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The Heart as an Endocrine Gland M A R C CANTIN AND JACQUES GENEST Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017 SUMMARY The sequence of atrial natriuretic factor (ANF) has been determined, as well as the complete structure of the rat and human complementary DNA and gene. ANF and ANF messenger RNA are present not only in atria but also in ventricles. The circulating form of ANF has been identified as the C-terminal of the molecule, ANF (Ser 99-Tyr 126). The isolated secretory granules of rat atrial cardiocytes contain only pro-ANF (Asn 1-Tyr 126). An enzyme (IRCM-SP1) has been isolated from heart atria and ventricles. This enzyme is highly specific in cleaving ANF (Asn 1-Tyr 126), to yield ANF (103-126), (102-126), and (99-126). In target cells, ANF produces a rise in cyclic guanosine 3',5'-monophosphate (cGMP) due to activation of participate guanylate cyclase, and inhibition of adenylate cyclase leading in some cases to a decrease in cyclic adenosine 3,5-monophosphate (cAMP). ANF produces relaxation of rabbit and rat aortic strips, inhibits steroidogenesis in both zona glomerulosa and zona fasciculata cells, and inhibits the release of arginine vasopressin from the isolated rat hypothalamohypophysial preparation in vitro but decreases AVP release in vivo only at pharmacological doses. In all forms of experimental hypertension, plasma levels of ANF are increased and, at some time periods, atrial levels are also decreased. The ventricular levels of immunoreactive ANF are also increased in renal hypertension. Infusion of ANF by mlnipumps decreases the blood pressure near control levels in several models of experimental hypertension. In card iomyo pat hie hamsters with heart failure, the atrial levels of immunoreactive ANF are decreased while the plasma and ventricular levels are increased. In humans, the mean plasma levels of ANF are not increased above control values in essential hypertension (for moderate increases in blood pressure), but they are increased in the aortic blood in renal hypertension. Plasma levels of immunoreactive ANF are increased hi paroxysmal tachycardia, valvular heart diseases, idiopathic cardiomyopathies, and coronary heart disease. (Hypertension 10 [Suppl I]: 1-118—1-121, 1987) KEY WORDS * atrial natriuretic factor physiology • physiopathology I T is now well established that the atria are endocrine glands secreting, in rats and humans, the 28-amino acid peptide atrial natriuretic factor (ANF)-(Ser 99-Tyr 126). ANF messenger RNA (mRNA) and immunoreactive ANF (irANF) are also present, although in much lower amounts, in ventricular cardiocytes. Biochemistry and Molecular Biology of ANF Isolation and Sequencing After the discovery by de Bold et al.1 that rat atrial extracts produce diuresis and natriuresis, we showed From the Laboratory of Pathobiology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. Supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension, and by grants from the National Research Council of Canada, the Ministere de la Science et de la Technologie du Que'bec, the Pfizer Company, and Bio-M6ga, Inc. Address for reprints: Marc Cantin, M.D., Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec H2W 1R7. molecular biology • biochemistry that the effects of the crude extracts are localized in the specific (now secretory) granules themselves.2 Early purification attempts established the polypeptide nature of ANF and the amino acid composition of short (C-terminal) peptides. 3 In June 1983 we isolated and sequenced, among others, a 33-amino acid peptide that is part of a larger molecule containing at least 106 amino acids.*~6 After its sequencing in our laboratory, the 33-amino acid peptide was synthesized by Merck Sharp & Dohme in September 1983." All our further work reported here has been done with this synthetic peptide. The C-terminal of human ANF has also been isolated and sequenced.7 Cloning the Complementary DNA and Gene of ANF The availability of atrial peptide sequence data prompted cloning of the complementary DNA (cDNA) and then of the gene for both rat and human ANF. Rat cDNA has been cloned.8 The rat gene has also been isolated and its structure determined.9 Human cDNA for A N F and the human gene10 have also been delineated. 1-118 ATRIAL NATRIURETIC FACTOR/Cantm and Genest 1-119 Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017 Chromosomal Localization of the ANF Gene Chromosomal assignment of the gene coding for human ANF was accomplished by in situ hybridization of a [3H]ANF probe to a human chromosome preparation and Southern blot analysis of somatic cell hybrid DNA with normal and rearranged chromosomes. The human ANF gene was mapped to the distal short arm of chromosome 1 in band 1P36. Southern blot analysis of hybrid mouse x Chinese hamster somatic cells was used to assign the mouse ANF gene to chromosome 4 . " bation with serum results in the formation of 11.7-kDa and 2.7-kDa peptides, which correspond to the N- and C-terminal portions of the molecule respectively. These results suggest that hydrolysis of the propeptide in serum is mediated by an enzyme activated during coagulation and thus has no physiological relevance. Atrial cardiocytes in culture contain and secrete large amounts of irANF in the medium." The irANF present in these cells is exclusively of the high Afr variety, as is most of what is secreted, with only about 15 to 25% being secreted as the low Mr form. Localization of Inununoreactive ANF in the Heart Up to now, it has been well established that the atria •are endocrine glands. Recent studies indicate that ventricular cardiocytes also harbor immunoreactive ANF (irANF) and contain and secrete it in the medium when cultured.12 Biochemical studies show that the irANF present in ventricular cardiocytes is, as in the atria, of the high-molecular-weight variety.12 Cardiac ventricular cardiocytes of control animals contain ANF messenger RNA (mRNA).13 Second Messengers In all target cells so far investigated, ANF produces a rise in cyclic guanosine 3',5'-monophosphate (cGMP) modulated by activation of paniculate guanylate cyclase,20'21 and an inhibition of adenylate cyclase leading in some cases to a decrease in cyclic adenosine 3',5'-monophosphate (cAMP). 2223 Circulating Form of ANF The circulating form of ANF has been isolated from plasma of morphine-treated rats as the C-terminal, active portion of the molecule, ANF (Ser 99-Tyr 126).14'15 Biosynthesis Secretory granules from rat atria were isolated by differential centrifugation and by a 53% Percoll gradient. Analysis of the ANF content in these isolated granules by high performance liquid chromatography (HPLC), amino acid analysis, and sequencing demonstrated that it was made up only of pro-ANF (Asn 1-Tyr 126). The precursor was present in all granules as demonstrated by immunocytochemistry using antibodies against synthetic N-terminal fragments of the propeptide.16 An enzyme (IRCM-SP1) has been isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific in cleaving ANF (Asn 1-Tyr 126) to yield ANF (103-126), (102-126), and (99-126) in that order of preference. The enzyme was nine times more abundant in atria than in ventricles per milligrams protein.17 ANF released by the isolated perfused heart (Langendorff preparation) was extracted from the perfusate by C l8 Sep-Pak cartridges and then isolated by immunoafflnity chromatography and reverse phase HPLC. About 500 fig of irANF was thus obtained and submitted to amino acid sequencing. The C-terminal Tyr was detected by radiolabeling. Identification of these residues indicated that the primary structure corresponds to ANF (Ser 99-Tyr 126).18 Injection of 125I-ANF (Glu 54—Tyr 126) in the perfusion fluid and analysis of the perfused material revealed no change of this relatively large peptide.18 When 125I-ANF (Asn 1-Tyr 126) is incubated in whole blood, plasma, or serum for different time intervals, the results indicate minimal cleavage of the propeptide in whole blood or plasma. Incu- Physiology Kidneys Receptors have been localized by radioligand-binding studies and by radioautography in the podocytes of visceral epithelial cells of glomeruli, the vasa recta bundles of the outer medulla, and the collecting duct cells of the inner medulla24'25; binding is present in glomeruli, thick ascending limbs of Henle, and collecting duct membranes, but not in proximal tubules.26 In glomeruli, a 50-fold increase of cGMP over basal levels occurs after exposure to ANF, while a two-fold increase occurs in the ascending limbs, and a threefold increase in collecting duct cells.21 ANF inhibits adenylate cyclase activity in glomeruli, collecting ducts, and loops of Henle, but not in proximal tubules.27 No metabolic change occurs in any isolated nephron segment after exposure to ANF. 28 Blood Vessels ANF produces a potent, dose-dependent, relaxing effect on rabbit and rat aortic strips.29'3? Adrenal Cortex ANF inhibits steroidogenesis prior to progesterone synthesis in both zona glomerulosa and zona fasciculata of the adrenal cortex.31"33 Arginine Vasopressin Release ANF inhibits the release of arginine vasopressin from the isolated rat hypothalamohypophysial preparation in vitro34 but does so in vivo only at pharmacological levels.33 Physiopathological Implications Animals Hypertension In all forms of experimental hypertension, plasma levels of ANF are increased and, at some time periods, 1-120 CATION TRANSPORT AND NATRIURETIC FACTORS atrial levels are decreased.36"38 The ventricular levels of irANF are also increased in renal hypertension, and typical secretory granules (as delineated by ultrastructural immunocytochemistry using an immunogold technique) appear in ventricular cardiocytes. Infusion of ANF (Arg 101-Tyr 126) with minipumps at 100 ng/hr for several days brings down the blood pressure of hypertensive animals to control levels in several models.39~*1 In the two-kidney, one clip model, only the saralasin-sensitive animals respond to ANF infusion by lowering of blood pressure.42 Congestive Heart Failure Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017 In cardiomyopathic hamsters, atrial levels of irANF are decreased while the plasma and ventricular levels are increased.43 ANF mRNA, which is present in moderate amounts in the ventricular cardiocytes of controls, is markedly increased in congestive heart failure. Typical secretory granules harboring irANF are present in the ventricular cardiocytes of hamsters with heart failure while they are absent in control animals.43 Humans Hypertension The mean plasma levels of ANF are not increased above control values in essential hypertension (for moderate increases in blood pressure), but they are increased in aortic blood in renal hypertension. Cardiac Diseases Plasma levels of irANF are increased in paroxysmal tachycardia, valvular heart diseases, idiopathic cardiomyopathies, and coronary heart diseases. It is now well established that the atria and possibly the ventricles as well are endocrine glands that secrete in the circulation a 28-amino acid peptide with farranging effects on the control of blood pressure and blood and extracellular fluid volume. The biosynthetic pathways of ANF in the heart, the factors controlling its release, and its physiological effects (as opposed to its pharmacological effects) remain to be determined. The renal effects of ANF are still not completely understood. The central nervous system effect of ANF as regards control of blood pressure and extracellular fluid volume still remains to be elucidated. The full clinical effects of ANF remain largely unknown. It is nevertheless almost certain that it will become a therapeutic agent in hypertension and in the clinical control of edematous states. Acknowledgments The work done on ANF at the Clinical Research Institute of Montreal has been from the start a collaborative effort primarily involving the members of the Multidisciplinary Research Group on Hypertension (Jacques Genest, Marc Cantin, Galtan Thibault, Raul Garcia, Jolanta Gutlcowska, Pavel Hamet, Andre1 De L6an, Ernesto Schiffrin, Otto Kuchel, Madhu B. Anand-Srivastava, Johanne Tremblay, Stephen Pang), members of the Laboratory of Molecular Endocrinology (Michel Chretien, Nabil G. Seidah, Claude Lazure), members of the Laboratory of Molecular Biology (Jacques Drouin, Mona Nemer), and Peter Schiller, and Ross Milne. SUPPL I HYPERTENSION, VOL 10, No 5, NOVEMBER 1987 References 1. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 1981;28:89-94 2. Garcia R, Cantin M, Thibault G, Genest J. Relationship of specific granules to the natriuretic and diuretic activity of rat atria. Experientia 1982;38:1071—1073 3. Thibault G, Garcia R, Seidah NG, et al. Purification of three rat atrial natriuretic factors and their amino acid sequence. FEBS Lett 1983;164:286-290 4. Seidah NG, Lazure C, Chretien M, et al. Amino acid sequence of homologous rat atrial peptides: natriuretic activity of native and synthetic forms. Proc Natl Acad Sci USA 1984;81: 2640-2644 5. Thibault G, Garcia R, Cantin M, et al. Primary structure of a high-Afr form of rat atrial natriuretic factor. FEBS Lett 1984; 167:352-356 6. Lazure C, Seidah NG, Chre'tien M, et al. Atrial pronatriodilatin: a precursor for natriuretic factor and cardiodilatin aminoacid sequence evidence. FEBS Lett 1984;172:80-86 7. Thibault G, Carrier F, Garcia R, et al. The human atrial natriuretic factor (hANF): purification and primary structure. Clin Invest Med 1984;7(suppl 2):59 8. Zivin RA, Condra JH, Dixon RAF, et al. Molecular cloning and characterization of DNA sequences encoding rat and human atrial natriuretic factor. Proc Natl Acad Sci USA 1984; 81:6325-6329 9. Argentin S, Nemer M, Drouin J, Scott GN, Kennedy RP, Dalvies PL. The gene for rat atrial natriuretic factor. J Biol Chem 1985;260:4568^571 10. Nemer M, Chamberland M, Sirois D, et al. Gene structure of human cardiac hormone precursor, pronatriodilatin. Nature 1984;312:654 11. Yang-Feng TL, Floyd-Smith G, Nemer M, Drouin J, Francke V. The pronatriodilatin gene is located on the distal short arm of human chromosome I and on mouse chromosome 4. Arri J Hum Genet 1985;37:1117-1128 12. Cantin M, Ding J, Thibault G, et al. Immunoreactive atrial natriuretic factor is present in both atria and ventricles. J Mol Cell Endocrinol 1987;52:105-113 13. Nemer M, Lavigne JP, Drouin J, Thibault G, Ganong M, Antakly T. Expression of atrial natriuretic factor gene in heart ventricular tissue. Peptides 1986;7:1147-1152 14. Thibault G, Lazure C, Schiffrin EL, et al. Identification of a biologically active circulating form of rat atrial natriuretic factor. Biochem Biophys Res Commun 1985;130:981-986 15. Cantin M, Genest J, Thibault G, et al. On the circulating form of atrial natriuretic factor. Trans Assoc Am Physiol 1985;98: 116-122 16. Thibault G, Garcia R, GutkowskaJ.etal. The propeptide (Asn 1-Tyr 126) is the storage form of rat atrial natriuretic factor. Biochem J 1987;241:265-272 17. Seidah NG, Cromlish JA, Hamelin J, Thibault G, Chre'tien M. Homologous IRCM-serine protease 1 from pituitary, heart atrium and ventricle: a common pro-hormone maturation enzyme? Biosci Rep 1986;6:835-844 18. Thibault G, Garcia R, Gutkowska J, et al. Identification of the released form of atrial natriuretic factor by the perfused heart. Proc Soc Exp Biol Med I986;182:137-141 19. Cantin M, Dagenais N, Salmi L, et al. Secretory pattern of atrial natriuretic factor (ANF) by cultured cardiocytes in right and left atria from newborn and adult rat. Clin Exp Hypertens 1985;A7(5&6):685-705 20. Hamet P, Tremblay J, Pang SC, et al. Cyclic GMP as mediator and biological marker of atrial natriuretic factor. J Hypertens 1986;4(suppl 2):S-49-S-55 21. Tremblay J, Gerzer R, Vinay P, Pang SC, Beliveau R, Hamet P. The increase of cGMP by atrial natriuretic factor correlates with die distribution of paniculate guanylate cyclase. FEBS Lett 1985;181:17-22 22. Anand-Srivastava MB, Vinay P, Genest J, Cantin M. Effect of atrial natriuretic factor on adenylate cyclase in various nephron segments. Am J Physiol 1986;251:F^tl7-F^24 23. Anand-Srivastava MB, Srivastava AK, Cantin M. Pertussis ATRIAL NATRIURETIC 24. 25. 26. 27. 28. 29. 30. Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017 31. 32. 33. toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase: involvement of Gi-guanine nucleotide regulatory protein. J Biol Chem 1987;262:4931-4934 Bianchi C, Gutkowska J, Thibault G, Garcia R, Genest J, Cantin M. Distinct localization of atrial natriuretic factor and angiotensin II in the glomerulus. Am J Physiol 1986;251: F594-F602 Bianchi C, Gutkowska J, Ballak J, Charbonneau C, Genest J, Cantin M. Localization of ll5I-atrial natriuretic factor (ANF) binding sites in rat renal medulla. Light and electron microscope radioautographic studies. J Histochem Cytochem 1987; 35:149-153 De L£an A, Vinay P, Cantin M. Distribution of atrial natriuretic factor receptor in dog kidney. FEBS Lett 1985;193:239-242 Anand-Srivastava MB, Vinay P, Genest J, Cantin M. Effect of atrial natriuretic factor on adenylate cyclase in various nephron segments. Am J Physiol 1986;251:F417-F423 Vinay P, Manillier C, Lalonde L, et al. Comparative effect of ANF and various diuretics on isolated nephron segments of dogs and rats. Kidney Int 1987;3:946-955 Garcia R, Thibault G, Cantin M, Genest J. Effect of a purified atrial natriuretic factor on rat and rabbit vascular strips and vascular beds. Am J Physiol 1984;247:R-34-R-39 Schiffrin EL, Chartier L, Thibault G, St-Louis J, Cantin M, Genest J. Vascular and adrenal receptors for atrial natnuretic factor (ANF) in the rat. Circ Res 1985;56:801-807 Chartier L, Schiffrin EL, Thibault G, Garcia R. Atrial natriuretic factor inhibits the effect of angiotensin H, ACTH and potassium on aldosterone secretion in vitro and angiotensin IIinduced steroidogenesis in vivo. Endocrinology 1984;115: 2026-2028 De Lean A, Racz K, Gutkowska J, Nguyen TT, Cantin M, Genest J. Specific receptor-mediated inhibition of synthetic atrial natriuretic factor of hormone-stimulated steroidogenesis in cultured bovine atrial cells. Endocrinology 1984;115:1636— 1638 Racz K, Kuchel O, Cantin M, De Uan A. Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex. FEBS Lett 1985;192:19-22 FAcroRJCantin and Genest 1-121 34. Januszewicz P, Thibault G, Garcia R, Gutkowska J, Genest J, Cantin M. Effect of synthetic atrial natriuretic factor on arginine vasopressin release by the rat hypothalamo-neurohypophyseal complex in organ culture. Biochem Biophys Res Commun 1986; 134:652-658 35. Januszewicz P, Larose P, Ong H, Gutkowska J, Genest J, Cantin M. Effect of atrial natriuretic factor on plasma vasopressin in conscious rats. Peptides 1986;7:989-993 36. Gutkowska J, Horky K, Lachance D, et al. Atrial natriuretic factor in spontaneously hypertensive rats. Hypertension 1986; 8(suppl I):I-137-I-140 37. Gutkowska J, Kuchel O, Racz K, Buu NT, Cantin M, Genest J. Increased plasma immunoreactive atrial natriuretic factor concentrations in salt sensitive Dahl rats. Biochem Biophys Res Commun 1986;136:411—416 38. Garcia R, Cantin M, Gutkowska J, Thibault G. Atrial natriuretic factor during development and reversal of one-kidney, one clip hypertension. Hypertension 1987;9:144—149 39. Garcia R, Thibault G, Gutkowska J, et al. Chronic infusion of low doses of atrial natriuretic factor (ANF Arg 101-Tyr 126) reduces blood pressure in conscious SHR without apparent changes in sodium excretion. Proc Soc Exp Biol Med 1985; 179:396-401 40. Garcia R, Gutkowska J, Genest J, Cantin M, Thibault G. Reduction of blood pressure and increased diuresis and natriuresis during chronic infusion of atrial natriuretic factor (ANF Arg 101-Tyr 126) in conscious one kidney, one clip hypertensive rats. Proc Soc Exp Biol Med 1985;179:539-545 41. Garcia R, Thibault G, Gutkowska J, Cantin M. Effect of chronic infusion of atrial natriuretic factor on plasma and urinary aldosterone, plasma renin activity, blood pressure and sodium excretion in 2K1C hypertensive rats. Clin Exp Hypertens 1986;A8:1127-1147 42. Garcia R, Gutkowska J, Cantin M, Thibault G. Renin dependency of the effect of chronically administered atrial natriuretic factor in two-kidney, one clip rat. Hypertension 1987;9:88-95 43. Ding J, Thibault G, Gutkowska J, et al. Cardiac and plasma atrial natriuretic factor (ANF) in experimental congestive heart failure. Endocrinology 1987;121:248-257 The heart as an endocrine gland. M Cantin and J Genest Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017 Hypertension. 1987;10:I118 doi: 10.1161/01.HYP.10.5_Pt_2.I118 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1987 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/10/5_Pt_2/I118 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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