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Phosphorylation State of pro-Atrial Natriuretic Factor in Rat Atrial Secretory Granules^*

Department of Heart and Hypertension Research (G.M.W., R.M.G.), Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195; and Division of Nuclear Medicine (A.J.F.) and Cellular and Molecular Research Laboratory (M.N.M., C.J.H.), Cardiac Unit Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts 02114

Address requests for reprints to: Gary M. Wildey, Department of Heart and Hypertension Research, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195.

Abstract

Recent studies have demonstrated that phosphorylation of atrial natriuretic factor (ANF) (99–126) in vitro modulates the bioactivity of this hormone. The potential physiological relevance of this observation was revealed in latter studies showing that endogenous proANF can be ³²PO₄-biosynthetically labeled by primary cultured atrial myocytes and by atrial appendage explants. The site and extent of proANF phosphorylation were different, however, in these two model systems. Whereas proANF extracted from atrial explants was phosphorylated on the bioactive, carboxy (C)-terminal portion of the molecule [ANF(99–126)], cultured atrial myocytes phosphorylated proANF on the amino (N)-terminal portion of the prohormone molecule [ANF(l–98)]. It was the goal of this study, therefore, to determine whether the bioactive region of proANF, ANF(99–126), is phosphorylated in vivo. ProANF was obtained by acid extraction of isolated rat atrial secretory granules followed by purification using reverse phase-HPLC. Analysis of purified ¹²⁵I-labeled proANF by isoelectric focusing (IEF) revealed two bands with isoelectric points of 5.3 and 5.0. The more acidic band comigrated on IEF gels with ³²PO₄-biosynthetically labeled proANF obtained from primary cultures of atrial myocytes, suggesting that this species of proANF represented endogenously phosphorylated proANF. The more acidic band accounted for only 15–25% of the total proANF found in the mature atrial secretory granule. The phosphorylation state of ANF(99–126) produced by thrombin cleavage of secretory granule proANF was examined using three complementary methods: 1) cation-exchange HPLC, 2) amino-terminal amino acid sequence analysis and 3) anti-ANF(99–105) antibody immunoreactivity. Evidence from these three independent approaches indicated that proANF is not phosphorylated on the C-terminal portion of the molecule in vivo. Therefore, phosphorylation is not a physiological regulator of ANF(99–126) bioactivity (Endocrinology 127: 2839–2848,1990)

Footnotes

^* This work was supported in part by NIH Grants, HL-35642, HL-38070, HL-19259, and CA-24432. This work was done during the tenure of a research fellowship (to G.M.W.) from the American Heart Association, Massachusetts Affiliate, Inc. (13–408–878).

Received June 4, 1990.