h-89 and Hypertension

We previously generated cytochrome P450 4F2 (CYP4F2) transgenic mice and showed high 20-hydroxyeicosatetraenoic acid (20-HETE) production, which resulted in an elevation of blood pressure. However, it was unclear whether 20-HETE affected glucose metabolism. We measured fasting plasma glucose, insulin, hepatic CYP4F2 expression, and 20-HETE production by hepatic microsomes, and hepatic 20-HETE levels in transgenic mice. We also assessed glycogen phosphorylase (GP) activity and the cAMP/protein kinase A (PKA)-phosphorylase kinase (PhK)-GP pathway, as well as expressions of insulin receptor substrate 1 and glucose transporters in vivo and in vitro. The transgenic mice had overexpressed hepatic CYP4F2, high hepatic 20-HETE and fasting plasma glucose levels but normal insulin level. The GP activity was increased and the cAMP/PKA-PhK-GP pathway was activated in the transgenic mice compared with wild-type mice. Moreover, these alterations were eliminated with the addition of N-hydroxy-N'-(4-butyl-2 methylphenyl) formamidine, which is a selective 20-HETE inhibitor. The results were further validated in Bel7402 cells. In addition, the transgenic mice had functional insulin signaling, and 20-HETE had no effect on insulin signaling in Bel7402 cells, excluding that the observed hyperglycemia in CYP4F2 transgenic mice resulted from insulin dysfunction, because the target tissues were sensitive to insulin. Our study suggested that 20-HETE can induce hyperglycemia, at least in part, through the cAMP/PKA-PhK-GP pathway but not through the insulin-signaling pathway.

Topics: Amidines; Animals; Blood Glucose; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytochrome P-450 Enzyme System; Glucose Transporter Type 1; Glycogen Phosphorylase; Hydroxyeicosatetraenoic Acids; Hyperglycemia; Hypertension; Insulin Receptor Substrate Proteins; Isoquinolines; Liver; Male; Mice; Mice, Transgenic; Phosphorylase Kinase; Phosphorylation; Signal Transduction; Sulfonamides

Alterations of calcium handling and other second messenger cascades including protein kinase C (PKC) and A (PKA) were suggested to be responsible for abnormal vascular function in spontaneously hypertensive rats (SHR). However, the relative contribution of these pathways to vasoconstriction is still not completely understood. We investigated the effect of Ro 31-8220 (PKC inhibitor) and H89 (PKA inhibitor) on vasoconstriction induced by 120 mM KCl or by addition of 10 microM noradrenaline (NA) in isolated femoral arteries of control Wistar rats and SHR. Moreover, we investigated these responses in the presence and absence of Ca(2+) ions in the incubation medium in order to assess the role of calcium influx in these contractions. We observed that while the vasoconstriction in the presence of calcium was not different between Wistar and SHR, the difference between constriction elicited by NA addition in the absence and presence of external calcium was larger in SHR. The inhibition of PKC had no effect on constrictions in SHR, but diminished constrictions in Wistar rats. PKA inhibition slightly enhanced constrictions in Wistar rats, but reduced them in the presence of calcium in SHR. We conclude that vasoconstriction elicited by adrenergic stimulation is more dependent on extracellular calcium influx in SHR compared to Wistar rats. Moreover, the activation of PKA contributes to this calcium-dependent vasoconstriction in SHR but not in Wistar. On the other hand, PKC activation seems to play a less important role in vasoconstriction in SHR than in Wistar rats.

Topics: Animals; Calcium; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Femoral Artery; Hypertension; In Vitro Techniques; Indoles; Isoquinolines; Male; Norepinephrine; Potassium Chloride; Protein Kinase C; Protein Kinase Inhibitors; Rats; Rats, Inbred SHR; Rats, Wistar; Sulfonamides; Vasoconstriction; Vasoconstrictor Agents

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