Abstract
It has been shown recently that alpha-adrenergic agonists can stimulate atrial natriuretic factor (ANF) expression in ventricular cardiac myocytes; however, little is known about the intracellular signals mediating this activation. The present study focused on the potential roles of calcium-regulated kinases and calcium influx in the alpha-adrenergic stimulation of ANF gene expression in ventricular myocardial cell cultures. Myocardial cells maintained for 48 h in serum-free medium supplemented with phenylephrine (PE) possessed up to 15-fold higher levels of ANF peptide and ANF mRNA than control cells. The removal of PE, or the addition of nifedipine, resulted in a rapid decline in ANF expression, suggesting that the sustained elevation of some intracellular messenger (e.g. calcium and/or phospholipid hydrolysis products) was required for the adrenergic response. The calcium channel agonist BAY K 8644 was capable of increasing ANF expression in a nifedipine-sensitive manner; however, unlike PE, it did not stimulate phosphoinositide hydrolysis. The protein kinase C inhibitor, H7, caused an approximate 75% reduction in PE-stimulated ANF expression, but had no effect on BAY K-stimulated expression. W7, a calcium/calmodulin inhibitor, completely blocked the effects of both PE and BAY K 8644. The addition of either H7 or W7 24 h after the PE addition resulted in a decline of ANF expression. These results indicate that alpha-adrenergic agonists augment ANF gene expression through at least two pathways, one that is H7-sensitive, perhaps involving the sustained activation of protein kinase C, and the other that is W7-sensitive, perhaps involving the sustained activation of calmodulin-regulated kinases. Further, it appears that BAY K 8644-mediated increases in ANF expression are independent of protein kinase C activation and dependent on calmodulin-regulated events.
Highlights
The a-Adrenergic Stimulation of Atrial Natriuretic Factor Expression in Cardiac Myocytes Requires CalciumInflux, Protein Kinase C, and Calmodulin-regulated Pathways*
In pursuing this hypothesis we have shown in the present debris from the dishes, samples were centrifuged, and labeled inositol study that a,adrenergic agonists influence atrialnatriuretic factor (ANF) expression through at least two pathways; one requires PI hydrolysis with the apparent sustained activationof PKC, and theother phosphate, which was isolated from the supernatant following ether extraction, was analyzed by anion exchange chromatography as described (Jones et al, 1988).For DAG determinations, the precipitate was extracted further with methanol as described (Bligh and Dyer, involves a calmodulin-regulated step, such as CaM kinase I1 1959)
We have investigated the potential roles of calcium influx, protein kinases, and calmodulin in aadrenergic-mediated increases of ANF expression in primary neonatal rat ventricular myocardial cell cultures
Summary
Medium ANF as a Measure of ANF Gene ExpressionPrimary neonatal ventricular myocytes release ANF constitutively such that little of the peptide is stored within the cells (Bloch et al, 1986). Changes in the level of ANF in the medium of ventricular myocyte cultures provide a reasonable estimate of changes in ANF mRNA levels.In many experiments discussed in this paper, the effects of a treatment on ANF expression were assessed initially by measuring changes in the levels of medium ANF. When cultures maintained in PE for 24 h were washed free of the agonist medium, ANF began to drop so that within 6 h it was only about half that observed in cultures remaining in P E (Fig. lA) This trend continued so that 12 hafter PE removal, medium ANF levels had declined to nearly control values. The finding that medium ANF levels reflected cellular levels indicated that PE removal affected total ANF production, and notsimply secretion This is consistent with previous findings that ventricular myocytes release ANF constitutively through a nonregulated pathway (Bloch et al, 1986). The kinetics of this decrease appeared to be slower than that for the peptide, which is perhaps consistent with a transcript
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