Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of G␤␥ subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/ reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a G␤␥-dependent manner by providing a scaffold composed of G␤␥ and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the G␤␥ inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-G␤␥ signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM. G-protein-coupled receptors (GPCRs)4 are signaling proteins on the cell surface responsible for mediating various ligands, such as hormones and neurotransmitters. Activation of cell surface GPCRs initiates nucleotide exchange on G␣ subunits, which leads to a conformational change of G␣␤␥ and subsequent transduction of signals to various intracellular effector molecules (1-3). In addition to such established signaling pathways, recent studies indicate the existence of a novel class of regulatory proteins for heterotrimeric G-proteins. These regulatory proteins may provide alternative signal processing via G␣␤␥, G␣, or G␤␥ subunits distinct from typical GPCR pathways, and identifying these mechanisms may uncover unrecognized roles of G-proteins beyond simple transducers of signals from GPCRs (4 -6).During the alteration of signaling pathways in disease states, such regulatory proteins may be involved in adaptation programs of cells to maintain homeostasis (7-11). Genetic modification of regulatory proteins for G-proteins leads to the development of cardiovascular dysfunction in mice including hypertension, maladaptive response to pressure overload, or altered baroreceptor reflex (9, 12, 13). Thus, such regulatory accessory proteins may be involved in the development of disease via either regulating GPCR-initiated signals or by undefined, alternative G-protein signaling pathways operating independent of the receptor.In our efforts to adaptation-specific sig...

We have demonstrated that chronic stimulation of the prostaglandin E 2 -cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, Prostaglandin E 2 (PGE 2 )3 is the most potent vasodilatory lipid mediator in the ductus arteriosus (DA), a fetal arterial connection between the pulmonary artery and the descending aorta (1). PGE 2 increases the intracellular concentration of cAMP, which activates cAMP-dependent protein kinase A (PKA), resulting in vasodilation in the DA (1, 2). In addition to its vasodilatory effect, our recent study has identified that chronic PGE 2 stimulation has another essential effect on DA development, namely intimal cushion formation (ICF) (3). Briefly, via EP4, a predominant PGE 2 receptor in the DA, the PGE 2 -cAMP-PKA stimulation up-regulates hyaluronic acid (HA) synthases, which increases HA production. Accumulation of HA then promotes smooth muscle cell (SMC) migration into the subendothelial layer to form intimal thickening. ICF then leads to luminal narrowing, helping adhesive occlusion of the vascular lumen and thus complete anatomical closure of the DA.A new target of cAMP, i.e. an exchange protein activated by cAMP, has recently been discovered; it is called Epac (4).

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The ductus arteriosus (DA), a fetal arterial connection between the pulmonary artery and the aorta, has a character distinct from the adjacent arteries. We compared the transcriptional profiles of the DA and the aorta of Wistar rat fetuses on embryonic day 19 (preterm) and day 21 (near-term) using DNA microarray analyses. We found that 39 genes were expressed 2.5-fold greater in the DA than in the aorta. Growth hormone (GH) receptor (GHR) exhibited the most significant difference in expression. Then, we found that GH significantly promoted migration of DA smooth muscle cells (SMCs), thus enhancing the intimal cushion formation of the DA explants. GH also regulated the expression of cytoskeletal genes in DA SMCs, which may retain a synthetic phenotype in the smooth muscle-specific cytoskeletal genes. Thus, the present study revealed that GH-GHR signal played a role in the vascular remodeling of the DA.