Rho kinase is involved in Ca2+ entry of rat penile small arteries

Tonic physiological activity of RhoA/Rho kinase contributes to the maintenance of penile flaccidity through its involvement in the Ca(2+) sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho kinase is also involved in the modulation of Ca(2+) entry induced by alpha(1)-adrenoceptor stimulation of penile arteries. Rat penile arteries were mounted in microvascular myographs for simultaneous measurements of intracellular Ca(2+) ([Ca(2+)](i)) and force. The Rho-kinase inhibitor Y-27632 markedly reduced norepinephrine-mediated electrically induced contractions and the increases in both [Ca(2+)](i) and tension elicited by the alpha(1)-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro-31-8220 reduced tension without altering the Phe-induced increase in [Ca(2+)](i). In the presence of nifedipine, Y-27632 still inhibited the non-L-type Ca(2+) signal and blunted Phe contraction. Y-27632 did not impair the capacitative Ca(2+) entry evoked by store depletion with cyclopiazonic acid but largely reduced the Ba(2+) influx stimulated by Phe in fura-2 AM-loaded arteries. The addition of Y-27632 to arteries depolarized with high KCl markedly reduced tension without changing [Ca(2+)](i). In alpha-toxin-permeabilized penile arteries stimulated with threshold Ca(2+) concentrations, Y-27632 inhibited the sensitization induced by either guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) or Phe in the presence of GTPgammaS. However, Y-27632 failed to alter contractions induced by a maximal concentration of free Ca(2+). These results suggest that Rho kinase, besides its contribution to the Ca(2+) sensitization of the contractile proteins, is also involved in the regulation of Ca(2+) entry through a nonselective cation channel activated by alpha(1)-adenoceptor stimulation in rat penile arteries.     

Mechanism of the negative inotropic effects of alpha 1-adrenoceptor agonists on mouse myocardium

This study was done to identify the mechanism of the alpha1-adrenoceptor (AR) mediated negative inotropic effects of phenylephrine (PE) on adult mouse myocardium. As reported by others, we also found that the nonselective alpha1AR agonist PE produced a negative inotropic effect on ventricular strips from adult mice that was inhibited by the alpha1AAR antagonist 5-methylurapidil (5MU) but not by the alpha1BAR antagonist chloroethylclonidine (CEC) or the alpha1DAR antagonist BMY 7378. The selective alpha1AAR agonist A61603 also produced a negative inotropic effect, which was antagonized by 5MU. Phorbol 12,13-dibutyrate (activator of all PKC isoforms) mimicked the negative inotropic responses to PE and A61603. The negative inotropic effects of PE were inhibited by bisindolylmaleimide (inhibitor of all PKC isoforms) but not by G? 6976 (inhibitor of Ca2+-dependent PKC). Rottlerin, an inhibitor of Ca2+-independent PKCdelta, antagonized the negative inotropic effects of PE and A61603. PE and A61603 increased the translocation of PKCdelta, which was prevented by rottlerin. These data suggest that the alpha1AR-mediated negative inotropy on adult mouse myocardium is signaled by Ca2+-independent PKCdelta.     

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).     

Contrasting inotropic responses to alpha1-adrenergic receptor stimulation in left versus right ventricular myocardium

The left ventricle (LV) and right ventricle (RV) have differing hemodynamics and embryological origins, but it is unclear whether they are regulated differently. In particular, no previous studies have directly compared the LV versus RV myocardial inotropic responses to alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation. We compared alpha(1)-AR inotropy of cardiac trabeculae from the LV versus RV of adult mouse hearts. As previously reported, for mouse RV trabeculae, alpha(1)-AR stimulation with phenylephrine (PE) caused a triphasic contractile response with overall negative inotropy. In marked contrast, LV trabeculae had an overall positive inotropic response to PE. Stimulation of a single subtype (alpha(1A)-AR) with A-61603 also mediated contrasting LV/RV inotropy, suggesting differential activation of multiple alpha(1)-AR-subtypes was not involved. Contrasting LV/RV alpha(1)-AR inotropy was not abolished by inhibiting protein kinase C, suggesting differential activation of PKC isoforms was not involved. However, contrasting LV/RV alpha(1)-AR inotropic responses did involve different effects on myofilament Ca(2+) sensitivity: submaximal force of skinned trabeculae was increased by PE pretreatment for LV but was decreased by PE for RV. For LV myocardium, alpha(1)-AR-induced net positive inotropy was abolished by the myosin light chain kinase inhibitor ML-9. This study suggests that LV and RV myocardium have fundamentally different inotropic responses to alpha(1)-AR stimulation, involving different effects on myofilament function and myosin light chain phosphorylation.     

Impaired Ca2+ handling in penile arteries from prediabetic Zucker rats: involvement of Rho kinase

Diabetes is associated with an increased vascular tone usually involved in the pathogenesis of diabetic cardiovascular complications such as hypertension, stroke, coronary artery disease, or erectile dysfunction (ED). Enhanced contractility of penile erectile tissue has been associated with augmented activity of the RhoA/Rho kinase (RhoK) pathway in models of diabetes-associated ED. The present study assessed whether abnormal vasoconstriction in penile arteries from prediabetic obese Zucker rats (OZRs) is due to changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and/or in myofilament Ca(2+) sensitivity. Penile arteries from OZRs and lean Zucker rats (LZRs) were mounted on microvascular myographs for simultaneous measurements of [Ca(2+)](i) and tension. The relationships between [Ca(2+)](i) and contraction for the α(1)-adrenergic vasoconstrictor phenylephrine (PE) were left shifted and steeper in OZRs compared with LZRs, although the magnitude of the contraction was similar in both groups. In contrast, the vasoconstriction induced by the thromboxane A(2) receptor agonist U-46619 was augmented in arteries from OZRs, and this increase was associated with an increase in both the sensitivity and maximum responses to Ca(2+). The RhoK inhibitor Y-27632 (10 μM) reduced the vasoconstriction induced by PE to a greater extent in OZRs than in LZRs, without altering Ca(2+). Y-27632 inhibited with a greater potency the contraction elicited by high KCl in arteries from OZRs compared with LZRs without changing [Ca(2+)](i). RhoK-II expression was augmented in arteries from OZRs. These results suggest receptor-specific changes in the Ca(2+) handling of penile arteries under conditions of metabolic syndrome. Whereas augmented vasoconstriction upon activation of the thromboxane A(2) receptor is coupled to enhanced Ca(2+) entry, a RhoK-mediated enhancement of myofilament Ca(2+) sensitivity is coupled with the α(1)-adrenergic vasoconstriction in penile arteries from OZRs.     

K+ depolarization induces RhoA kinase translocation to caveolae and Ca2+ sensitization of arterial muscle

KCl causes smooth muscle contraction by elevating intracellular free Ca2+, whereas receptor stimulation activates an additional mechanism, termed Ca2+ sensitization, that can involve activation of RhoA-associated kinase (ROK) and PKC. However, recent studies support the hypothesis that KCl may also increase Ca2+ sensitivity. Our data showed that the PKC inhibitor GF-109203X did not, whereas the ROK inhibitor Y-27632 did, inhibit KCl-induced tonic (5 min) force and myosin light chain (MLC) phosphorylation in rabbit artery. Y-27632 also inhibited BAY K 8644- and ionomycin-induced MLC phosphorylation and force but did not inhibit KCl-induced Ca2+ entry or peak ( approximately 15 s) force. Moreover, KCl and BAY K 8644 nearly doubled the amount of ROK colocalized to caveolae at 30 s, a time that preceded inhibition of force by Y-27632. Colocalization was not inhibited by Y-27632 but was abolished by nifedipine and the calmodulin blocker trifluoperazine. These data support the hypothesis that KCl caused Ca2+ sensitization via ROK activation. We discuss a novel model for ROK activation involving translocation to caveolae that is dependent on Ca2+ entry and involves Ca2+-calmodulin activation.     

Rho-associated protein kinase is involved in establishing the cardiomyocyte contractile phenotype

We have shown that Y-27632, an inhibitor of Rho-associated kinase (ROCK), delays sarcomere assembly in cultured rat neonatal cardiomyocytes pretreated with angiotensin II. Y-27632 affects the beat rate of cardiomyocytes, but this effect is only observed at high cell density and, therefore, seems to be related to impairment of cell-cell interactions. Consistent with this suggestion, ROCK2 is localized in myofibrillar Z-disks of human myocardium as well as in intercalated disks. We propose that ROCK participates in maturation of the myocardial contractile system through phosphorylation of its molecular targets in Z-disks and in intercalated disks.     

Effect of exercise on augmented aortic vasoconstriction in the db/db mouse model of type-II diabetes

We evaluated the effects of exercise on the vascular constrictor responses to alpha-adrenergic stimulation in the db/db mice. Twenty male db/db and their age-matched wild-type (WT) mice were exercised (1 hour/day, five days a week). Mice were anesthetized 7 weeks later, thoracic aortae were mounted in wire myograph and constrictor responses to phenylephrine (PE, 1 nM-10 microM) were obtained. Citrate synthase activity measured in the thigh adductor muscle was significantly increased in db/db mice that were exercise trained. Maximal force generated by PE was markedly greater in db/db aortae and exercise did not attenuate this augmented contractile response. Vessels were incubated with inhibitors of nitric oxide synthase (L-NAME, 200 microM), endothelin receptors (bosentan, 10 microM), protein kinase C (PKC) (calphostin C, 5 microM), cyclooxygenase (indomethacin, 10 microM) or Rho-kinase (Y-27632, 0.1 microM). Only calphostin-C normalized the augmented PE-induced constriction in db/db and db/db- exercised mice to that observed in WT (p<0.05). Cumulative additions of indolactam, a PKC activator, induced significantly greater constrictor responses in aortic rings of db/db mice compared to WT and exercise did not affect this response. Our data suggest that the augmented vasoconstriction observed in the aorta of db/db mice is likely due to increased PKC activity and that exercise do not ameliorate this increased PKC-mediated vasoconstriction.     

Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.     

Acute and chronic NOS inhibition enhances alpha(2)- adrenoreceptor-stimulated RhoA and Rho kinase in rat aorta

We demonstrated that arteries from rats made hypertensive with chronic nitric oxide (NO) synthase (NOS) inhibition (N(omega)-nitro-L-arginine in drinking water, LHR) have enhanced contractile sensitivity to alpha(2)-adrenergic receptors (alpha(2)-AR) agonist UK-14304 compared with arteries from normotensive rats (NR). NO may regulate vascular tone in part through suppression of RhoA and Rho kinase (ROK). We hypothesized that enhanced RhoA and ROK activity augments alpha(2)-AR contraction in LHR aortic rings. Y-27632 eliminated UK-14304 contraction in LHR and NR aortic rings. The order of increasing sensitivity to Y-27632 was the following: endothelium-intact NR, LHR, and endothelium-denuded NR. UK-14304 stimulated RhoA translocation to the membrane fraction in LHR and denuded NR but not in intact NR aorta. Basally, more RhoA was present in the membrane fraction in denuded NR than in intact NR or LHR aorta. Relaxation to S-nitroso-N-acetyl-penicillamine and Y-27632 in denuded ionomycin-permeabilized rings was greater in NR than in LHR. Together these studies indicate alpha(2)-AR contraction depends on ROK activity more in NR than LHR aorta. Additionally, endogenous NO may regulate RhoA activation, whereas chronic NOS inhibition appears to cause RhoA desensitization.     

Rho-associated protein kinase is involved in establishing the contractile phenotype of cardiomyocytes

It has been shown that Y-27632, an inhibitor of Rho-associated kinase, delays sarcomere assembly in rat neonatal cardiomyocytes pretreated with angiotensin II. Y-27632 affects the beat rate of cardiomyocytes; however, this effect is only observed at high cell density and, therefore, seems to be related to the formation of gap junctions between adjacent cardiomyocytes. Consistent with this suggestion, we established that Rho-associated kinase is localized in myofibrillar Z-discs of human myocardium and intercalated discs, the structures enriched in gap junctions. We propose that Rho-associated kinase participates in the maturation of the myocardial contractile system through phosphorylation of its molecular targets in Z-discs and intercalated discs.     

RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells

Protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) mediates some agonist-induced smooth muscle contraction by suppressing the myosin phosphatase in a phosphorylation-dependent manner. The physiologically relevant kinases that phosphorylate CPI-17 remain to be identified. Several previous studies have shown that some agonist-induced CPI-17 phosphorylation in smooth muscle tissues was attenuated by the Rho kinase (ROCK) inhibitor Y-27632, suggesting that ROCK is involved in agonist-induced CPI-17 phosphorylation. However, Y-27632 has recently been found to inhibit protein kinase C (PKC)-, a well-recognized CPI-17 kinase. Thus the role of ROCK in agonist-induced CPI-17 phosphorylation remains uncertain. The present study was designed to address this important issue. We selectively activated the RhoA pathway using inducible adenovirus-mediated expression of a constitutively active mutant RhoA (V14RhoA) in primary cultured rabbit aortic vascular smooth muscle cells (VSMCs). V14RhoA caused expression level-dependent CPI-17 phosphorylation at Thr38 as well as myosin phosphatase phosphorylation at Thr853. Importantly, we have shown that V14RhoA-induced CPI-17 phosphorylation was not affected by the PKC inhibitor GF109203X but was abolished by Y-27632, suggesting that ROCK but not PKC was involved. Furthermore, we have shown that the contractile agonists thrombin and U-46619 induced CPI-17 phosphorylation in VSMCs. Similarly to V14RhoA-induced CPI-17 phosphorylation, thrombin-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X, but it was blocked by inhibition of RhoA with adenovirus-mediated expression of exoenzyme C3 as well as by Y-27632. Taken together, our present data provide the first clear evidence indicating that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. protein kinase C; signal transduction; adenovirus     

Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway

Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+ sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the alpha1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (< or =3 microM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in alpha-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+ activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+ sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.     

Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C

It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca(2+) sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca(2+) sensitization were both attenuated by 10(-6) mol Y-27632 /L, a ROCK inhibitor, 10(-6) mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca(2+) sensitization in alpha-toxin-permeabilized bronchial smooth muscle. In this case, the Ca(2+) sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca(2+) sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

RhoA-Rho kinase mediates synergistic ET-1 and phenylephrine contraction of rat corpus cavernosum

Maintenance of the detumescent state of the penis is believed to involve the actions of several vasoconstrictors. However, our mechanistic understanding of any synergistic vasoconstrictor influences is extremely limited. We tested the hypothesis that a vasoconstrictor combination of endothelin (ET-1) and phenylephrine (PE) augments the constrictor responses in rat corporal cavernosal tissues by a mechanism involving the RhoA-Rho kinase pathway. Independently, ET-1 (1 nM-30 microM) and PE (100 nM-100 microM) both caused dose-dependent contractions of isolated rat cavernosal tissues. In combination, ET-1 (30 nM) augmented the contractile effect of PE and shifted the calculated EC50 for PE (90 +/- 12 to 45 +/- 5 microM). The active stress generated by cavernosal strips during the ET-1 + PE combined stimulation (4.9 +/- 0.2 mN/mm2) was greater than the combined stress generated with ET-1 (0.4 +/- 0.1 mN/mm2) or PE (3.3 +/- 0.2 mN/mm2) stimulations alone. Blockade of ETA receptors (30 nM; A-127722) reversed the augmented stress generation and the Rho-kinase inhibitor Y-27632 differentially and dose-dependently relaxed the tissue. The combined constrictor effect was associated with a fourfold increase of RhoA in the membrane faction of the tissue homogenates. We conclude that the ET-1 + PE combination potentiate vasoconstriction through mutual activation of the RhoA-Rho kinase pathway. The interactions of these agonists likely play important roles in the maintenance of the flaccid state and contribute to some forms of erectile dysfunction.     

Effects of dexmedetomidine on the release of glial cell line-derived neurotrophic factor from rat astrocyte cells

Dexmedetomidine (DEX) has been found to improve neuronal survival after transient global or focal cerebral ischemia in rats. Astrocyte cells may possess beneficial properties that promote neuronal recovery by secreting neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF). The purpose of this study was to investigate the effects of DEX on GDNF release from astrocytes and the possible mechanisms involved. Astrocyte cells were treated with DEX, and GDNF level in the conditioned media was determined by ELISA assay. The expression of CREB, p-CREB and PKCα was analyzed by Western blotting to explore the mechanisms involved in GDNF release. Our results showed that DEX stimulated GDNF release in a time- and dose-dependent manner; and this stimulation was blocked by the α2-adrenoreceptor antagonist yohimbine, but not by α1-adrenoreceptor antagonist prasozin, demonstrating that DEX induced GDNF release likely acts via activating the α2A adrenoreceptor. In addition, DEX-stimulated GDNF release was also blocked by the universal PKC inhibitor Ro-318220 and PKCα/β inhibitor G? 6976, but not by PKCδ inhibitor rottlerin and PKCβ inhibitor LY333531. Interestingly, DEX also activated CREB phosphorylation, which was inhibited by Ro-318220, G? 697 and ERK kinase inhibitor PD98059. Silencing CREB by siRNA decreased the DEX-stimulated GDNF release. In addition, the membrane translocation of PKCα was enhanced following DEX treatment. Furthermore, we found that DEX stimulated GDNF release rescued neurons against OGD-induced neurotoxicity; this effect was partly abolished by GDNF antibody. Thus, through α2A adrenergic receptors, DEX may activate astrocytes, and promote GDNF release to protect neurons after stroke, and this signaling is possibly dependent on PKCα and CREB activation.     

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.     

Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension

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

Pregnancy reduces RhoA/Rho kinase and protein kinase C signaling pathways downstream of thromboxane receptor activation in the rat uterine artery

During pregnancy, reduced vascular responses to constrictors contribute to decreased uterine and total vascular resistance. Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor that exerts its actions via diverse signaling pathways, and its biosynthesis increases in preeclampsia. In this study, we hypothesized that maternal vascular responses to TxA(2) will be attenuated via Rho kinase, PKC, p38 MAPK, and ERK1/2 signaling pathways. Isolated ring segments of uterine and small mesenteric arteries from late pregnant (19-21 days) and virgin rats were suspended in a myograph, and isometric force was measured. Pregnancy did not affect uterine and mesenteric artery responses to the TxA(2) analog U-46619 (10(-9)-10(-5) M), but transduction signals associated with these contractions were different between pregnant and nonpregnant rats. Inhibition of Rho kinase (10(-6) M Y-27632) reduced sensitivity to U-46619 in virgin uterine vessels but did not inhibit these contractions in pregnant uterine arteries and had no effect on mesenteric vessels. Treatment of arterial segments with a PKC inhibitor (10(-6) M bisindolylmaleimide I) reduced U-46619-induced contractions in virgin uterine and mesenteric arteries and in pregnant mesenteric arteries. Pregnant uterine arteries, however, were unresponsive to PKC inhibition. Inhibition of ERK1/2 (10(-5) M PD-98059) and p38 MAPK (10(-5) M SB-203580) reduced U46619-induced contractions in nonpregnant vessels and in pregnant uterine and mesenteric vessels. These data suggest that normal pregnancy does not affect uterine and mesenteric contractile responses to TxA(2) but reduces the contribution of Rho kinase and PKC signaling pathways to these contractions in the uterine vasculature. In contrast, the role of ERK1/2 and p38 MAPK in U-46619-induced uterine contractions remains unchanged with pregnancy. TxA(2)-associated transduction signals and its regulators might present potential targets for the development of new treatments for preeclampsia and other pregnancy-associated vascular diseases.