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Title: Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage

Abstract

The localization of mRNA encoding preproatrial natriuretic peptide was investigated in tissue sections and cultures of rat heart and in sections of human right atrial appendage using the technique of in situ hybridization with /sup 32/P- and /sup 35/S-labeled RNA probes. Rat atrial natriuretic peptide (ANP) transcripts were demonstrated in numerous atrial myocytes and, to a lesser extent, in ventricular myocytes in both tissue sections and newborn rat heart cultures. These findings are consistent with those obtained by RNA blot analysis of rat heart total RNA, indicating that a single prepro-ANP transcript of approx. 900 nucleotides was present in the ventricles as well as the atria. Using a /sup 35/S-labeled RNA probe for human ANP mRNA, ANP transcripts were also localized to the majority of myocytes in the human right atrial appendage. Only background levels of autoradiographic labeling were obtained when RNA probes identical to the coding sequence of rat or human ANP mRNA were used. A close correlation was found between the distribution of ANP immunoreactivity and prepro-ANP mRNA in these preparations. These results provide unequivocal evidence for the expression of the ANP gene in the rat ventricles, as well as the atria, because myocytes in these tissues havemore » been established as the sites of both ANP localization and precursor biosynthesis. The combined use of cardiac cultures and in situ hybridization may be of value in future studies investigating the regulation of ANP synthesis in cardiac myocytes.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Royal Postgraduate Medical School, London (England)
OSTI Identifier:
7143466
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States); Journal Volume: 84:19
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; HEART; AUTORADIOGRAPHY; CYTOCHEMISTRY; MESSENGER-RNA; HYBRIDIZATION; CELL CULTURES; IMMUNOLOGY; MAN; MUSCLES; PEPTIDES; PHOSPHORUS 32; RATS; SULFUR 35; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOCHEMISTRY; BODY; CARDIOVASCULAR SYSTEM; CHEMISTRY; DAYS LIVING RADIOISOTOPES; EVEN-ODD NUCLEI; ISOTOPES; LIGHT NUCLEI; MAMMALS; NUCLEI; NUCLEIC ACIDS; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; ORGANS; PHOSPHORUS ISOTOPES; PRIMATES; PROTEINS; RADIOISOTOPES; RNA; RODENTS; SULFUR ISOTOPES; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques; 550301 - Cytology- Tracer Techniques

Citation Formats

Hamid, Q., Wharton, J., Terenghi, G., Hassall, C.J.S., Aimi, J., Taylor, K.M., Nakazato, H., Dixon, J.E., Burnstock, G., and Polak, J.M. Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage. United States: N. p., 1987. Web. doi:10.1073/pnas.84.19.6760.
Hamid, Q., Wharton, J., Terenghi, G., Hassall, C.J.S., Aimi, J., Taylor, K.M., Nakazato, H., Dixon, J.E., Burnstock, G., & Polak, J.M. Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage. United States. doi:10.1073/pnas.84.19.6760.
Hamid, Q., Wharton, J., Terenghi, G., Hassall, C.J.S., Aimi, J., Taylor, K.M., Nakazato, H., Dixon, J.E., Burnstock, G., and Polak, J.M. 1987. "Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage". United States. doi:10.1073/pnas.84.19.6760.
@article{osti_7143466,
title = {Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage},
author = {Hamid, Q. and Wharton, J. and Terenghi, G. and Hassall, C.J.S. and Aimi, J. and Taylor, K.M. and Nakazato, H. and Dixon, J.E. and Burnstock, G. and Polak, J.M.},
abstractNote = {The localization of mRNA encoding preproatrial natriuretic peptide was investigated in tissue sections and cultures of rat heart and in sections of human right atrial appendage using the technique of in situ hybridization with /sup 32/P- and /sup 35/S-labeled RNA probes. Rat atrial natriuretic peptide (ANP) transcripts were demonstrated in numerous atrial myocytes and, to a lesser extent, in ventricular myocytes in both tissue sections and newborn rat heart cultures. These findings are consistent with those obtained by RNA blot analysis of rat heart total RNA, indicating that a single prepro-ANP transcript of approx. 900 nucleotides was present in the ventricles as well as the atria. Using a /sup 35/S-labeled RNA probe for human ANP mRNA, ANP transcripts were also localized to the majority of myocytes in the human right atrial appendage. Only background levels of autoradiographic labeling were obtained when RNA probes identical to the coding sequence of rat or human ANP mRNA were used. A close correlation was found between the distribution of ANP immunoreactivity and prepro-ANP mRNA in these preparations. These results provide unequivocal evidence for the expression of the ANP gene in the rat ventricles, as well as the atria, because myocytes in these tissues have been established as the sites of both ANP localization and precursor biosynthesis. The combined use of cardiac cultures and in situ hybridization may be of value in future studies investigating the regulation of ANP synthesis in cardiac myocytes.},
doi = {10.1073/pnas.84.19.6760},
journal = {Proc. Natl. Acad. Sci. U.S.A.; (United States)},
number = ,
volume = 84:19,
place = {United States},
year = 1987,
month =
}
  • We investigated the receptor localization of atrial natriuretic peptide (ANP) in the rat kidney, by in vitro macro- and micro-autoradiography (ARG) of (/sup 125/I)-ANP using nonfixed frozen sections. First, we examined the optimum conditions for ARG with respect to the effects of polyethylenimine (PEI), thickness of section, incubation time and degradation of (/sup 125/I)-ANP. Saturation experiments of (/sup 125/I)-ANP using rat kidney sections revealed the presence of high affinity binding sites of (/sup 125/I)-ANP in the renal cortex (Kd = 0.52 nM). Macro-autoradiograms showed that the dense grains representing specific binding sites of (/sup 125/I)-ANP were distributed in the cortexmore » in a punctate pattern. Using micro-autoradiography, the localization of the dense grains on the emulsion was compared with the staining pattern of the same section subjected to double staining. In the renal cortex, the dense grains were observed on the glomerulus, blood vessels and proximal tubules. Dense grains were also observed in the mesangium area of glomeruli, the inside wall of blood vessels (especially endothelium), and the inside wall of proximal tubules (possibly brush border). These results suggested that the physiological action of ANP in the kidney is mediated by its receptors. Also, ARG was useful for accurately detecting the action sites of ANP.« less
  • The distribution of atrial natriuretic peptide (ANP) clearance receptors in rat kidney was investigated by in vitro autoradiography using des(Gln18,Ser19,Gly20,Leu21,Gly22)-ANP-(4- 23) (C-ANP) and 125I-Tyr0-ANP-(5-25) as relatively specific ligands of this receptor. Alpha-125I-ANP (100 pM) bound reversibly but with high affinity to glomeruli, outer medullary vasa recta bundles, and inner medulla. C-ANP (10 microM) inhibited greater than 60% of this glomerular binding but did not inhibit the binding of alpha-125I-ANP to medullary tissues. Alpha-125I-ANP also bound reversibly to the renal arteries up to the glomerulus. This arterial binding was only partly inhibited by 10 microM C-ANP. In the presence of 10more » microM C-ANP, increasing concentrations of alpha-125I-ANP bound to a residue of glomerular sites with apparent dissociation constants of 0.82 +/- 0.16 to 2.73 +/- 1.20 nM at different cortical levels. 125I-Tyr0-ANP-(5-25) bound significantly to glomeruli and intrarenal arteries but not to vasa recta bundles or inner medulla. This glomerular binding also occurred with nanomolar dissociation constants. It was completely inhibited by 1 microM alpha-ANP and 10 microM C-ANP, but not by unrelated peptides such as gastrin. These results suggest that renal ANP clearance receptors are restricted in vivo to the glomeruli and renal arterial system of the rat.« less
  • Atrial natriuretic peptides (ANP) have recently been identified in both heart and CNS. These peptides possess potent natriuretic, diuretic, and vasorelaxant activities, and are all apparently derived from a single prohormone. Specific ANP binding sites have been characterized in the adrenal zona glomerulosa and kidney cortex, and one study reported ANP binding sites in the CNS. However, a detailed examination of the localization of ANP binding sites throughout the brain has not been reported. In this study, quantitative autoradiography was employed to examine the distribution of ANP receptors in the rat CNS. The binding of (3-/sup 125/I-iodotyrosyl28) rat ANP-28 tomore » binding sites in the rat CNS was saturable, specific for ANP-related peptides, and displayed high affinity (Kd = 600 pM). When the relative concentrations of ANP binding sites were determined throughout the rat brain, the highest levels of ANP binding were localized to the circumventricular organs, including the area postrema and subfornical organ, and the olfactory apparatus. Moderate levels of ANP binding sites were present throughout the midbrain and brain stem, while low levels were found in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. The presence of ANP binding sites in the subfornical organ and the area postrema, regions considered to be outside the blood-brain barrier, suggests that peripheral ANP levels may regulate some aspects of CNS control of salt and water balance. The possible functions of ANP binding sites in other regions of the rat brain are not known, but, like many other peptides, ANP may act as a neurotransmitter or neuromodulator at these loci.« less
  • The autoradiographic localization of (/sup 125/I)endothelin-1 (ET-1) and (/sup 125/I)atrial natriuretic peptide (ANP) after i.v. administration has been investigated in rats. Labeled peptides are rapidly distributed to tissues and peripheral organs. After administration of (/sup 125/I)ET-1 (1-21), the highest enrichment of radioactivity was found in the lung, kidney, liver, adrenal gland, and heart. After administration of (/sup 125/I)ANP (1-28), the highest levels of radioactivity could be observed in the kidney, adrenal gland, and endocardium. Compared to blood levels in both cases, a relative enrichment of radioactivity is also found in the vascular wall of the aorta. For ANP, no distributionmore » of radioactivity in the lung could be observed. Other organs, especially the kidney and the adrenal gland, showed a similar distribution pattern with respect to substructures.« less
  • Some, though not all studies, have indicated that atrial natriuretic peptide (ANP) can bind to adrenal medullary cells. ANP-like immunoreactivity (ANP-LI) has also been identified in catecholamine-secreting cells. Together, these findings suggest that ANP may be taken up and/or synthesized in the adrenal medulla. The present study was designed to ascertain, by in situ hybridization, whether adrenal chromaffin cells could synthesize ANP, to define by an in vivo ultrastructural autoradiographic approach, whether ANP could, in fact, bind to rat adrenal medulla cells, to determine whether there was a cellular (noradrenaline (NA) vs. adrenaline (A)) selectivity in the binding process, andmore » to establish whether extracellular (125I)ANP could be internalized by these cells. The cellular and subcellular distribution of endogenous ANP-LI was also investigated in both cell types by cryoultramicrotomy and immunocytochemical approaches. The in situ hybridization studies indicate the presence of mRNA to ANP in about 15% of adrenal medullary cells. Intravenous injection of (125I)ANP resulted in a 3-fold, preferential and specific radiolabeling of A-as compared to NA-containing cells. In A-containing cells, plasma membranes were significantly labeled 2 and 5 min post injection; cytoplasmic matrix, mitochondria, and secretory granules throughout the time course studied (1-30 min post injection). Lysosomes, rough endoplasmic reticulum, Golgi apparatus, and nuclei were not labeled. ANP-LI was identified in both NA- and A-containing cells; in the former, it was almost exclusively localized in secretory vesicles, in the latter it was detected in plasma membranes, cytoplasmic matrix, nuclear euchromatin, some mitochondria and relatively fewer granules than in NA-containing cells.« less