Processing of the atrial natriuretic factor propeptide by atrial cardiocytes as revealed by immunocryoultramicrotomy.

Antibodies were raised against three fragments of the rat ANF molecule: C-terminal atrial natriuretic factor (ANF)-(101-126), N-terminal ANF-(11-37), and the putative cleavage site of the ANF propeptide, ANF-(94-103). These antibodies were purified by affinity chromatography and revealed a major band (17K) corresponding to the propeptide by Western blot analysis. Antibodies against ANF-(94-103) were used in a RIA. All of the peptides that possess this region (98-99) were able to displace iodinated pro-ANF from the antibody. However, peptides that have only one part of the fragment, such as ANF-(99-126) or ANF-(1-98), failed to displace pro-ANF; human ANF-(79-98) (100 pmol) demonstrated about 0.01% cross-reactivity. Immunohistochemical studies revealed that all atrial cardiocytes are reactive with the three antibodies. Immunocryoultramicrotomy revealed that the propeptide travels, uncleaved, from the Golgi complex to immature granules and mature secretory granules. These results indicate that cleavage of the ANF propeptide does not occur at these sites.

Atrial natriuretic factor in Purkinje fibers of rabbit heart.

The Purkinje fibers of the rabbit false tendons (chordae tendineae spuriae) are endocrine cells containing immunoreactive atrial natriuretic factor (ANF) and ANF messenger RNA (mRNA). These cells, as visualized by immunocryoultramicrotomy, contain immunoreactive ANF in their secretory granules and their Golgi complex and exhibit ANF mRNA, as visualized by in situ hybridization with an ANF complementary RNA probe. The content of immunoreactive ANF and ANF mRNA of the Purkinje fibers is midway between that of atrial and ventricular working cardiocytes. High-pressure liquid chromatography analysis of immunoreactive ANF using antibodies against the C-terminal and N-terminal moieties of the molecule indicates that part of immunoreactive ANF contained in Purkinje fibers is the propeptide [Asn1,Tyr126]ANF whereas part was nonspecifically cleaved into C-terminal and N-terminal ANF. The chordae tendineae spuriae exhibit binding sites for ANF (Kd:approximately 1.0 nM; Bmax:approximately 2.3 fmol/mg). ANF profoundly decreases basal and stimulated (epinephrine, dopamine, isoproterenol, and forskolin) adenylate cyclase activity and cyclic adenosine monophosphate (AMP) levels. ANF has little effect on norepinephrine-stimulated adenylate cyclase activity or on norepinephrine-stimulated cyclic AMP levels. ANF produces only a slight increase in guanylate cyclase activity and cyclic guanosine monophosphate levels at high (10(7)-10(6) M) concentrations. These results suggest an autocrine function for ANF in the modulation of the impulse in the peripheral conduction cells (Purkinje fibers) of the rabbit through changes in second messenger levels.

Atrial natriuretic factor in the vena cava and sinus node.

We investigated the localization of atrial natriuretic factor (ANF) mRNA and of immunoreactive ANF in the vena cava and sinus node of rat and, for comparative purposes, in atria and ventricles. In situ hybridization with an ANF cRNA probe revealed that the supradiaphragmatic portion of the inferior vena cava contains almost as much mRNA as the atria, whereas the levels were less in the superior vena cava and higher than in ventricles in the sinus node. Immunoreactive ANF (high Mr form) was found to be 22 times less abundant in the supradiaphragmatic vena cava and 148 times less abundant in the superior vena cava than in atrial cardiocytes. The wall of the supradiaphragmatic portion of the vena cava and the valve (eustachian valve) that separates the atrial cavity from that of the vein are made up of atrial-like cardiocytes containing secretory granules. The subendothelial area of the superior vena cava also contains atrial-like cardiocytes with secretory granules, whereas the outer portion of the vein is made up of "transitional cells" without or with only a few secretory granules. Secretory granules in the vena cava and nodal cells, as well as transitional cells, contain immunoreactive ANF. With immunocryoultramicrotomy, virtually all cells, whether atrial-like, transitional, or nodal, and even those without secretory granules, were found to contain immunoreactive ANF in their Golgi complex and in secretory vesicles in the vena cava and in the sinus node.

Immuno-electron microscopy of atrial natriuretic factor secretory pathways in atria and ventricles of control and cardiomyopathic hamsters with heart failure.

The secretory pathways of atrial natriuretic factor have been investigated in atrial and ventricular cardiocytes of control and cardiomyopathic Syrian hamsters in severe congestive heart failure with four antibodies: a monoclonal antibody (2H2) against rat synthetic atrial natriuretic factor (101-126), which is directed against region 101-103 of rat atrial natriuretic factor (99-126), and polyclonal, affinity-purified antibodies produced in rabbits against synthetic C-terminal atrial natriuretic factor (101-126), synthetic N-terminal atrial natriuretic factor (11-37) or the putative cleavage site of atrial natriuretic factor (98-99): atrial natriuretic factor (94-103). Application of the immunogold technique on thin frozen sections (immunocryoultramicrotomy) revealed an identical picture with the four antibodies. In atria of both control and cardiomyopathic hamsters where atrial natriuretic factor secretion is regulated, the atrial natriuretic factor propeptide travels, uncleaved, from the Golgi complex to immature and mature secretory granules. In ventricles of control hamsters, where secretion is constitutive, the atrial natriuretic factor propeptide travels from the Golgi complex to secretory vesicles. In the ventricles of hamsters with severe congestive heart failure, the Golgi complex is larger, secretory vesicles more abundant and a few secretory granules are present in approximately 20% of cardiocytes. Here again, the peptide travels uncleaved in all these pathways. These results reveal the pathways of secretion of atrial natriuretic factor in atrial and ventricular cardiocytes and indicate that the propeptide is not cleaved intracellularly.

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles.

A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.