Heterotrimeric G protein G alpha13-induced induction of cytokine mRNAs through two distinct pathways in cardiac fibroblasts.

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.

Caveolae-independent activation of protein kinase A in rat neonatal myocytes.

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.

Galpha12/13-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts.

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.