Physiology, New Endocrine System , Cardiac Hormones

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(New Endocrine Systems) Cardiac Hormones:

Introduction: Recent advances in physiology, during the last two decades, have established an endocrine role of the heart in addition to its normal function as a pump. It is now well known that both atria and ventricles are capable of producing natriuretic peptides. Since the discovery of atrial natriuretic factor by DeBold and his coworkers in 1981, a vast amount of research has been performed on cardiac natriuretic peptides concerning their synthesis , release and physiological activities. These natriuretic factors are of special interest both physiologically and clinically, because they participate in the regulation of cadiovascular and renal homeostatic mechanisms. They are thought to be involved in the regulation of body fluid volume and electrolyte balance.

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The principal actions of these endogenous natriuretic factors are natriuresis, diuresis and hypotension. They exert these effects by altering renal haemodynamics, inhibition of tubular reabsorption of Na+ and water indirectly through inhibition of vasopressin release and antagonizing the effects of renin angiotensin - aldosterone system. Their direct vasorelaxant properties contribute mainly to the hypotensive effect. The release of cardiac natriuretic peptides is enhanced mainly by expansion of the extracellular fluid volume and atrial distension. Their release is increased manyfold in oedematous disorders such as congestive heart failure and chronic renal failure.

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Historical account: Morphologic observations showed that the atrial myocytes contain structures resembling secretory granules , located at the nuclear pole near the mitochondria and Golgi apparatus . Their endocrine function was not demonstrated until De Bold and his colleagues in 1978, described the structural similarities between atrial granules and the secretory granules that exist or seen in other endocrine tissues, and speculated that atrial granules could contain a hormone . De Bold's initial experiments demonstrated a change in the number and density of these granules with alteration in salt and water intake. Thus, it seemed likely that these granules were intimately involved in the control of ECF volume. These investigators did further work and they prepared an extract of rat cardiac atria and injected that intravenously into bioassay rats.

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The atrial extract produced dramatic natriuresis and diuresis (DeBold et al. 1981). At the beginning, the term atrial natriuretic factor has been commonly used to describe the active substance in the extract, until its chemical structure was identified, thereafter it was known as atrial natriuretic peptide (ANP). Another Physiologically important natriuretic factor named brain natriuretic peptide (BNP), was first discovered in porcine brain by Sudoh et al. in 1988.New reports have shown that significant amount of this natriuretic peptide is also synthesized in and released into the circulation from the heart. It exerts physiological and pharmacological actions very similar to that of ANP, such as natriuresis, diuresis and hvpotension. Recent evidences suggest that BNP and ANP act in a dual natriuretic peptide system involved in circulatory homeostasis.

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Synthesis and Chemistry of ANP and BNP: The purification of ANP from atrial tissue coincided with the period when molecular biologic techniques were being applied routinely to the identification of the structure of proteins (recombinant DNA Studies). These techniques therefore greatly expedited the identification of the structure of circulating ANP and its precursor in the atria. Both ANP and BNP have a common core peptide sequence. This was used to make the complementary DNA (cDNA) probes to identify the mRNA for ANP and thus the whole sequence of the precursor molecule

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In both rat and man the ANP mRNA comprises 0.5-3 % of the total atrial mRNA indicating that it is a major transcription product. The human mRNA codes for a 151 amino acids (precursor) peptide. This precursor was called Prepro-ANP. The entire sequence of human ANP prohormone, has now been identified. Pro-ANP consists of 126 amino acids and it is an abundant storage form of the peptide. The ANP prohormone in the cardiac tissue is cleaved into two fragments, both of which enter the circulation a C-terminal 28 amino acid peptide (ANP 99-126) derived from the COOH terminus of the prohormone and an N-terminal fragment (1-98). The cleaved fragment of the pro-ANP from 99 – 126 (thereafter known as ANP) is biologically active.

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Until recently, information on BNP in man has been scarce, mainly because human BNP immunoreactivity was not detected with early antisera which were raised against porcine or rat BNP. The BNP prohormone (1-108 amino acid) in human was recently elucidated. BNP has been isolated from the human atrium and found to comprise 32 amino acid residues (77-108 amino acid).This peptide is derived from C-terminus of the pro-BNP molecule, and shows remarkable similarity to the structure of ANP. A third polypeptide, CNP, is present in the human brain but apparently not in the human heart. ANP,BNP, and CNP are encoded by different genes.

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Human ANP,BNP and CNP. Top: Single – letter codes for amino acid residues aligned to show common sequences. Bottom: Shape of molecules. Note that one cysteine is the Cterminal amino acid residue in CNP, so there is no C-terminal extention from the 17member ring.

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Storage and release of ANP and BNP: ANP is primarily formed, stored and secreted from atrial myocytes. However, a larger amount of ANP prohormone has been identified in atrial tissue and it is therefore, the most likely storage form. It has been reported that ANP is stored in atrial cardiocytes as ANP precursor and cleavage of this precursor occurs during the process of secretion to yield the active ANP. Whereas, BNP was first isolated form brain, but it is also found in the circulation and in highest amounts in the heart ventricles, especially in pathophysiologic states. BNP is formed by cleavage of human BNP (hBNP) precursor before secretion and stored in cardiocytes and regarded as a major storage form both in atria and ventricles.

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The most effective stimuli that cause ANP release are salt loading and ECF volume expansion. Several maneuvers including, blood volume expansion, mechanical distension of the atria and increase in central blood volume induced by head-out water immersion or head down tilt have been shown to raise ANP levels. In addition, elevated circulating levels of the peptide have been found in pathologic states associated with increased atrial filling pressure leading to increased atrial wall stretch such as in congestive heart failure, renal failure and primary aldosteronism. This means that atrial stretch or distension is the prime factor leading to ANP secretion. The proANP is cleaved by a membrane bound protease and released into the circulation as 28 amino acid peptide.

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Regarding the BNP, it has been reported that BNP level represents only 2-3% of that of ANP level in the porcine atrial extract. BNP levels in human atria and ventricles in the normal heart have been measured. The tissue BNP levels in the ventricle was found to be much lower (7.2%) than that in the atrium on weight basis. These findings raised the possibility that the ventricles produce and secrete a considerable amount of BNP. Plasma levels of BNP mainly reflect the degree of ventricular overload or ventricular muscle damage. This indicates that BNP is expressed in ventricular myocytes in response to haemodynamic stress such as in congestive heart failure, dilated cardiomyopathy and acute myocardial infarction. Furtheremore, high levels of BNP were found in human plasma after

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dietary sodium loading and associated with significant increase in urinary

sodium excretion. This indicates that in addition to ANP, BNP may be a new and important natriuretic peptide which regulates sodium homeostasis in man during increased sodium intake. However, a fall in plasma levels of ANP and BNP can be induced by measures like furosemide administration, sodium depletion and during dehydration.

Atrial Reflexes That Activate the Kidneys—The “Volume Reflex.” Stretch of the atria also causes significant reflex dilation of the afferent arterioles in the kidneys. And still other signals are transmitted simultaneously from the atria to the hypothalamus to decrease secretion of antidiuretic hormone. The decreased

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afferent arteriolar resistance in the kidneys causes the glomerular capillary pressure to rise, with resultant increase in filtration of fluid into the kidney tubules. The diminution of antidiuretic hormone diminishes the reabsorption of water from the tubules. Combination of these two effects — increase in glomerular filtration and decrease in reabsorption of the fluid increases fluid loss by the kidneys and reduces an increased blood volume back toward normal. Atrial stretch caused by increased blood volume also elicits a hormonal effect on the kidneys—release of ANP that adds still further to the excretion of fluid in the urine and return of blood volume toward normal.

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Half-life , metabolism and clearance of cardiac peptides : The half-life of the circulating ANP is short, and it has been demonstrated that injection of ANP into animals or human volunteers revealed that its half-life was on the order of 1 – 4 min. The metabolic clearance rate is high and the volume of distribution of the peptide is large. Synthetic avian ANP was given by iv infusion at various rates to Pekin ducks and the associated plasma concentrations of immunoreactive ANP (irANP) were measured by radioimmunoassay, the results indicated that ANP has an elimination half-time of 1-2 min and is rapidly removed from the circulation. The data on human irANP pharmacokinetics are rather uniform: the distribution volume appears to be 10-17 liters, and plasma half-

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life of 2.5 - 4.5 minutes. Pharmacokinetic studies in anaesthetized rabbits have revealed a short biological half-life of 1-2 minute for ANF applied exogenously. The receptors of cardiac natriuretic peptides are transmembrane proteins. A large body of evidences indicate that, ANP and BNP exert their biological activities through these receptors. Both peptides stimulate guanylate cyclase enzyme, which catalyzes the conversion of intracellular GTP to cGMP. For instance, stimulation of cGMP formation in vascular smooth muscles mediates ANP's vasorelaxant activity, cGMP probably in turn alters the calcium mobilization in vascular smooth muscles resulting in vasodilatation. The clearance of ANP and BNP from the circulation is mediated by two major distinct pathways: binding to receptors and specific

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degradative enzymatic pathway by neutral endopeptidase enzyme, which is present in high concentrations on brush borders of proximal tubules and in the lung. Neutral endopeptidase inactivates both ANP and BNP. Furthermore, the clearance rate of cardiac nartiuretic peptides is significantly reduced and their half-life is prolonged in the presence of endopeptidase inhibitors.

Physiological actions of ANP and BNP: Both ANP and BNP exert similar physiological actions such as natriuretic, diuretic and hypotensive effects. Renal effects: The renal actions of cardiac natriuretic peptides include, effects on the renal

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vasculature, the glomerular mesangial cells, renin secretion and tubular sodium reabsorption. It has been demonstrated that intrarenal administration of synthetic ANP markedly increases the urine volume and sodium excretion with associated increase in GFR and decreased tubular reabsorption of sodium. Injection of the atrial extract into bioassay rats was associated with significant increases in urine flow rate, Na+ excretion (20 folds) and K+ excretion. Micropuncture studies on euvolemic Munich-wistar rats demonstrated that ANP infusion decreases the arterial resistance in the glomerular afferent arteriole in a dose dependant manner, while leaving the resistance of the efferent arteriole unchanged or increased. This effect of ANP, makes the ANP system as unique and different from classic renal vasodilators such as bradykinins and acetylcholine. Thus an increased GFR is likely to occur by the selective effect of

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ANP on glomerular microcirculation. An increase in GFR occurs as a result of a rise in glomerular capillary hydrostatic pressure from afferent arteriolar vasodilation and efferent arteriolar vasoconstriction. Moreover, small changes or increase in GFR (such as 5%), which certainly may escape detection, can produce a significant increase in urine output and alteration in sodium excretion, independent of changes in tubular sodium handling. The natriuretic effect of ANP may also be mediated in part, by renal specific tubular mechanisms rather than purely by effects on the intrarenal haemodynamics. There is no clear understanding mechanism showing the direct effect of ANP on proximal tubules and loops of Henle. In spite of the presence of natriuretic peptide receptors on proximal tubular cells, it has been found that atrial natriuretic peptides, modulate the

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proximal tubular transport of sodium and water only in the presence of angiotensin II . Moreover, there is no demonstrable direct ANP effect on isolated proximal tubular cells. Atrial natriuretic peptides do not appear to affect the proximal tubular transport directly, but act via specific receptors on the basolateral and luminal membranes to raise the intracellular cGMP levels and inhibit the angiotensin 11 stimulated transport. Regarding the effects of ANP on the loops of Henle and renal medullary washout, animal experiments have shown the loss of medullary solute gradient after ANP infusion. Moreover, all human studies have demonstrated a marked fall in urinary osmolality by administration of ANP, which indicates an increase in medullary washout that can dissipate the medullary hyperosmolality and contribute to the diuresis. Although, the mechanism of

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action of ANP on the distal tubules and cortical collecting ducts is not clear, it has been reported that ANP is capable of inhibiting the action of vasopressin in this segment of the nephron. In inner medullary collecting ducts (IMCD), receptors for ANP have been characterized, and cGMP was identified as an intracellular second messenger. The sodium transport across IMCD cells occurs through amiloride-sensitive Na+ channels in the apical membrane. It has been suggested that cGMP may directly inhibit these channels by phosphorylation independent mechanism and also by a cGMP- dependant protein kinase phosphorylation step. Thus the reduced entry of Na will indirectly inhibit the Na+-K+ ATPase pump on the basolateral surface of these cells. These actions of ANP clearly favour natriuresis and diuresis.

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The postulated mechanism of action of cardiac natriuretic peptides on the IMCD cells. GC-A = Guanylate Cyclase-linked receptor type A. PKG = Protein Kinase G.

2.Effects on renin - angiotensin - aldosterone

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system and vasopressin secretion:

Infusion of auriculin (an early name of ANP) into normal dogs produced a marked and sustained supperssion of renin secretion and plasma renin values. The fall in renin secretion may be due to ANP's renal haemodynamic actions.This effect on renin secretion is also thought to be due to inhibition of sympathetic tone to the Juxtaglomerular renin secreting cells. The inhibitory effect of the peptide is much more pronounced in innervated than denervated kidneys. However, it is important to note that in cultured juxtaglomerular cells ANP also inhibits renin secretion. Hence, ANP inhibits the first step in sodium-retaining renin - angiotensin-aldosterone system. Consequently, it reduces the circulating levels and the vasoconstrictor effects of angiotensin II and the sodium retaining

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effects of aldosterone and angiotensin II . Moreover, ANP decreases the responsiveness of zona glomerulosa cells to stimuli that normally increase the aldosterone secretion . It has been reported that ANP is capable of inhibiting the action of vasopressin on distal tubules and cortical collecting ducts. Meanwhile, it has been shown that synthetic ANP can inhibit dehydration and haemorrhage-induced ADH release in vivo. Also, it has been suggested that ANP directly acts on the posterior pituitary to inhibit ADH secretion and the effect is partly mediated by the increased production of cGMP and decreased production of cAMP. On other hand, the production of ANP within the brain is thought to be involved locally in the control of ADH release.

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ANP, was observed to have a significant effect on AVP-induced decrease in urine output and sodium excretion in patients with congestive heart failure (CHF). Co-infusion of ANP with AVP in patients with CHF ,enhanced both the urine flow rate and Na+ excretion without change in the systemic arterial blood pressure. Cardiovascular effects of ANP and BNP: Cardiac natriuretic peptides proved to be potent vasorelaxant agents. ANP has a vasorelaxant effect on renal vasculature, and on large arteries and vascular beds and also on non-vascular smooth muscles. There is some evidence to indicate that renal arteries are more sensitive than other arterial tissues to the vasodilator effect of ANP. Although, ANP opposes the vasoconstriction induced by all hormonal and pharmacologic agents, but its antagonism is greater to

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angiotensin II than to norepinephrine induced contraction of rabbit aorta. Direct negative inotropic effect, of the peptide on the cardiac myocytes has not been reported, and its hypotensive action is partly attributed to the stimulation of parasympathetic and inhibition of sympathetic discharges into the heart. Moreover, the hypotensive effect of ANP is reduced markedly after vagatomy. In spite of the inhibitory effect of ANP on the sympathetic activity, it has been shown that intense activation of the sympathetic system resulted in a significant reduction in the level of the circulating ANP. The antagonistic relationship between ANP and the renin - angiotensin system in the central nervous system as well as in the periphery, affects the modulation of the body fluids, blood pressure and baroreflex responses. Hence, the

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cardiovascular effects ANP can summarized in the following points:

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1. Reduction in the peripheral resistance (afterload). 2. Reduction in the venous return and atrial filling pressure (preload). 3. Stimulation of the parasympathetic activity (vagi). 4-Reduction in the cardiac output All or most of these effects are secondary to inhibition or stimulation of the mechanisms regulating the cardiovascular system.

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Effects on the brain: The distribution of ANP and its receptors in the brain are localized in the regions identified to be intimately involved in the ECF volume and pressure homeostasis such as the supraoptic, paraventricular nuclei, nucleus of tractus solitareus, the area postrema and chnoroid plexus. It has been reported that intracerebroventricular injection of both ANP and BNP antagonize the central effects of angiotensin II. Moreover, they bind to the supraoptic and paraventricular nuclei and directly inhibit the AVP release. The regional distribution or BNP in procine brain, and highest concentrations of immunoreactive BNP (irBNP) were found in the hypothalamus, medulla, pons and spinal cord.

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