atrial-natriuretic-factor and cobaltous-chloride

Mitogen-activated protein kinases (MAPKs) constitute one of the most important intracellular signalling pathways. In particular, the p38-MAPK subfamily is known to be activated under various stressful conditions, such as mechanical or oxidative stress. Furthermore, cobalt chloride (CoCl2) has been shown to mimic hypoxic responses in various cell lines and cause overproduction of reactive oxygen species (ROS). In the current study, we investigated the effect of CoCl2 on p38-MAPK signalling pathway in the perfused Rana ridibunda heart. Immunoblot analysis of the phosphorylated, and thus activated, form of p38-MAPK revealed that maximum phosphorylation was attained at 500 micromol l(-1) CoCl2. A similar profile was observed for MAPKAPK2 and Hsp27 phosphorylation (direct and indirect p38-MAPK substrates, respectively). Time course analysis of p38-MAPK phosphorylation pattern showed that the kinase reached its peak within 15 min of treatment with 500 micromol l(-1) CoCl2. Similar results were obtained for Hsp27 phosphorylation. In the presence of the antioxidants Trolox or Lipoic acid, p38-MAPK CoCl2-induced phosphorylation was attenuated. Analogous results were obtained for Hsp27 and MAPKAPK2. In parallel, mRNA levels of the ANP gene, a hormone whose transcriptional regulation has previously been shown to be regulated by p38-MAPK, were examined (semi-quantitative ratiometric RT-PCR). CoCl2 treatment significantly increased ANP mRNA levels, whereas, in the presence of antioxidants, the transcript levels returned to basal values. All the above data indicate that CoCl2 stimulates compensatory mechanisms involving the p38-MAPK signalling cascade along with ANP.

Topics: Animals; Atrial Natriuretic Factor; Cobalt; Heart; Heat-Shock Proteins; In Vitro Techniques; MAP Kinase Signaling System; Myocardium; p38 Mitogen-Activated Protein Kinases; Perfusion; Ranidae

Norepinephrine has growth-promoting effects in cardiac myocytes. The present study in cultured neonatal rat cardiac myocytes tested the hypothesis that norepinephrine also stimulates expression of vascular endothelial growth factor (VEGF), an important angiogenic factor. As assessed by polymerase chain reaction cardiac myocytes and non-myocytes expressed all three isoforms of rat VEGF, with the short isoform (VEGF 121 ) preferentially expressed in non-myocytes. When cardiac myocytes were stimulated with 1 micro M norepinephrine for 24 h in the presence or absence of the specific alpha - and beta -adrenoceptor antagonists prazosin and propranolol, respectively, VEGF mRNA levels and splice variant pattern did not change, whereas atrial natriuretic peptide mRNA levels increased 3 to 4-fold. CoCl(2) increased VEGF mRNA levels in cardiac myocytes five-fold. When cardiac myocytes were cultured with conditioned medium from non-myocytes that had been stimulated with norepinephrine for 24 h VEGF mRNA increased 2-fold. The increase was blocked by antibodies neutralizing TGF beta. These data suggest that norepinephrine stimulates myocardial angiogenesis by a paracrine mechanism that involves cardiac non-myocytes and TGF beta.

Topics: Adenylyl Cyclases; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Alternative Splicing; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cells, Cultured; Cobalt; Culture Media, Conditioned; Gene Expression Regulation; Genetic Variation; Myocytes, Cardiac; Norepinephrine; Paracrine Communication; Prazosin; Propranolol; Protein Isoforms; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A

We investigated whether impairment of myocardial energy metabolism attenuates cardiac function and increases cardiac endothelin-1 (ET-1) gene expression in rats. Three weeks after commencing administration of cobalt chloride (CoCl2), an inhibitor of mitochondrial function, the peak positive first derivative of left ventricular (LV) pressure, an indicator of myocardial contractility, was significantly decreased in the CoCl2-treated rats. LV end-diastolic pressure and right ventricular systolic pressure were increased in the CoCl2-treated rats. Echocardiography showed that fractional shortening was significantly decreased in the CoCl2-treated rats. Myocardial expressions of acyl-CoA synthase mRNA, an enzyme involved in fatty acid utilization, was markedly decreased in the CoCl2-treated rats. Under such conditions, myocardial expression of preproendothelin-1 mRNA and atrial natriuretic peptide (ANP) mRNA, molecular markers of heart failure, was markedly increased in the CoCl2 rats. In conclusion, the data suggest that impairment of myocardial energy metabolism causes hemodynamic abnormality and increases molecular markers of heart failure (ET-1, ANP mRNA). These data suggest that myocardial energy metabolism is one of the factors involved in the upregulation of ET-1 gene expression in the failing heart.

Topics: Animals; Atrial Natriuretic Factor; Cobalt; Endothelin-1; Endothelins; Energy Metabolism; Heart Failure; Male; Myocardium; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger