Developmental expression of peptidylglycine alpha-amidating monooxygenase (PAM) in primary cultures of neonatal rat cardiocytes: a model for studying regulation of PAM expression in the rat heart.

Developmental Expression of Peptidylglycine a-Amidating Monooxygenase (PAM) in Primary Cultures of Neonatal Rat Cardiocytes: A Model for Studying Regulation of PAM Expression in the Rat Heart Jean-Yves Maltese and Betty A. Eipper Department of Neuroscience Johns Hopkins University School Baltimore, Maryland 21205 of Medicine INTRODUCTION Primary cultures of neonatal rat atrial and ventricular cardiomyocytes were used to investigate the expression of peptidylglycine a-amidating monooxygenase (PAM), a bifunctional enzyme required for the production of cY-amidated neuroendocrine peptides. The use of assays for the individual enzymes, peptidylglycine n-amidating monooxygenase (PHM) and peptidyl-a-hydroxyglycine a-amidating lyase (PAL), demonstrated that the levels of expression observed in vitro approximated those observed in viva. Both in viva and in vitro, atrial and ventricular PAL activity greatly exceeded PHM activity. Atrial and ventricular cardiomyocytes secreted PHM and PAL activity at a constant rate throughout the culture period. lmmunofluorescence studies localized PAM proteins to the perinuclear region, with intense punctate staining. Both in viva and in vitro, PAM mRNAs encoding integral membrane proteins predominated throughout the neonatal period, with PAM-l mRNA becoming more prevalent after the first week in culture. Although PAM-2 mRNA decreased in prevalence in viva at the time when PAM-l expression increased, levels of PAM-2 mRNA remained elevated throughout 2 weeks in vitro. Western blot analysis demonstrated intact PAM-l and PAM-2 proteins in atrial cultures, with the prevalence of PAM1 increasing in older cultures. Atrial cardiomyocytes secreted only bifunctional PAM proteins. Many of the features of PAM expression, processing, and storage that are unique to cardiomyocytes as opposed to endocrine cells are faithfully replicated by primary atrial and ventricular cultures. (Molecular Endocrinology 6: 1996-2006, 1992) ome-8809/92/1998-2008$03.00/0 Molecular Endocrmology CopyrIght 0 1992 by The Endocrme Peptidylglycine a-amidating monooxygenase (PAM; EC 1.14.17.3) is involved in the posttranslational processing of many peptide hormones and is commonly associated with the secretory granules of endocrine cells and neurons (1, 2). PAM catalyzes the formation of CYamidated peptides from peptide precursor molecules with a COOH-terminal glycine. It has recently been shown that peptide cu-amidation is a two-step reaction catalyzed by the sequential action of two enzymatic domains contained within the PAM precursor (3-6). The first enzyme, peptidylglycine cu-hydroxylating monooxygenase (PHM), produces an oc-hydroxylated intermediate in the presence of copper, ascorbate, and molecular oxygen. The second enzyme, peptidyl-a-hydroxyglycine cu-amidating lyase (PAL), cleaves the peptidylcY-hydroxyglycine intermediate to form the cu-amidated peptide and glyoxylate; this reaction can occur spontaneously under nonphysiological conditions. The two catalytic domains of the bifunctional PAM protein can be separated by endoproteolysis and can act independently (7). A single complex gene encodes PAM in the rat (8). Tissue-specific alternative splicing can generate at least seven forms of PAM mRNA (9-11). Two major forms of PAM mRNA (rPAM-1 and -2) have been characterized in the adult rat atrium (9, 10). Both forms encode bifunctional PAM precursor proteins with an NH,-terminal signal sequence, followed by the PHM and PAL catalytic domains and a single putative transmembrane domain near the COOH-terminus. PAM-l contains a noncatalytic domain (exon A) that separates the PHM and PAL domains, while PAM-2 lacks this domain. The various PAM proteins are subjected to tissue-specific posttranslational modifications that amplify the degree of diversity generated from the PAM gene (1 l-1 4). For Socmty 1998 Developmental Expression of PAM 1999 example, in pituitary cells, exon A serves as the site for endoproteolytic cleavages separating PHM from PAL (15). In the adult atrium, the majority of the PAM proteins remain bifunctional and membrane associated (7). During fetal and neonatal development, the prevalence of rPAM-1 and -2 mRNAs in the rat cardiac atrium and ventricle varies dramatically (16). While rPAM-2 mRNA is the most abundant form from postnatal days 1-3, approximately equal amounts of both forms of PAM mRNA are present on postnatal day 5; rPAM-1 mRNA becomes more abundant by postnatal day 7 and is the major form of PAM mRNA in the adult rat atrium. Less PAM mRNA is found in the ventricle than in the atrium throughout most of development, but changes in the ratio of PAM-1 to PAM-2 mRNA follow the same time course in both tissues. Although the atrium of the heart has higher levels of PAM activity and mRNA than any other tissue (17-l 9), the atrium is not known to contain high levels of Namidated peptide, and the function of PAM in this tissue is not clear. Based on subcellular fractionation of adult rat atria, most of the PAM activity is recovered in the secretory granule-enriched fractions containing atrial natriuretic factor (ANF) (12). ANF, the major peptide hormone synthesized in the heart atrium, is stored in these atrial granules as a prohormone and is cleaved at the time of secretion (20-22); neither the prohormone nor the active form of ANF is a-amidated. Primary cultures prepared from neonatal cardiac atria have served as a useful model system for studying ANF expression in cardiomyocytes (23-25). We have used a similar primary culture system to determine if the developmental regulation of PAM expression observed in viva is mimicked in culture. Our earlier studies on PAM expression in cultured cardiomyocytes preceded the elucidation of the bifunctional nature of the enzyme; in the earlier studies, secretion of PAM activity by cultured atrial cardiomyocytes was found to be responsive to glucocorticoids and (Bu&AMP (26). In this study we compared levels of PHM and PAL activities in cell cultures of various ages with the levels of enzyme activity in tissue extracts prepared from pups of the same age. The forms of PAM mRNA in cultures of different ages were identified by reverse transcription/ polymerase chain reaction (PCR) in order to determine whether the developmentally regulated switch from a predominance of PAM-2 mRNA to a predominance of PAM-l mRNA occurred in culture. The PAM proteins stored in the cultured cardiomyocytes were localized by immunofluorescence, and the forms of PAM protein in cell extracts and spent media were characterized by Western blot analysis. RESULTS PHM and PAL Activities in Rat Atrium in Viva and in Primary Cell Culture and Ventricle Our earlier studies indicated that expression activity in atrium and ventricle varied during of PAM develop- ment (16). Since peptide Lu-amidation is now known to involve the sequential action of two enzymes contained within the bifunctional PAM protein, we separately measured these two a (...truncated)