Effects of resistance training on arterial compliance and plasma endothelin-1 levels in healthy men.

Arterial compliance (AC) is an index of the elasticity of large arteries. Endothelial dysfunction has been reported to result in reduced arterial compliance, which represents increased arterial stiffness. A reduction in AC is elicited by high-intensity resistance training, however the mechanisms are obscure. Because a single bout of resistance exercise causes a transient increase in circulating plasma endothelin-1 in humans, some vasoconstrictors may play a role in the mechanisms. The present study aimed to investigate whether resistance training-induced decrease in AC is associated with changes in circulating vasoconstrictors levels in young men. Young sedentary men were assigned to control (n=5) or training (n=9) groups. The training group performed four-week high-intensity resistance training (weight training exercise; three sessions/week). We measured AC and plasma levels of endothelin-1, angiotensin II, and norepinephrine before and after intervention. Resistance training significantly decreased AC, whereas the changes in plasma levels of neither endothelin-1, nor angiotensin II, nor norepinephrine were significantly different between the control and the training groups. Moreover, we found no significant correlations between changes in circulating plasma levels (endothelin-1, angiotensin II, and norepinephrine) and in the AC. Despite of no alteration of the resting circulating plasma levels (endothelin-1, etc.), we cannot exclude a possibility that the tissue/local concentrations of vasoconstrictors (endothelin-1, etc.) around the vessels might be increased and also involved in a reduction of AC in the training group. Taken together, the present results suggest that circulating vasoconstrictors (endothelin-1, etc.) in plasma are not involved in a reduction in AC by the resistance training.

Effects of Cold Stimulation on Mitochondrial Activity and VEGF Expression in vitro.

We aimed to clarify the effects of cold stimulation at various temperatures on mitochondrial activity and vascular endothelial growth factor (VEGF) expression in vitro. Human fibroblast, human mesenchymal stem cell, and rat skeletal muscle myoblast cell lines were used. For each cell type, cells were divided into 4 groups and stimulated in various cold temperatures (0, 4, 17 and 25°C) 3 times for 15 min each by placement on crushed ice or floating on cold water set at each temperature. Control cells were subjected to warm water at 37°C. Factors related to mitochondrial activity, mitochondrial DNA copy numbers, and VEGF expression were analyzed 24 h after the last cold stimulation. In all cell types, significant increases of factors related to mitochondrial activity and mitochondrial DNA copy numbers were seen in the 4°C and 17°C-stimulated cells compared with control cells. In rat skeletal muscle cells stimulated at 4°C, VEGF expression significantly increased compared to the control cells. Our data suggest that cold stimulation at certain temperatures promotes mitochondrial activity, biogenesis and VEGF expression.

Effect of urotensin II on PC12 rat pheochromocytoma cells.

Urotensin II (U-II), initially identified as a cyclic peptide from fish urophysis, acts both as a strong vasoconstrictor and vasodilator in the vasculature via its receptor, G-protein coupled receptor 14. In addition, U-II and its receptor are co-expressed in the adrenal medulla, as well as in human pheochromocytomas, suggesting that this peptide may have some function in chromaffin cells. However, the precise role of U-II in these cells is unknown. In the present study, we initially demonstrate that U-II and its receptors mRNA are co-expressed in the rat pheochromocytoma cell line PC12. Moreover, U-II has not effect on tyrosine hydroxylase (TH), the rate-limiting enzyme involved in the biosynthesis of catecholamine, in terms of enzyme activity or at the mRNA level. However, U-II does induce an increase in the phosphorylation of TH specifically at Ser31 without affecting phosphorylation at the two other sites (Ser19 and Ser40). U-II also markedly activates extracellular signal-regulated kinases (ERKs) and p38, but not Jun N-terminal kinase. Blockade of the epidermal growth factor (EGF) receptor by AG1478 significantly reduces activation of ERK, suggesting that EGF receptor transactivation could act upstream of the ERK pathway in PC12 cells. Furthermore, U-II significantly increases dopamine secretion from PC12 cells. Finally, we show that U-II induced significant DNA synthesis in a ERKs and P38 mitogen-activated protein kinase-dependent manner. The results obtained indicate that U-II may exert its effects as a neuromodulator in chromaffin cells.

Smectic-C*alpha phase with two coexistent helical pitch values and a first-order smectic-C*alpha to smectic-C* transition.

Previous results from Kundu using dielectric relaxation have suggested a reentrant antiferroelectric-ferroelectric-antiferroelectric transition in the compound LN36. Our comprehensive studies of this compound using differential optical reflectivity, nonadiabatic scanning calorimetry, null transmission ellipsometry, and resonant x-ray diffraction show that in fact LN36 exhibits the usual phase sequence for chiral smectic liquid crystals: SmA*-SmC*alpha-SmC*-SmC*FI1-SmC*A . Moreover, the SmC*alpha-SmC* transition is a first-order transition, characterized by a discontinuous change in the helical pitch. At temperatures just above the SmC*alpha-SmC* transition, two different values for the helical pitch are simultaneously observed for the first time.

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside stimulates tyrosine hydroxylase activity and catecholamine secretion by activation of AMP-activated protein kinase in PC12 cells.

The activity of AMP-activated protein kinase (AMPK) is regulated by the metabolic and nutritional state of the cell. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is transformed into riboside monophosphate (ZMP) via phosphorylation by adenosine kinase inside the cell and exerts it effect by stimulating AMPK. AICAR significantly induces an increase in AMPK activity in a dose- and time-dependent manner in the rat pheochromocytoma cell line PC12. In addition, compound C, an AMPK inhibitor, as well as 5'-amino-5'-dAdo, an adenosine kinase inhibitor, inhibits the AICAR-induced AMPK activity. AICAR significantly stimulates tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamine) activity and the corresponding mRNA level, which closely matches with the TH protein level. In addition, AICAR provokes a rapid and long-lasting increase in the phosphorylation of TH at Ser19, Ser31 and Ser40. AICAR also markedly activates ERKs, JNK and p38. The MEK-1-inhibitor (PD-098059) causes a partial, but significant, inhibition of AICAR-induced TH enzyme activity by phosphorylation of Ser31 without affecting phosphorylation at the two other sites. By contrast, neither the JNK-inhibitor nor the p38-inhibitor affects TH enzyme activity and phosphorylation. Similarly, PD-098059 partially, but significantly, inhibits the AICAR-induced increase in the TH mRNA level. Furthermore, AICAR increases the level of cAMP in PC12 cells. The present study also shows that H89, a protein kinase A inhibitor, abolishes the AICAR-induced increase in the level of TH mRNA, as well as the corresponding enzyme activity and Ser40 phosphorylation. Finally, AICAR significantly increases dopamine secretion from PC12 cells. These findings indicate that AICAR activates catecholamine synthesis and secretion through AMPK activation in chromaffin cells.

Quasi-two-dimensional Ising critical behavior of de Vries liquid crystals observed in the heat capacity and dielectric response.

Heat capacity and dielectric measurements have been made on a liquid crystal compound exhibiting de Vries type of smectic-A{*} (Sm-A{*}) phase. Heat capacity shows a significant anomaly which is almost symmetric for above and below the Sm-A{*}-Sm-C{*} transition temperature. The transition was found to be very weakly first order. The critical exponent gamma determined from the dielectric data lies 1.8+/-0.2. The present heat capacity data as well as former data for another compound of de Vries type have been analyzed in detail. It was found that the heat capacity data for both compounds are fitted well with a logarithmic divergence except in the immediate vicinity of the transition. These results agree with an expectation that de Vries Sm-A{*}-Sm-C-{*} transition can exhibit quasi-two-dimensional Ising critical behavior.

Effects of exercise on adiponectin and adiponectin receptor levels in rats.

Adiponectin reportedly reduces insulin-resistance. Exercise has also been shown to lessen insulin-resistance, though it is not known whether exercise increases levels of adiponectin and/or its receptors or whether its effects are dependent on exercise intensity and/or frequency. Catecholamine levels have been shown to increase during exercise and to fluctuate based on exercise intensity and duration. In light of this information, we examined the effects of exercise on catecholamine, adiponectin, and adiponectin receptor levels in rats. Our data showed that blood adiponectin levels increased by 150% in animals that exercised at a rate of 30 m/min for 60 min 2 days per week, but not 5 days, per week; no such increase was observed in rats that exercised at a rate of 25 m/min for 30 min. The effects of exercise on adiponectin receptor mRNA were variable, with adiponectin receptor 1 (AdipoR1) levels in muscle increasing up to 4 times while adiponectin receptor 2 (AdipoR2) levels in liver fell to below half in response to exercise at a rate of 25 m/min for 30 min 5 days per week. We also observed that urinary epinephrine levels and plasma lipids were elevated by exercise at a rate of 25 m/min for 30 min 2 days per week. Exercise frequency at a rate of 25 m/min for 30 min correlated with AdipoR1 and AdipoR2 mRNA expression in the muscle and liver, respectively (r=0.640, p<0.05 and r=-0.808, p<0.0005, respectively). Urinary epinephrine levels correlated with AdipoR2 mRNA expression in liver tissues (r=-0.664, p<0.05) in rats that exercised at a rate of 25 m/min for 30 min. Thus, exercise may regulate adiponectin receptor mRNA expression in tissues, which might cause increases in glucose uptake and fatty acid oxidation in the muscle. The effect of exercise on adiponectin levels depends on the specific conditions of the exercise.

Appearance of a liquid crystalline nematic-isotropic critical point in a mixture system of rod- and bent-shaped molecules.

Heat capacity measurements have been made on a liquid-crystal mixture system formed from bent-shaped molecule 1,3-phenylene bis[4-(4-8-alkoxyphenyliminomethyl)benzoates] (P-8-O-PIMB) and rod-shaped molecule n-pentyl-cyanobiphenyl (5CB). The obtained results can be understood assuming that the addition of P-8-O-PIMB molecules to the 5CB system affects as a field conjugate to the nematic order parameter. The B(X)-B(4) transition can be viewed as the nematic-isotropic transition of 5CB, which is embedded in a framework of the B(4) structure of P-8-O-PIMB molecules. The present mixture system offers a rare example of the nematic-isotropic critical point, whose critical behavior has not been studied yet in detail.

Smectic-Calpha*-smectic-C* phase transition and critical point in binary mixtures.

We have investigated the smectic-Calpha*-smectic-C* (SmCalpha*-SmC*) transition in a series of binary mixtures with resonant x-ray diffraction, differential optical reflectivity, and heat capacity measurements. Results show that the phases are separated by a first-order transition that ends at a critical point. We report the observation of such a critical point. We have proposed the appropriate order parameter and obtained values of two critical exponents associated with this transition. The values of the critical exponents suggest that long-range interactions are present in the SmCalpha*-SmC* critical region.

Stimulation of catecholamine biosynthesis via the PKC pathway by prolactin-releasing peptide in PC12 rat pheochromocytoma cells.

We have previously shown that prolactin-releasing peptide (PrRP) stimulates catecholamine release from PC12 cells (rat pheochromocytoma cell line). However, it is not known whether PrRP also affects catecholamine biosynthesis. Thus, we examined the effect of PrRP on catecholamine biosynthesis in PC12 cells. PrRP31 (>10 nM) and PrRP20 (>100 nM) significantly increased the activity and expression level of tyrosine hydroxylase (TH), a rate-limiting enzyme, in catecholamine biosynthesis. However, the PrRP20-stimulated TH activity was markedly weaker than that of PrRP31. PrRP31 (>1 nM) and PrRP20 (>10 nM) significantly induced an increase in the level of PKC activity. Both Ro 32-0432 (a protein kinase C inhibitor) and H89 (a protein kinase A inhibitor) effectively suppressed the PrRP31 (100 nM)-induced TH mRNA level. Next, we examined the effect of PrRP on mitogen-activated protein kinases (MAPKs). PrRP31 (1 microM) significantly induced an increase in the activity of extracellular signal-related kinases (ERKs) and the stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK). In contrast to ERKs and JNK, PrRP31 did not affect P38 MAPK activity. Consistent with these findings, pretreatment of cells with the MEK-1-inhibitor, PD-98059 (50 microM), significantly inhibited the PrRP31 (100 nM)-induced increase in TH mRNA. These results indicate that PrRP stimulates catecholamine synthesis through both the PKC and PKA pathways in PC12 cells.

Calorimetric investigations of liquid-crystal compounds exhibiting almost no layer-shrinkage behavior through the smectic-A-smectic- C* transition.

Heat-capacity measurements have been made on liquid-crystal compounds exhibiting almost no layer-shrinkage (NLS) behavior through the Sm-A-Sm- C(*) phase transition. The transition was found to be second order for two of the substances studied. It was found that the heat-capacity anomaly accompanying a second-order Sm-A-Sm- C(*) transition with NLS behavior is quite similar to that observed for typical antiferroelectric liquid crystals of the 4-(1-methylheptyloxycarbonyl)phenyl 4'-octyloxybiphenyl-4-carboxylate (MHPOBC) group, showing three-dimensional (3D) XY behavior in the vicinity of the transition. On the other hand, for one compound which shows a weakly first-order transition, the anomaly is almost symmetric above and below T(c) , with a significant fluctuation effect in the Sm-A phase. For this compound, the critical behavior of the heat-capacity anomaly is almost tricritical in the immediate vicinity of T(c) , while away from T(c) the behavior can be explained with the 3D XY model. This suggests that the underlying transition with the 3D XY critical behavior is driven to almost being tricritical but remaining weakly first order. No indication of low-dimensional character in the critical behavior was found in both cases.

Experimental investigations of one liquid-crystal compound exhibiting the no-layer-shrinkage effect near the Sm-A-Sm- C(*) transition.

Three experimental probes have been employed to investigate the nature of the smectic- A -smectic- C ( Sm-A-Sm- C(*) ) phase transition of one liquid-crystal compound showing almost no layer-shrinkage effect through the transition. Results from both x-ray diffraction and optical studies indicate that the compound exhibits a crossover behavior of different molecular packing arrangements within the bulk Sm-A phase window. The calorimetry results show a significant critical anomaly near the Sm-A-Sm- C(*) transition, although it was found to be weakly first order.

Angiotensin subtype-2 receptor (AT2 ) negatively regulates subtype-1 receptor (AT1 ) in signal transduction pathways in cultured porcine adrenal medullary chromaffin cells.

BACKGROUND: Two distinct types of angiotensin II (AngII) receptors, AT1 and AT2, have been cloned. We have shown previously that stimulation of AT2 reduces intracellular cyclic guanosine monophosphate (cGMP) levels in cultured porcine chromaffin cells in which AT2 is the predominantly expressed receptor. However, it has not been determined whether AT1 or AT2 affects signal transduction pathways involving mitogen-activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs) in chromaffin cells. Also, it is unclear whether cGMP/protein kinase G (PKG) is involved in the regulation of MAPKs and STATs in these cells. DESIGN: Chromaffin cells were derived from porcine adrenal medulla. The effects of AngII alone (representing physiological conditions), AngII plus CV-11974 (an AT1 antagonist, which simulates specific AT2 stimulation), AngII plus PD 123319 (an AT2 antagonist, which simulates specific AT1 stimulation), and 8-Br-cGMP (a membrane-permeable cGMP analogue) alone on MAPKs (ERKs, JNK, p-38 MAPK) and STATs (STATs 1, 3 and 5) activity were measured. METHODS: Phosphorylated MAPKs (extracellular signal-related kinases (ERKs), c-jun N-terminal kinase (JNK) and p38 MAPK) and STATs (STATs 1, 3 and 5) were measured by immunoprecipitation-Western blot analysis (IP-Western blot). RESULTS: AT1 stimulation markedly increased expression of ERKs, JNK, p38 MAPK via Ca2+-dependent protein kinase C (PKC) isoforms (cPKC), as well as STATs 1, 3 and 5 in cultured porcine chromaffin cells. In contrast, AT2 stimulation markedly decreased the expression of these signaling molecules. Also, 8-Br-cGMP alone induced increases in ERKs, JNK, p38 MAPK, and STATs 1, 3 and 5. Because AT2 inhibits cGMP production, we speculate that AT2 may act to suppress cGMP production, which in turn reduces the activity of both MAPKs and STATs in chromaffin cells. CONCLUSION: AT2 negatively regulates AT1 in signal transduction pathways in chromaffin cells.

Leptin stimulates catecholamine synthesis in a PKC-dependent manner in cultured porcine adrenal medullary chromaffin cells.

We have previously shown that murine recombinant leptin directly stimulates catecholamine synthesis through the long form of the leptin receptor (Ob-Rb) expressed in cultured porcine chromaffin cells. Additionally, we found that leptin activates IP3 production after PLC activation. It is well established that activation of PLC elicits IP3 production as well as an increase in diacylglycerol, a compound that stimulates PKC. Therefore, we investigated the involvement of PKC in leptin-induced catecholamine synthesis. Leptin was found to induce significant increases in PKC activity in a dose-dependent manner (1, 10, and 100 nM); chelation of extracellular Ca(2+) by EDTA abolished this PKC stimulatory activity. We also confirmed by Western blot analysis that leptin (at 100 nM) induced significant increases in Ca(2+)-dependent PKC alpha, -beta(I), and -gamma expression. The activity of the rate-limiting enzyme tyrosine hydroxylase (TH) in the biosynthesis of catecholamine is regulated at the transcriptional and posttranscriptional levels. TH enzyme activity and TH mRNA levels induced by 100 nM leptin were significantly inhibited by the PKC inhibitor Ro 32-0432 as well as by EDTA. In addition, increases in TH protein and intracellular catecholamine content stimulated by leptin were completely inhibited by Ro 32-0432. Leptin markedly activated ERKs and, to a lesser extent, JNK; these stimulatory effects on ERKs and JNK were completely inhibited by Ro 32-0432 as well as EDTA. In contrast, leptin did not activate P38 MAPK. Similar to leptin, PMA activated ERK and JNK. Nicardipine and omega-conotoxin GVIA, each at 1 microM, were effective at inhibiting leptin-induced TH enzyme activity, TH mRNA accumulation, PKC activity, and ERK activity. Leptin increased activating protein-1 DNA-binding activity, and this was diminished by Ro 32-0432 as well as EDTA, similar to the reduction of TH mRNA levels. In addition, using supershift analysis, we documented the involvement of c-Fos and, to a lesser extent, c-Jun in leptin-induced activating protein-1 activity. These results indicate that leptin stimulates Ca(2+)-dependent PKC isoform-dependent catecholamine synthesis in porcine chromaffin cells. Previously, we had shown that leptin stimulated cAMP. The present study also showed that H89 (a PKA inhibitor) moderately, but significantly, inhibited leptin-induced ERK and TH mRNA. Consistent with this finding, leptin is shown here to activate novel PKC epsilon, which is assumed to stimulate Raf, upstream of ERKs, via cAMP, supporting the suggestion that Ca(2+)-independent novel PKC may also play some physiological role in regulating catecholamine synthesis.

Activation of angiotensin II subtype 2 receptor induces catecholamine release in an extracellular Ca(2+)-dependent manner through a decrease of cyclic guanosine 3',5'-monophosphate production in cultured porcine adrenal medullary chromaffin Cells.

We have previously demonstrated that CGP 42112 (AT(2) agonist > or =1 nM) markedly reduces catecholamine biosynthesis through AT(2), which is the major angiotensin II (AngII) receptor subtype in cultured porcine chromaffin cells. Also, we have shown that CGP 42112 (> or =1 nM) induces a reduction in cGMP production in these cells. The present study showed that AngII reduced cGMP production via AT(2) in a manner similar to that found with CGP 42112. AngII (1 nM) significantly increased catecholamine secretion from cultured porcine adrenal medullary chromaffin cells. The stimulation was significantly inhibited by PD 123319 (AT(2) antagonist). The stimulation was moderately, but significantly, attenuated by CV-11974 (AT(1) antagonist, > or =10 nM), suggesting an involvement of AT(1). Moreover, CGP 42112 (> or =10 nM) markedly increased catecholamine release from these cells. The stimulation by CGP 42112 was abolished by PD 123319, whereas CV-11974 had no effect, indicating that this response is also mediated by AT(2). We further examined whether extracellular Ca(2+) is involved in the stimulatory effect of AT(2) on catecholamine secretion. Removal of external Ca(2+) significantly suppressed either AngII plus CV-11974 (100 nM; which simulates specific AT(2) stimulation) or CGP 42112- induced catecholamine secretion. AngII plus CV-11974 or CGP 42112 caused a sustained increase in intracellular Ca(2+) ([Ca(2+)](i)), as determined in fura-2-loaded chromaffin cells in an extracellular Ca(2+)-dependent manner. In the presence of EGTA, the subsequent addition of AngII with CV-11974 and CGP 42112 did not cause any increase in [Ca(2+)](i) levels. Consistent with this finding, CGP 42112 (10 nM to 1 microM) did not alter inositol triphosphate (IP(3)) production, a messenger for mobilization of Ca(2+) from intracellular storage sites. In addition, the intracellular Ca(2+) chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethylester (BAPTA) did not affect CGP 42112-induced catecholamine release. We tested whether a decrease in cGMP was the cause of the stimulatory effect of AT(2) on catecholamine secretion. Pretreatment with 8-bromo-cGMP (1 mM) prevented the stimulatory effect of AngII plus CV-11974 and CGP 42112 on both catecholamine secretion and [Ca(2+)](i). When 8-bromo-cGMP was added after application of AngII plus CV-11974 or CGP 42112, [Ca(2+)](i) induced by these agents was gradually reduced toward the baseline values. Similarly, guanylin completely abolished the AngII- plus CV-11974-induced increase in both NE secretion and [Ca(2+)](i). The Ca(2+) channel blockers, nicardipine and omega-conotoxin G VIA, at 1 microM in both cases, were also effective in inhibiting AT(2) stimulation-induced secretion. On the other hand, neither T-type voltage-dependent Ca(2+) channel blockers, flunarizine, nor Ni(2+) affected catecholamine release caused by AT(2) stimulation. These findings demonstrate that AT(2) stimulation induces catecholamine secretion by mobilizing Ca(2+) through voltage-dependent Ca(2+) channels without affecting intracellular pools and that these effects could be mediated by a decrease in cGMP production.

Ca(2+) mobilization, tyrosine hydroxylase activity, and signaling mechanisms in cultured porcine adrenal medullary chromaffin cells: effects of leptin.

Leptin acts as a satiety factor, but there is also evidence that it affects energy expenditure. Leptin's effects are mediated by its receptors, which function as activators of a Janus family of tyrosine kinases-signal transducer and activator of transcription (JAK-STAT) pathway. We have previously shown that murine recombinant leptin markedly induces both the release of catecholamine and tyrosine hydroxylase (TH) (rate-limiting enzyme in the biosynthesis of catecholamine)-messenger RNA (mRNA) levels, probably through Ob-Rb expressed in cultured porcine chromaffin cells. In the present study, we examined the effect of leptin on Ca(2+) mobilization, TH enzyme activity, and signaling. Ca(2+) channel blockers, nicardipine and omega-Conotoxin GVIA, each at 1 microM, were effective in inhibiting leptin-induced catecholamine secretion. When intracellular Ca(2+) ([Ca(2+)](i)) was measured in fura 2-loaded chromaffin cells, leptin was found to cause a sustained increase of Ca(2+) by mobilizing Ca(2+) from both extra- and intracellular pools. Additionally, leptin significantly stimulated inositol 1.4.5-triphosphate IP(3) production in a dose-dependent manner. TH-activity is regulated by both TH enzyme activity and increased TH-mRNA levels accompanied by increased TH protein synthesis. Leptin (>/=1 nM) significantly stimulated TH enzyme activity and increased the TH protein level, indicating that it stimulates catecholamine biosynthesis. In addition, removal of external Ca(2+) completely inhibited leptin (100 nM)-induced TH enzyme activity. Leptin (>/=1 nM) caused an increase in the activity of mitogen-activated protein kinases (MAPKs) that was accompanied by increased phosphorylation of STAT-3 and -5, but not STAT-1. Moreover, MAPK activity evoked by leptin(100 nM) and TH-mRNA caused by leptin (10 nM) were inhibited by 50 and 30 microM of PD-98059 (the MAP kinase kinase-1 inhibitor), respectively. These findings indicate that leptin activates voltage-dependent Ca(2+) channels (VDCC), presumably L-type and N-type Ca(2+) channels, as well as phospholipase C, and suggest that leptin-induced catecholamine secretion is mainly mediated by activation of VDCC. In addition, leptin stimulates the JAK-STAT pathway as well as increasing the levels of TH-mRNA levels through the MAPK pathway in porcine chromaffin cells.

RGS14, a GTPase-activating protein for Gialpha, attenuates Gialpha- and G13alpha-mediated signaling pathways.

Regulator of G protein signaling (RGS) proteins are a family of approximately 20 proteins that negatively regulate signaling through heterotrimeric G protein-coupled receptors. The RGS proteins act as GTPase-activating proteins (GAPs) for certain Galpha subunits and as effector antagonists for Gqalpha. Mouse RGS14 encodes a 547-amino-acid protein with an N-terminal RGS domain, which is highly expressed in lymphoid tissues. In this study, we demonstrate that RGS14 is a GAP for Gialpha subfamily members and it attenuates interleukin-8 receptor-mediated mitogen-activated protein kinase activation. However, RGS14 does not exhibit GAP activity toward Gsalpha or Gqalpha nor does it regulate Gsalpha- or Gqalpha-mediated signaling pathways. Although RGS14 does not act as a GAP for G12/13alpha, it impairs c-fos serum response element activation induced by either a constitutively active mutant of G13alpha (G13alphaQ226L) or by carbachol stimulation of muscarinic type 1 receptors. An RGS14 mutant (EN92/93AA), which does not block Gialpha-linked signaling, also inhibits serum response element activation. RGS14 localizes predominantly in the cytosol, but it can be recruited to membranes by expression of G13alphaQ226L. Although RGS14 is constitutively expressed in lymphoid cells, agents that activate B or T lymphocytes further enhance its levels. Taken together, our results suggest that signals generated after lymphocyte activation may via RGS14 directly impinge on Gialpha- or G13alpha-mediated cellular processes in lymphocytes, such as adhesion and migration.

kappa-Opioid inhibits catecholamine biosynthesis in PC12 rat pheochromocytoma cell.

It was reported that nicotine-induced dopamine release in the rat pheochromocytoma cell line, PC12 cells, was inhibited by kappa-opioid. However, it is not known whether inhibition of catecholamine biosynthesis is involved in the inhibitory mechanisms of kappa-opioids in PC12 cells. U-69593 (a kappa-opioid agonist: >/=100 nM) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH, a rate-limiting enzyme in biosynthesis of catecholamine) enzyme activity and TH mRNA levels. These inhibitory effects were completely reversed by naloxone and nor-binaltorphimine dihydrochloride (nor-BNI), a specific kappa-antagonist, whereas pertussis toxin (PTX) only partially reversed this inhibitory effect. Also, U-69593 (>/=100 nM) significantly inhibited the nicotine-induced increase of cAMP production. This inhibitory effect was completely reversed by naloxone and nor-BNI, whilst only partially reversed by PTX. Moreover, U-69593 (>/=100 nM) significantly inhibited the nicotine-induced increase of both the TH protein level and intracellular catecholamine levels. These results indicate that the anti-cholinergic actions of kappa-opioid can be explained partially by its inhibition of both TH enzyme activity and TH synthesis, through suppression of the cAMP/protein kinase A pathway. It would also appear that the PTX-sensitive G-protein mediates the inhibitory effect of this pathway, at least in part.

Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells.

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.

Angiotensin-II subtype 2 receptor agonist (CGP-42112) inhibits catecholamine biosynthesis in cultured porcine adrenal medullary chromaffin cells.

Angiotensin II subtype 2 receptor (AT(2)-R) is abundantly expressed in adrenal medullary chromaffin cells. However, the physiological roles of AT(2)-R in chromaffin cells remain to be clarified. Therefore, we investigated the effects of CGP42112 (AT(2)-R agonist) on catecholamine biosynthesis in cultured porcine adrenal medullary cells. We initially confirmed AT(2)-R was predominantly expressed in porcine adrenal medullary cells by [(125)I]-Ang II binding studies. CGP42112 (>==1 nM) significantly inhibited cGMP production from the basal value. Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the biosynthesis of catecholamine, and its activity is regulated by both TH-enzyme activity and TH-synthesis. CGP42112 (>==1 nM) significantly inhibited TH-enzyme activity from the basal value. These inhibitory effects of CGP42112 on TH-enzyme activity and-cGMP production were abolished by PD123319 (AT(2)-R antagonist) while CV-11974 (AT(1)-R antagonist) was ineffective. We also tested whether decrease of cGMP is involved in the inhibitory effect of CGP42112 on TH-enzyme activity. Pretreatment of 8-Br-cGMP (membrane-permeable cGMP analogue) prevented the inhibitory effect of CGP 42112 on TH-enzyme activity. Similar to that of TH-enzyme activity, CGP42112 (>==1 nM) significantly reduced TH-mRNA and TH-protein level from the basal value, and these inhibitory effects were abolished by PD123319 but not CV-11974. These findings demonstrate that CGP 42112 reduces both TH-enzyme activity and TH-synthesis and that these inhibitory effects could be mediated by decrease of cGMP production.