[Show abstract][Hide abstract] ABSTRACT: Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP(3)) is responsible for Ca(2+) increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP(3) is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.
The EMBO Journal 12/2006; 25(22):5305-16. DOI:10.1038/sj.emboj.7601417 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clathrin is a major component of clathrin-coated pits and serves as a binding scaffold for endocytic machinery through the binding of a specific sequence known as the clathrin-binding motif. This motif is also found in cellular signaling proteins other than endocytic components, including G protein-coupled receptor kinase 2 (GRK2), which phosphorylates G protein-coupled receptors and promotes uncoupling of receptor-G protein interaction. However, the functions of clathrin in the regulation of GRK2 are unknown. Here we demonstrated that overexpression of GRK2 mutated at the clathrin-binding motif with alanine (GRK2-5A) results in inhibition of phosphorylation and internalization of the beta2-adrenergic receptor (beta2AR). However, the interaction of beta2AR with GRK2-5A is the same as that of wild type GRK2 as determined by bioluminescence resonance energy transfer. Furthermore, GRK2-5A phosphorylates rhodopsin essentially to the same extent as wild type GRK2 in vitro. Depletion of the clathrin heavy chain using small interference RNA inhibits agonist-induced phosphorylation and internalization of beta2AR. Thus, clathrin works as a regulator of GRK2 in cells. These results indicate that clathrin is a novel player in cellular functions in addition to being a component of endocytosis.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of constitutively active (CA)-G alpha(13) significantly increased the expression of interleukin (IL)-1 beta and IL-6 mRNAs and proteins in rat cardiac fibroblasts. IL-1 beta mRNA induction by CA-G alpha(13) was suppressed by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, but not by BAPTA-AM, an intracellular Ca2+ chelator. In contrast, IL-6 mRNA induction by CA-G alpha(13) was suppressed by BAPTA-AM but not by DPI. However, both IL-1 beta and IL-6 mRNA induction was suppressed by nuclear factor kappa B (NF-kappa B) inhibitors. The CA-G alpha(13)-induced NF-kappa B activation was suppressed by DPI and BAPTA-AM, but not C3 toxin and the Rho-kinase inhibitor Y27632. IL-6 mRNA induction by CA-G alpha(13) was suppressed by SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), an inhibitor of receptor-activated nonselective cation channels, and the expression of CA-G alpha(13) increased basal Ca2+ influx. These results suggest that G alpha(13) regulates IL-1 beta mRNA induction through the reactive oxygen species-NF-kappa B pathway, while it regulates IL-6 mRNA induction through the Ca2+-NF-kappa B pathway.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of constitutively active (CA)-G alpha13 significantly increased the expression of interleukin (IL)-1beta and IL-6 mRNAs and proteins in rat cardiac fibroblasts. IL-1beta mRNA induction by CA-G alpha13 was suppressed by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, but not by BAPTA-AM, an intracellular Ca2+ chelator. In contrast, IL-6 mRNA induction by CA-G alpha13 was suppressed by BAPTA-AM but not by DPI. However, both IL-1beta and IL-6 mRNA induction was suppressed by nuclear factor kappaB (NF-kappaB) inhibitors. The CA-G alpha13-induced NF-kappaB activation was suppressed by DPI and BAPTA-AM, but not C3 toxin and the Rho-kinase inhibitor Y27632. IL-6 mRNA induction by CA-G alpha13 was suppressed by SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), an inhibitor of receptor-activated nonselective cation channels, and the expression of CA-G alpha13 increased basal Ca2+ influx. These results suggest that G alpha13 regulates IL-1beta mRNA induction through the reactive oxygen species-NF-kappaB pathway, while it regulates IL-6 mRNA induction through the Ca2+-NF-kappaB pathway.
Journal of Pharmacological Sciences 06/2006; 101(2):144-50. · 2.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Beta-arrestin mediates desensitization and internalization of beta-adrenergic receptors (betaARs), but also acts as a scaffold protein in extracellular signal-regulated kinase (ERK) cascade. Thus, we have examined the role of beta-arrestin2 in the betaAR-mediated ERK signaling pathways. Isoproterenol stimulation equally activated cytoplasmic and nuclear ERK in COS-7 cells expressing beta1AR or beta2AR. However, the activity of nuclear ERK was enhanced by co-expression of beta-arrestin2 in beta2AR-but not beta1AR-expressing cells. Pertussis toxin treatment and blockade of Gbetagamma action inhibited beta-arrestin2-enhanced nuclear activation of ERK, suggesting that beta-arrestin2 promotes nuclear ERK localization in a Gbetagamma dependent mechanism upon receptor stimulation. beta2AR containing the carboxyl terminal region of beta1AR lost the beta-arrestin2-promoted nuclear translocation. As the carboxyl terminal region is important for beta-arrestin binding, these results demonstrate that recruitment of beta-arrestin2 to carboxyl terminal region of beta2AR is important for ERK localization to the nucleus.
[Show abstract][Hide abstract] ABSTRACT: Cardiomyocytes express both beta(1)- and beta(2)-adrenergic receptors, and these receptors play a differential role in chronotropic and inotropic effects of the heart. Caveolae are known as an important regulator of G-protein-coupled receptor signaling. In the present report, we examined whether caveolae have a role in beta-adrenergic receptor-stimulated cAMP production and protein kinase A activation in neonatal myocytes. Isoproterenol-stimulated cAMP production was mediated by beta(1)- and beta(2)-subtypes, which depends on the receptor number of each subtype. However, protein kinase A activation was exclusively mediated by the beta(1)-subtype. Disruption of caveolae by methyl-beta-cyclodextrin treatment did not affect the relative contribution of subtypes to isoproterenol-stimulated cAMP production. beta(1)-Subtype-mediated protein kinase A activation was also not affected by the disruption of caveolae. These results suggest that beta(1)-adrenergic receptor-mediated protein kinase A activation is compartmentalized and independent of caveolae.
[Show abstract][Hide abstract] ABSTRACT: Angiotensin II (Ang II) activates multiple signaling pathways leading to hyperplasia of cardiac fibroblasts. Reactive oxygen species (ROS) produced by Ang II stimulation are assumed to play pivotal roles in this process. Here, we show that ROS mediate Ang II-induced activation of nuclear factor of activated T cells (NFAT) in rat cardiac fibroblasts. Ang II-induced NFAT activation was suppressed by diphenyleneiodonium (an NADPH oxidase inhibitor), dominant negative (DN)-Rac, DN-p47(phox), and an inhibitor of Galpha(12/13) (Galpha(12/13)-specific regulator of G protein signaling domain of p115RhoGEF, p115-regulator of G protein signaling (RGS)). Stimulation of Ang II receptor increased the intracellular ROS level in a Rac- and p47(phox)-dependent manner. Because p115-RGS suppressed Ang II-induced Rac activation, Ang II receptor-coupled Galpha(12/13) mediated NFAT activation through ROS production by Rac activation. Ang II-induced nuclear translocation of the green fluorescent protein (GFP)-tagged amino-terminal region of NFAT4 (GFP-NFAT4) was suppressed by p115-RGS or BAPTA but not by diphenyleneiodonium. The expression of constitutively active (CA)-Galpha(12/13), CA-G translocation alpha(13), or CA-Rac increased the nuclear of GFP-NFAT4. These results suggest that NFAT activity is regulated by both Ca(2+)-dependent and ROS-dependent pathways. Furthermore, activation of c-Jun NH(2)-terminal kinase (JNK) induced by Ang II stimulation is required for NFAT activation because Ang II-induced NFAT activation was inhibited by SP600125, a selective JNK inhibitor. These results indicate that Ang II stimulates the nuclear translocation and activation of NFAT by integrated pathways including the activation of Galpha(12/13), Rac, NADPH oxidase, and JNK and that Galpha(12/13)-mediated ROS production is essential for NFAT transcriptional activation.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of
ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among
three MAPKs, c-Jun NH2-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium,
inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of
the Gα12/13-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Gα12/13, but not by an RGS domain specific for Gαq. Constitutively active Gα12- or Gα13-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium.
Angiotensin II receptor stimulation rapidly activated Gα13, which was completely inhibited by the Gα12/13-specific RGS domain. Furthermore, the Gα12/13-specific but not the Gαq-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated
ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by
Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin
II-induced Rho activation was inhibited by Gα12/13-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not
affect angiotensin II-induced JNK activation cascade. These results suggest that Gα12/13-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.
[Show abstract][Hide abstract] ABSTRACT: beta-Adrenergic stimulation enhances the activity of L-type Ca(2+) channels through mechanisms mediated by adenosine 3'5'-cyclic monophosphate (cAMP) and protein kinase A in cardiac myocytes. However, in smooth muscle cells, the effect of beta-adrenoceptor stimulation on the L-type Ca(2+) channel activity has been controversial, and the exact mechanism is still unclear. The present study was aimed at elucidating the effect of beta-adrenergic stimulation upon the activity of L-type Ca(2+) channels in guinea-pig detrusor smooth muscle cells. Isoproterenol (0.1-1 microM) inhibited Ba(2+) currents through L-type Ca(2+) channels (I(Ba)). Isoproterenol (0.1 microM) shifted the steady-state inactivation curve to negative voltages by 11 mV without affecting activation curves. The stimulation of cAMP-mediated signal transduction pathway by forskolin, 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP), or the intracellular application of cAMP also mimicked the effects of isoproterenol on I(Ba), which was blocked by the inhibition of protein kinase A. These results indicate that, in detrusor smooth muscles, the stimulation of beta-adrenoceptors exerts negative modulation of L-type Ca(2+) channels via cAMP/protein kinase A-dependent mechanism.
European Journal of Pharmacology 06/2003; 470(1-2):9-15. DOI:10.1016/S0014-2999(03)01762-X · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Detrusor muscle relaxes upon activation of beta-adrenoceptors on smooth muscle cells. However, the mechanism of relaxation following the stimulation of beta-adrenoceptors remains unclear. In order to clarify the mechanism, we investigated the involvement of ion channels in bladder relaxation. In guinea-pig isolated bladder strips precontracted by high-K(+), isoproterenol caused concentration-dependent relaxation. The relaxation caused by isoproterenol (1 microM) was larger in 30 mM K(+) than in 120 mM K(+) (54.2+/-8.0% and 18.2+/-4.1% of papaverine-induced relaxation, respectively, n=4). Iberiotoxin (100 nM) inhibited the isoproterenol-induced relaxation (vehicle 69.5+/-8.0% vs. iberiotoxin 24.9+/-6.2%, respectively, n=5). Whole-cell patch-clamp recording revealed that isoproterenol as well as forskolin increased the iberiotoxin-sensitive K(+) currents, and this increase was abolished by protein kinase inhibitor. These results suggest that the isoproterenol-induced relaxation of guinea-pig bladder smooth muscle is mainly mediated by facilitation of BK(Ca) channels subsequent to the activation of the cAMP/protein kinase A pathway.
European Journal of Pharmacology 10/2000; 404(1-2):231-8. DOI:10.1016/S0014-2999(00)00606-3 · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the contribution of Ser203 of the human β2-adrenoceptor (β2-AR) to the interaction with isoprenaline. The affinity of (−)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (−)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type β2-AR and S207A-β2-AR and even lower affinities for S203A-β2-AR and S204A-β2-AR. By contrast, an (−)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type β2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (−)-isoprenaline was increased, by about 120 fold, for each alanine-substituted β2-AR mutant. These results suggest that Ser203 of the human β2-AR is important for both ligand binding and receptor activation.
British Journal of Pharmacology (1999) 128, 272–274; doi:10.1038/sj.bjp.0702813
British Journal of Pharmacology 10/1999; 128(2):272-4. DOI:10.1038/sj.bjp.0702813 · 4.84 Impact Factor