M. Ballak

Radioautographic Localization of 125I-Atrial Natriuretic Factor Binding Sites in the Brain

Rats were injected through the carotid artery (cephalad direction) with 18.9 mu Ci of either 125I-Arg 101-Tyr 126 atrial natriuretic factor alone or together with an excess of unlabeled hormone. At 2 min after injection, all rats were fixed in vivo by perfusion and serial sections of the whole brain were processed for light microscope radioautography. The radioautographic reaction produced by 125I-atrial natriuretic factor alone was localized in all circumventricular organs (except the subcommissural organ), the smooth muscle cells and endothelial cells of arteries, arterioles, veins, venules, the endothelial cells of intraparenchymal capillaries and the epithelial cells of the choroid plexus. In rats which received 125I-atrial natriuretic factor plus an excess of unlabeled hormone, the radioautographic reaction was reduced by 70-90%. Binding sites are thus localized in regions of the brain, some of them involved in the central monitoring of blood pressure and osmolarity. In addition, the presence of binding sites in the cerebral vasculature and in the epithelium of the choroid plexus suggests that circulating ANF may play a role in the control of cerebral blood flow and in the production of vertebrospinal fluid.

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

SummaryA 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.

Corticotropin (ACTH) and the N-terminal fragment of pro-opiomelanocortin are located in the same granules in cells of rat pituitary gland.

The localization of corticotropin (ACTH) and of the N-terminal fragment (NTF) of pro-opiomelanocortin were assessed by light and electron microscope immunochemistry. Using the unlabeled technique of Sternberger at the light microscope level, a strongly positive reaction for both NTF and ACTH antisera was observed in all secretory cells of the pars intermedia. In the pars distalis, immunostaining with ACTH antiserum was localized in stellate cells dispersed throughout the lobe. As observed in serial, consecutive sections, the NTF antiserum stained exactly the same cells. At the electron microscope level, using the protein A-gold technique, all the secretory granules of the cells of the pars intermedia showed a positive reaction with both antisera. In the pars distalis, after exposure to either of the antisera, gold particles were found over secretory granules of typical stellate corticotrophs. Making use of the possibility of reacting both faces of a fine section with gold particles of different sizes, it was found that the same secretory granules in all the cells of the pars intermedia and in corticotrophs of the pars distalis contained particles of both sizes. These results indicate that ACTH and NTF are contained in the same secretory granules and released together in the circulation where NTF is found in high amounts.

Desensitization of the stimulatory A2 adenosine receptor-adenylate cyclase system in vascular smooth muscle cells from rat aorta

We have previously shown that adenylate cyclase present in rat aorta vascular smooth muscle cells can be stimulated by adenosine, its analogs and other agonists. In the present studies, we have examined the effect of preexposure of aorta vascular smooth muscle cells to N-ethylcarboxamide adenosine (NECA) on adenylate cyclase activity stimulated by NECA and other agonists. The vascular smooth muscle cells, when exposed to NECA, resulted in a concentration- and time-dependent loss of NECA-stimulated adenylate cyclase activity. NECA stimulated adenylate cyclase activity by about 120% in control cells, which was decreased to 20% in cells pretreated with 50 microM NECA for 30 min at 37 degrees C. However, GTP-, isoproterenol-, and forskolin-sensitive adenylate cyclase activities were not affected by such treatment, suggesting that NECA treatment of the cells resulted in homologous desensitization. Similarly, the exposure of the cells to isoproterenol resulted in the desensitization of isoproterenol-stimulated adenylate cyclase activity without affecting the NECA-stimulated adenylate cyclase activity. Furthermore, when NECA-treated cells were washed free of agonist, the desensitized state was reversed and the cells regained about 75% responsiveness to NECA stimulation of adenylate cyclase.

Ultrastructural cytochemistry of atrial muscle cells: IX. Reactivity of specific granules in cultured cardiocytes

Abstract A technique for the culture of neonatal (2- to 3-day-old) rat cardiocytes is described. With this technique, ventricular cardiocytes started beating earlier and lived longer, atrial cardiocytes degenerating after 10 days of culture. Specific granules, mostly of the A type, were present in atrial but not ventricular cardiocytes at all time intervals. These specific granules were argentaphobic when stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The core of these granules was moderately positive after staining fine sections with phosphotungstic acid at low pH. A similar reaction was shown in both atria and ventricle by the cell coat, residual bodies (C-granules) and Z-discs. The Golgi complex was more extensively stained by phosphotungstic acid in atrial than in ventricular cultured cardiocytes or in atrial and ventricular cardiocytes of young (3- and 13-day-old) rats in situ. Multivesicular bodies with a dense core, identical to those already noted in the cells of various endocrine glands, and thought to be crinophagic, were present, as in atrial cardiocytes in situ, in cultured atrial but not ventricular cardiocytes. They were silver negative. Their dense core, as in situ, reacted to phosphotungstic acid but their matrix, contrary to classical multivesicular bodies without a dense core, did not. Numerous, small, phosphotungstic acid-positive vesicles were present in cultured atrial and, to a lesser extent, in cultured ventricular cardiocytes. Vesicles of the same type were rare in either atrial of ventricular cardiocytes of young rats in situ. Glycogen, as revealed by the periodic acid Schiff and Thiery techniques, was equally abundant in cardiocytes in situ and in 1-day-old cultures of cultured cardiocytes from atria and ventricle. These results indicate that specific granules of cultured atrial cardiocytes are probably made up, as in situ, of glycoproteins.

Secretory Patterns of Atrial Natriuretic Factor (ANF) By Culture Cardiocytes of Right and Left Atrium From Newborn and Adult RS

Atrial cardiocytes from newborn (2-5 day old) and adult rats were cultured and the secretory patterns of atrial natriuretic factor (ANF) from isolated right and left atrial cells were investigated by radioimmunoassay. Newborn atrial cardiocytes from the left atrium consistently secreted larger amounts of ANF than those from the right with a peak on the 6th day and a decrease up to the 12th day. In contrast, adult atrial cardiocytes secreted much less ANF and this decreased to very low levels from the 3rd day up to the 12th day in culture although ANF was present in measurable amounts in these cells.

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

SummaryThe 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 ∼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.

Synthesis and migration of proteins and glycoproteins in juxtaglomerular cells of sodium-deficient rats

SummarySections of juxtaglomerular cells from sodium-deficient rats were subjected to radioautography after a single intravenous injection of L-tyrosine3,5 3H or of L-fucose 3H to identify the sites of synthesis and to follow the migration of newly-formed proteins and glycoproteins. As early as 2 min after injection of L-tyrosine 3H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 60 min, much of the label had migrated from the RER to the Golgi complex. Some radioactivity was already present over specific granules by 2 min but a peak was reached at 4h. The label over myofilaments was evident at all time intervals, indicating a certain incorporation of tyrosine into their contractile and/or structural proteins. The label over the cell surface peaked at 4h. After injection of L-fucose 3H, there was an early and important relative specific radioactivity in the Golgi complex at 5 min with a peak at 20 min and a decrease thereafter. The label increased slightly but steadily in secretory granules and cell surface to reach maxima at 4 h. A low level of radioactivity was recorded in mitochondria at all time intervals. After injection of both fucose 3H and tyrosine 3H, the label was detected at relatively low levels in the cytosol. These results suggest that renin, as the major secretory glycoprotein of juxtaglomerular cells, is synthetized in the RER, packaged in the Golgi complex and found relatively rapidly in newly-formed secretory granules. Part of the fucose and tyrosine labels is also associated with the thick cell coat of these cells.

DNA Synthesis and Mitotic Activity in Adult Atrial Cardiocytes in Culture

Trypsin-dissociated adult rat atrial cardiocytes were exposed to [3H]thymidine for sequential 24-hr periods from day 3 to day 13 of culture. Following preparation for ultrastructural autoradiography, 1000 cells (at each daily interval) were examined with an electron microscope. Maximal incorporation occurred on day 5 when 63% of the cells were labeled. Small secondary peaks of incorporation occurred on days 8 and 11 following previous exposure to fresh serum. Mitotic activity never exceeded 0.5% of all cardiocytes examined. DNA synthesis and mitoses occurred only in immature cardiocytes characterized by subsarcolemmal filaments and Z-bands with or without specific granules; more mature cells were never labeled.