Download The Heart as an Endocrine Gland

The Heart as an Endocrine Gland
M A R C CANTIN AND JACQUES GENEST
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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
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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
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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
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The heart as an endocrine gland.
M Cantin and J Genest
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Hypertension. 1987;10:I118
doi: 10.1161/01.HYP.10.5_Pt_2.I118
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Copyright © 1987 American Heart Association, Inc. All rights reserved.
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