Abstract
Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.
Highlights
Atrial natriuretic factor (ANF)’ is a peptide hormone demyocytes as a prohormone (ANF-(1-126)) and is cose- rivedfrom cardiac atria that is involved in salt and water cretionally processed to the circulating ANF-related balance aswell as theregulation of blood pressure (reviewed peptides,ANF-(1-98) and ANF-(99-126).Recently, by Baxter et al, 1988)
ANF secretion was stimulated ap- planted within the atrial chamb(eLredsome et al, 1986), and proximately 4-fold after a 1-h incubation of the cul- constriction of the aorta and pulmonary artery (Edwards et tures with the phorbolester 12-0-tetradecanoylphor- al., 1988) all increase the circulatinglevels of ANF
Ditions.,incubating theculturesformore ablates theincrease in ANFlevels caused by volume than 1 h with TPA resulted in a blunted secretory expansion (Eskay et al, 1986), and evidence for a humoral response to further TPA challenge anda 40-50% de- ANF-releasing agentof pituitary origin has been obtainedby creaseinthequantity of ANF inthe cells
Summary
Effects of PKC Activators on ANF Secretion-Phorbol esters, which bind to andactivate protein kinase C (PKC),have been widely reported to affect the secretion rate of endocrine studies, adrenergic agonists have had effects on regulating cells, presumably by substitutingfor diacylglycerol in the ANF secretion, the relative roles of a- and P-adrenergic activation have not been clarified. The active phorbol ester 12-O-tetradecanoylphorbol13-acetate (TPA) was tested for the ability to elicit ANF secretion from the primary atrial cultures. Such a model system could beof value in determining what compounds can alter ANF secretion and/or ANF biosynthesis atthe level of the cardiocyte. In re- (Shields et al, 1988), it was of interest to determine if the cent studies it was shown that when maintained in glucocor- processing mechanism could be overwhelmed by the 4-fold ticoid-containing serum-free media, primary neonatal atrial increased secretion rate induced by TPA. The goal of the present study was to utilize this process- difference was observed in the ability of the cells to secrete ing capable primary culture system to determine whether primarily ANF-(99-126) (Fig. 1, B and C). Tory to subsequent challenge with phorbol esters (Stojilkovic et al, 1988b).This effect was observed with the primary
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