] eflux, respectively. NO-induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the PKG inhibitor (RP)-8-Br-PET-cGMP-S, suggesting the involvement cGMP and PKG. Additionally, the time course for NO-induced decreases in TM cell volume correlated with NO-induced increases in outflow facility, suggesting that the NO-induced alterations in cell volume may influence outflow facility. eye; signal transduction; cGMP; protein kinase G; soluble guanylate cyclase

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-[Tyr.sup.418], FAK-[Tyr.sup.397]-[Tyr.sup.576]-[Tyr.sup.925], and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 [micro]mol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at [Tyr.sup.397] and [Tyr.sup.576] but not FAK-[Tyr.sup.925] or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of N[H.sub.2]-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK[Tyr.sup.925] phosphorylation under static conditions, it nevertheless exhibited increased FAK-[Tyr.sup.925] phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-[Tyr.sup.925]-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting. migration; cyclic strain; intestine; mechanotransduction; signaling

Neurokinin 3 receptor (NK3R) signaling has an integral role in the stimulated oxytocin (OT) and vasopressin (VP) release in response to byperosmolarity and hypotension. Peripheral injections of cholecystokinin (CCK) receptor agonists for the CCK-A (sulfated CCK-8) and CCK-B (nonsulfated CCK-8) receptors elicit an OT release in rat. It is unknown whether NK3R contributes to this endocrine response. Freely behaving male rats were administered an intraventricular pretreatment of 250 or 500 pmol of SB-222200, a specific NK3R antagonist, or 0.15 M NaCl before an intraperitoneal or intravenous injection of CCK-8 (nonsulfated or sulfated) or 0.15 M NaCl. Blood samples were taken before intraventricular treatment and 15 min after intraperitoneal or intravenous injection, and plasma samples were assayed for OT and VP concentration. Intraperitoneal injection of both nonsulfated and sulfated CCK-8 significantly increased plasma OT levels and had no effect on plasma VP levels. Intravenous injection of sulfated CCK-8 stimulated an increase in plasma OT levels and did not alter plasma VP levels. However, intravenous injection of nonsulfated CCK-8 stimulated a significant increase in plasma levels of both OT and VP. No other studies have demonstrated CCK-8-stimulated release of VP in rat. NK3R antagonist did not alter baseline levels of either hormone. However, pretreatment of NK3R antagonist significantly blocked the CCK-stimulated release of OT in all CCK treatment groups and blocked VP release in response to intravenous injection of nonsulfated CCK-8. Therefore, central NK3R signaling is required for OT and VP release in response to CCK administration. vasopressin; oxytocin

We investigated the expression of the proinflammatory cytokine interleukin (IL)-17 in cardiac fibroblasts and its induction by high glucose (HG). Our results show that primary mouse cardiac fibroblasts (mCFs) secrete low basal levels of IL-17 and that HG (25 mM D-glucose) as opposed to low glucose (5 mM D-glucose + 20 mM mannitol) significantly enhances its secretion. HG induces IL-17 mRNA expression by both transcriptional and posttranscriptional mechanisms. HG induces phosphoinositide 3-kinase [PI3K; inhibited by adenoviral (Ad).dominant negative (dn)PI3Kp85], Akt (inhibited by Ad.dnAkt1), and ERK (inhibited by PD-98059) activation and induces IL-17 expression via PI3K [right arrow] Akt [right arrow] ERK-dependent signaling. Moreover, mCFs express both IL-17 receptors A and C, and although IL-17RA is upregulated, HG fails to modulate IL-17RC expression. Furthermore, IL-17 stimulates net collagen production by mCFs. Pretreatment with the phytoalexin resveratrol blocks HG-induced PI3K-, Akt-, and ERK-dependent IL-17 expression. These results demonstrate that 1) cardiac fibroblasts express IL-17 and its receptors; 2) HG upregulates IL-17 and IL-17RA, suggesting a positive amplification loop in IL-17 signaling in hyperglycemia; 3) IL-17 enhances net collagen production; and 4) resveratrol can inhibit these HG-induced changes. Thus, in hyperglycemic conditions, IL-17 may potentiate myocardial inflammation, injury, and remodeling through autocrine and paracrine mechanisms, and resveratrol has therapeutic potential in ameliorating this effect. signal transduction; proinflammatory cytokines

Shear stress is a pathophysiologically relevant mechanical signal in cartilage biology and tissue engineering. Cyclooxygenase-2 (COX-2) is a pivotal proinflammatory enzyme, which is induced by mechanical loading-derived shear stress in chondrocytes. In the present study, we investigated the transcriptional machinery and signaling pathway regulating shear-induced COX-2 expression in human chondrocytic cells. Deletion and mutation analyses of the human cox-2 promoter reveal that the CCAAT/enhancer-binding protein (C/EBP) and activator protein-1 (AP-1) predominantly contribute to the shear-induced cox-2 promoter activity. Supershift assays disclose that C/EBP[beta], but not C/EBP[alpha] or C/EBP[delta], binds to the C/EBP site, whereas c-Jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBP[beta] expression by c-Jun, and the critical roles of both c-Jun and C/EBP[beta] in shear-induced COX-2 synthesis. Our studies also indicate that Rac and, to a lesser extent, Cdc42 transactivate MEKK1, which is, in turn, responsible for activation of mitogen-activated protein kinase kinase 7 (MKK7). MKK7 regulates c-Jun N[H.sub.2]-terminal kinase 2 activation, which, in turn, triggers the phosphorylation of c-Jun that controls shear-mediated COX-2 upregulation in chondrocytes. Reconstructing the signaling network regulating shear-induced COX-2 expression and inflammation may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic interventions for arthritic disorders. mechanobiology; signal transduction; shear stress; cyclooxygenase; enhancer-binding protein; mitogen-activated protein kinase kinase; c-Jun N[H.sub.2]-terminal kinase

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBEI) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase I (ASKI), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASKI-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity. airway epithelial cells; injury; thioredoxin; mitogen-activated protein kinase

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detmsor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladde,' detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the [Ca.sup.2+] indicator fluo 4AM. ICC fired [Ca.sup.2+] transients in response to stimulation by carbachol (1/10 [micro]M). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 [micro]M), an [M.sub.3] receptor antagonist, but not by the [M.sub.2] receptor antagonist methoctramine (1 [micro]M). The source of [Ca.sub.2+] underlying the carbachol transients in isolated 1CC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 [micro]M) or [Ni.sup.2+] (30-100 [micro]M) to block [Ca.sup.2+] channels or the removal of external [Ca.sup.2+] reduced the amplitude of the carbachol transients. Application of ryanodine (30 [micro]M) or tetracaine (100 [micro]M) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 [micro]M), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 [micro]M) caused a significant reduction and Xestospongin C (1 [micro]M) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a [Ca.sup.2+] indicator showed distinctively different patterns of spontaneous [Ca.sup.2+] events in smooth muscle ceils and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing [Ca.sup.2+] transients. confocal microscopy; c-Kit

Magnesium, an essential intracellular cation, is critically involved in many biochemical reactions involved in the regulation of vascular tone and integrity. Decreased magnesium concentration has been implicated in altered vascular reactivity, endothelial dysfunction, vascular inflammation, and structural remodeling, processes important in vascular changes and target organ damage associated with hypertension. Until recently, very little was known about mechanisms regulating cellular magnesium homeostasis, and processes controlling transmembrane magnesium transport had been demonstrated only at the functional level. Two cation channels of the transient receptor potential melastatin (TRPM) cation channel family have now been identified as magnesium transporters, TRPM6 and TRPM7. These unique proteins, termed chanzymes because they possess a channel and a kinase domain, are differentially expressed, with TRPM6 being found primarily in epithelial cells and TRPM7 occurring ubiquitously. Vascular TRPM7 is modulated by vasoactive agents, pressure, stretch, and osmotic changes and may be a novel mechanotransducer. In addition to its magnesium transporter function, TRPM7 has been implicated as a signaling kinase involved in vascular smooth muscle cell growth, apoptosis, adhesion, contraction, cytoskeletal organization, and migration, important processes involved in vascular remodeling associated with hypertension and other vascular diseases. Emerging evidence suggests that vascular TRPM7 function may be altered in hypertension. This review discusses the importance of magnesium in vascular biology and implications in hypertension and highlights the transport systems, particularly TRPM6 and TRPM7, which may play a role in the control of vascular magnesium homeostasis. Since the recent identification and characterization of [Mg.sup.2+]-selective transporters, there has been enormous interest in the field. However, there is still a paucity of information, and much research is needed to clarify the exact mechanisms of magnesium regulation in the cardiovascular system and the implications of aberrant transmembrane magnesium transport in the pathogenesis of hypertension and other vascular diseases. calcium; cations; signal transduction; mechanotransduction; vascular smooth muscle; ion channels

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how [Ca.sup.2+], despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of [Ca.sup.2+] sources and targets. capacitative calcium entry; caveolae; endothelial cells: endothelial nitric oxide synthase; mathematical model

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-[beta]1 and of a-smooth muscle actin and [beta]ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-[beta]1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS. reactive oxygen species; transforming growth factor-[beta]; [beta]ig-h3; [alpha]-smooth muscle actin; oxidative stress

We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates C1 secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-N[H.sub.2] and 2-furoyl-LIGRLON [H.sub.2]. Short-circuit current ([I.sub.sc]) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-N[H.sub.2] or 2-furoyl-LIGRLO-N[H.sub.2] caused a concentration-dependent change in Isc that was significantly reduced in [Cl.sup.-]-free buffer and by the intracellular [Ca.sup.+] blockers thapsigargin and BAPTA-AM, but not by the [Ca.sup.+] channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated [C1.sup.-] transport. PAR-2 activation was associated with increases in cAMP and intracellular [Ca.sup.+]. Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-N[H.sub.2]-induced increases in [l.sub.sc]. Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated [I.sub.sc]. We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial [Cl.sup.-] secretion via cAMP- and [Ca.sup.+]-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity. ion transport; proteinases; signal transduction; protein kinase; cyclo-oxygenase; mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2

Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 [micro]M azide resulted in 48 [+ or -] 3% and 56 [+ or -] 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 [+ or -] 39 pmol/min/mg (mean [+ or -] SE) in untreated cells. This increased to 669 [+ or -] 69 and 823 [+ or -] 83 pmol/min/mg in cells treated with 50 and 75 [micro]M azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.

Signal transducer and activator of transcription (STAT) 3 is involved in mediating a broad range of biological processes, including cell survival, proliferation, and immune response. Recent evidence has indicated that STAT3 in cardiomyocytes can be activated by ischemicoxidative stress and exerts cardioprotection in the ischemic heart. There is no information, however, regarding the effect of endothelial cell-derived STAT3 on the myocardial response to ischemia-reperfusion (I/R) injury. We hypothesized that the ablation of the STAT3 gene in endothelial cells would worsen postischemic myocardial function by affecting capillary network integrity, suppressing antiapoptotic signaling. Isolated hearts from wild-type and endothelial cell STAT3 knockout (STAT3KO) mice were subjected to 20 min of global ischemia followed by 60 min of reperfusion. Endothelial cell STAT3 deficiency decreased recovery of myocardial function in response to I/R, which was associated with higher levels of LDH release, decreased activation of myocardial STAT3, and elevated p38 MAPK activation in STAT3 endothelial knockout (KO) hearts. In addition, although no significant apoptosis was observed in wild-type and KO hearts, our results showed more expression of myocardial caspase-8 and more apoptosis in the myocardium around the capillary in STAT3KO mice subjected to I/R. Furthermore, endothelial cell STAT3 ablation resulted in increased myocardial expression of IL-6 and suppressor of cytokine signal 3. This study demonstrates that endothelial cell-derived STAT3 plays an important role in postischemic myocardial function. ischemia-reperfusion; signal transducer and activator of transcription 3; myocardial function

O'Connor PM, Cowley AW Jr. Vasopressin-induced nitric oxide production in rat inner medullary collecting duct is dependent on V2 receptor activation of the phosphoinositide pathway. Am J Physiol Renal Physiol 293: F526-F532, 2007. First published May 16, 2007; doi: 10.1152/ajprenal.00052.2007.--We previously reported that arginine vasopressin (AVP) stimulates the production of nitric oxide (NO) in inner medullary collecting duct (IMCD) via activation of V2 receptors (V2R) and the mobilization of intracellular [Ca.sup.2+]. The aim of this study was to determine the pathway(s) through which this response is mediated. IMCDs were dissected from male Sprague-Dawley rats and intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and NO production were measured using a fluorescence imaging system. AVP (100 nmol/l) produced a rapid increase [[[Ca.sup.2+]].sub.i] of 381 [+ or -] 78 nmol/l that was followed by a significant increase of NO production (166 [+ or -] 61%). The specific nonpeptide V2R antagonist OPC31260 (1 [micro]M), but not the V1R antagonist OPC21268 (1 [micro]M), inhibited the increase in [[[Ca.sup.2+]].sub.i] (up to 91 [+ or -] 5%) and abolished the NO response to AVP. Both the phospholipase C inhibitor U73112 (3 [micro]M) and the inositol (1,4,5) tri-phosphate 3 receptor blocker 2-APB (75 [micro]M) reduced the peak [[[Ca.sup.2+]].sub.i] response to AVP (by 65 [+ or -] 9 and 59 [+ or -] 15%, respectively) and abolished the NO response. Although forskolin (100 [micro]M; an activator of adenylyl cyclase) elicited a moderate increase in [[[Ca.sup.2+]].sub.i], neither preincubation with the adenylyl cyclase inhibitor 2'-5'-dideoxyadenosine (50 [micro]M) nor the protein kinase A (PKA) inhibitor PK[A.sub.14-22] (100 [micro]M) significantly inhibited peak [[[Ca.sup.2+]].sub.i] in response to AVP. IMCD [[[Ca.sup.2+]].sub.i] responses to AVP were reduced by 72 [+ or -] 8% when incubated in [Ca.sup.2+]-free media and could be completely abolished by preincubation with the [Ca.sup.2+]-ATPase inhibitor thapsigargin. We conclude that AVP-induced NO production in IMCD is dependent on V2R activation of the phosphoinositide pathway and the mobilization of [Ca.sup.2+] from both intracellular and extracellular pools. antidiuretic hormone; calcium; kidney; signal transduction; rats

The JAK/STAT pathway is activated in vitro by angiotensin II (ANG II) and endothelin-1 (ET-1), which are implicated in the development of diabetic complications. We hypothesized that ANG II and ET-1 activate the JAK/ STAT pathway in vivo to participate in the development of diabetic vascular complications. Using male Sprague-Dawley rats, we performed a time course study [days 7, I4, and 28 after streptozotocin (STZ) injection] to determine changes in phosphorylation of JAK2, STAT1, and STAT3 in thoracic aorta using standard Western blot techniques. On day 7 there was no change in phosphorylation of JAK2, STAT1, and STAT3. Phosphorylation of JAK2, STAT1, and STAT3 was significantly increased on days 14 and 28 and was inhibited by treatment with candesartan (A[T.sub.1] receptor antagonist, 10 mg * [kg.sup.-1] * [day.sup.-1] orally in drinking water), atrasentan (ETA receptor antagonist, 10 mg * [kg.sup.-1] [day.sup.-1] orally in drinking water), and AG-490 (JAK2 inhibitor, 5 mg * [kg.sup.-1] * [day.sup.-1] intraperitoneally). On day 28, treatment with all inhibitors prevented the significant increase in systolic blood pressure (SBP; tail cuff) of STZ-induced diabetic rats (SBP: 157 [+ or -] 9.0, 130 [+ or -] 3.3, 128 [+ or -] 6.8, and 131 [+ or -] 10.4 mmHg in STZ, STZ-candesartan, STZ-atrasentan, and STZ-AG-490 rats, respectively). In isolated tissue bath studies, diabetic rats displayed impaired endothelium-dependent relaxation in aorta (maximal relaxation: 95.3 [+ or -] 3.0, 92.6 [+ or -] 7.4, 76.9 [+ or -] 12.1, and 38.3 [+ or -] 13.1% in sham, sham + AG-490, STZ + AG-490, and STZ rats, respectively). Treatment of rats with AG-490 restored endothelium-dependent relaxation in aorta from diabetic rats at 14 and 28 days of treatment. These results demonstrate that JAK2 activation in vivo participates in the development of vascular complications associated with STZ-induced diabetes. vascular smooth muscle cells; type 1 diabetes; angiotensin II; Janus-activated kinase 2; signal transducer and activator of transcription

Angiotensin (ANG) II exerts a negative modulation on insulin signal transduction that might be involved in the pathogenesis of hypertension and insulin resistance. ANG-(1-7), an endogenous heptapeptide hormone formed by cleavage of ANG I and ANG II, counteracts many actions of ANG II. In the current study, we have explored the role of ANG-(1-7) in the signaling crosstalk that exists between ANG II and insulin. We demonstrated that ANG-(1-7) stimulates the phosphorylation of Janus kinase 2 (JAK2) and insulin receptor substrate ORS)-1 in rat heart in vivo. This stimulating effect was blocked by administration of the selective ANG type 1 (ATe) receptor blocker losartan. In contrast to ANG II, ANG-(1-7) stimulated cardiac Akt phosphorylation, and this stimulation was blunted in presence of the receptor Mas antagonist A-779 or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The specific JAK2 inhibitor AG-490 blocked ANG(1-7)-induced JAK2 and IRS-1 phosphorylation but had no effect on ANG-(1-7)-induced phosphorylation of Akt, indicating that activation of cardiac Akt by ANG-(1-7) appears not to involve the recruitment of JAK2 but proceeds through the receptor Mas and involves PI3K. Acute in vivo insulin-induced cardiac Akt phosphorylation was inhibited by ANG II. Interestingly, coadministration of insulin with an equimolar mixture of ANG II and ANG-(1-7) reverted this inhibitory effect. On the basis of our present results, we postulate that ANG(1-7) could be a positive physiological contributor to the actions of insulin in heart and that the balance between ANG II and ANG-(1-7) could be relevant for the association among insulin resistance, hypertension, and cardiovascular disease. angiotensin II; Janus kinase; insulin receptor substrate-1; signal transduction

The present study was undertaken to determine whether neuropeptide Y (NPY) induces proliferation of rat aortic endothelial cells (RAECs). Since NPY increased the permeability of RAEC monolayers to large molecules via the NPY [Y.sub.3] receptor, RAEC proliferation has been evaluated in terms of NPY-receptor subtypes and also intracellular mechanisms. RAECs were incubated with gases containing 20, 15, or 10% [O.sub.2] and a certain amount of [N.sub.2], depending on the [O.sub.2] content in 5% C[O.sub.2] incubators. NPY ([10.sup.-9]-[10.sup.-6] M) increased the RAEC numbers under hypoxic conditions, such as 15 or 10% [O.sub.2]. Peptide YY elicited no proliferative effect on RAEC, and NPY-(18-36) inhibited the NPY-induced increase in cell number, suggesting that NPY increases the RAEC count through the NPY [Y.sub.3] receptor. Pertussis toxin, U-73122, GF-109203X, myristorylated autocamtide-2-related inhibitory peptide, and wortmannin inhibited the NPY-induced proliferation of RAEC concentration dependently. DY9760e little affected the proliferation caused by NPY. ML-9 and imatinib actually enhanced the NPY-induced proliferation of cells. These results indicated that the NPY [Y.sub.3] receptor is coupled with [G.sub.i] protein, and that NPY-induced increases in RAEC proliferation are mediated by phospholipase C-protein kinase C and/or phosphatidylinositol 3-kinase pathways. In intracellular [Ca.sup.2+]-calmodulin-dependent pathways, calmodulin-dependent protein kinase II partly participates in the NPY-induced cell proliferation. Regarding the previously reported effect of NPY on the permeability of RAEC monolayers to large molecules, it is probable that protein kinase C and phosphatidylinositol 3-kinase pathways are activated for both permeability and cell proliferation induced by NPY under hypoxia, relevant to new insights into the roles of NPY in ischemia-hypoxia. protein kinase C; phosphatidylinositol 3-kinase; phospholipase C; calmodulin-dependent protein kinase

Although insulin resistance is recognized as a potent and prevalent risk factor for coronary heart disease, less is known as to whether insulin resistance causes an altered cardiac phenotype independent of coronary atherosclerosis. In this study, we investigated the relationship between insulin resistance and cardiac contractile dysfunctions by generating a new insulin resistance animal model with rats on high cholesterol-fructose diet. Male Sprague-Dawley rats were given high cholesterol-fructose (HCF) diet for 15 wk; the rats developed insulin resistance syndrome characterized by elevated blood pressure, hyperlipidemia, hyperinsulinemia, impaired glucose tolerance, and insulin resistance. The results show that HCF induced insulin resistance not only in metabolic-response tissues (i.e., liver and muscle) but also in the heart as well. Insulin-stimulated cardiac glucose uptake was significantly reduced after 15 wk of HCF feeding, and cardiac insulin resistance was associated with blunted Akt-mediated insulin signaling along with glucose transporter GLUT4 translocation. Basal fatty acid transporter FATP1 levels were increased in HCF rat hearts. The cardiac performance of the HCF rats exhibited a marked reduction in cardiac output, ejection fraction, stroke volume, and end-diastolic volume. It also showed decreases in left ventricular end-systolic elasticity, whereas the effective arterial elasticity was increased. In addition, the relaxation time constant of left ventricular pressure was prolonged in the HCF group. Overall, these results indicate that insulin resistance reduction of cardiac glucose uptake is associated with defects in insulin signaling. The cardiac metabolic alterations that impair contractile functions may lead to the development of cardiomyopathy. Akt; glucose transporter; cardiac dysfunction; high cholesterol-fructose diet; insulin resistance