h-89 and Cardiomegaly

cAMP responsive element binding protein (CREB) is a stimulus induced transcription factor with possible relevance for the pathophysiology of the heart. In the present study, we provide evidence that the hypertrophic agonist, phenylephrine (PE), promotes phosphorylation of CREB in adult rat cardiac myocytes through alpha(1)- and beta-adrenergic receptors. PE-induced phosphorylation of CREB was partially inhibited by Ro318220 and H89, which were shown to be potent inhibitors of mitogen- and stress-activated protein kinase-1 (MSK1) activation, implicating the involvement of this kinase in the response. Similar results were obtained when cardiac myocytes were treated with the inhibitors of ERK1/2 and p38 MAPK pathways. In addition, inhibition of protein kinase A by RpcAMP reduced phosphorylation of CREB, suggesting that this pathway is also involved. Furthermore, PE stimulation was accompanied by an increase in CRE-binding activity, which was reduced by drugs that prevented phosphorylation of CREB. An enhanced CBP/phospho-CREB complex formation was also observed, suggesting recruitment of CBP to phosphorylated CREB. These results suggest that PE stimulates phosphorylation and DNA binding activity of CREB in adult rat ventricular myocytes through multiple signaling pathways involving ERK1/2, p38 MAPK, MSK1 and PKA. The same pathways seem to regulate atrial natriuretic peptide (ANF) mRNA expression, a highly conserved marker gene of cardiac hypertrophy, suggesting that the PE-stimulated activation of CREB is likely to play an important role in the hypertrophic response.

Topics: Adrenergic Agonists; Adrenergic alpha-1 Receptor Agonists; Animals; Atrial Natriuretic Factor; Cardiomegaly; CREB-Binding Protein; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; Immunoprecipitation; Indoles; Isoquinolines; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phosphorylation; Rats; Receptors, Adrenergic, alpha-1; Receptors, Adrenergic, beta; Ribosomal Protein S6 Kinases, 90-kDa; RNA, Messenger; Signal Transduction; Sulfonamides; Trans-Activators

Production of heat shock protein 70 (HSP70) in the heart is induced by hemodynamic stress, but its intracellular signal transduction system has not been elucidated well.. To investigate the hypothesis that protein kinase A (PKA)-dependent and protein kinase C (PKC)dependent systems are involved in the pressure-induced expression of HSP70 mRNA in perfused adult rat heart. Isolated tetrodotoxin-arrested Sprague-Dawley rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. Aortic pressure in rats of the pressure-overloaded group was elevated from 60 to 120 mmHg for 2-120 min. cAMP contents and rates of synthesis of protein were measured by radioimmunoassay and the incorporation of [14C]-phenylalanine into total heart protein, respectively. Expression of HSP70 mRNA was determined by Northern blot analysis.. Elevation of aortic pressure significantly increased cAMP content after 2 min of perfusion (by 41%), significantly increased rates of synthesis of protein during the second hour of perfusion (by 41%), and induced expression of HSP70 mRNA maximally after 60 min of perfusion (2.7-fold the control value). Exposure to glucagon, forskolin or 1 -methyl-3-isobutylxanthine mimicked increases in these parameters caused by elevation of aortic pressure. Administration of a selective PKA inhibitor, H-89, significantly prevented induction of increases in expression of HSP70 mRNA and rates of synthesis of protein by a high pressure overload and exposure to agents that increase cAMP content. Furthermore, administration of phorbol ester induced expression of HSP70 mRNA. Administration of a PKC inhibitor, calphostin C, significantly prevented induction of increases in expression of HSP70 mRNA by a pressure overload and by exposure to phorbol ester.. These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Blood Pressure; Cardiomegaly; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Gene Expression; Glucagon; HSP70 Heat-Shock Proteins; Hypertension; In Vitro Techniques; Isoquinolines; Male; Myocardium; Naphthalenes; Perfusion; Protein Biosynthesis; Protein Kinase C; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Sulfonamides

The aim was to determine whether proto-oncogene c-fos expression and acceleration of protein synthesis by acute pressure overload to the heart were coupled with a cAMP- and protein-kinase-A-dependent system in adult rat heart.. Isolated adult rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. In the pressure-overloaded group, aortic pressure was raised from 60 to 120 mmHg for the time indicated. Agents that increase cAMP were added to the perfusate at an aortic pressure of 60 mmHg. Furthermore, a selective protein kinase A inhibitor (H-89) or a selective protein kinase C inhibitor (calphostin C) was administered before the elevation of aortic pressure or the addition of the agents. cAMP content or rates of protein synthesis were measured by RIA or the incorporation of [14C]phenylalanine into total heart protein, respectively. c-fos mRNA expression was determined by Northern blot analysis.. Elevation of aortic pressure in beating hearts and arrested hearts increased cAMP content at 2 min of perfusion by 36 and 41%, induced c-fos mRNA expression at 30-60 min of perfusion by 4.8- and 2.0-fold, and accelerated rates of protein synthesis during the 2nd hour of perfusion by 39 and 41% over control levels, respectively. Glucagon, forskolin or IBMX mimicked increases in these parameters by elevated aortic pressure. H-89 prevented these changes by elevated pressure overload or exposure to forskolin or IBMX in arrested hearts. On the other hand, calphostin C prevented the pressure-induced increases in c-fos expression and protein synthesis rates in arrested hearts.. These results suggest that c-fos expression induced by acute pressure overload may be coupled with increased cAMP content and protein kinase A activity in addition to increased protein kinase C activity in adult rat heart.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Blotting, Northern; Cardiomegaly; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Gene Expression; Genes, fos; Glucagon; Isoquinolines; Male; Perfusion; Phosphodiesterase Inhibitors; Pressure; Protein Biosynthesis; Protein Kinase C; Rats; Rats, Sprague-Dawley; Sulfonamides