Alterations in binding sites for atrial natriuretic factor in kidneys and adrenal glands of Dahl hypertension-sensitive rats

Binding sites for atrial natriuretic factor (ANF) were studied in kidneys and adrenal glands of 7- and 10-week-old male Dahl hypertension-sensitive (S/JR) and hypertension-resistant (R/JR) rats by quantitative autoradiography. Binding sites for 125I-ANF-28 in kidney were highly localized and of high density in the glomeruli; binding sites were less concentrated in the renal papilla. In adrenal gland, binding sites for 125I-ANF-28 were highly concentrated in the zona glomerulosa, but were of a very low density in the inner adrenal cortex. At 7 weeks of age, the maximum binding capacity (Bmax) for 125I-ANF-28 in kidney glomeruli was increased by 21% in S/JR rats compared with R/JR rats. From 7 to 10 weeks of age, decreases in Bmax for 125I-ANF-28 in glomeruli occurred, with no apparent difference between strains. Strain or age differences in the affinity constant (Ka) for 125I-ANF did not occur in the kidney. In adrenal zona glomerulosa, the Bmax for 125I-ANF-28 binding was similar for S/JR and R/JR rats at 7 weeks of age. At 10 weeks of age, however, Bmax for 125I-ANF-28 in adrenal zona glomerulosa was increased by 19% in S/JR rats compared with age-matched R/JR controls. These findings suggest that alterations may occur in ANF binding sites in kidney and adrenal gland of S/JR rats in response to the sharp increase in blood pressure that is characteristic of rats of this strain.     

Localization of specific binding sites for atrial natriuretic factor in peripheral tissues of the guinea pig, rat, and human

Specific, high affinity atrial natriuretic factor (ANF) binding sites were identified and localized by autoradiographic techniques in peripheral tissues of the guinea pig, rat, and human. In the guinea pig kidney, high concentrations of ANF binding sites were located in the glomerular apparatus, outer medulla, and small renal arteries. Other peripheral tissues containing ANF binding sites included the zona glomerulosa of the adrenal cortex, the smooth muscle layer of the aorta and gallbladder, the lung parenchyma, the posterior lobe of the pituitary, the ciliary body of the eye, and the leptomeninges and choroid plexus of the brain. The distribution of ANF binding sites in the rat and human kidney was nearly identical to those seen in the guinea pig kidney; high concentrations were present in the glomerular apparatus, outer medulla, and small renal arteries. These results are consistent with earlier physiological and pharmacological studies that suggested that ANF plays a functional role in the regulation of extracellular fluid volume and blood pressure. There appears to be little species variation in the location and concentration of renal ANF binding sites, suggesting that, at least in the kidney, the results in experimental animals are relevant to the actions of ANF in humans. The finding that ANF binding sites were stable and present in high concentrations in human postmortem kidneys further suggests that these tissues may be amenable to testing for the involvement of ANF receptor dysfunction in diseases such as hypertension and congestive heart failure.     

Atrial natriuretic factor: Physiologic actions and implications in congestive heart failure

Summary Atrial natriuretic factor (ANF) represents a newly recognized hormone of cardiac origin. This peptide is synthesized by the myocardial cells of both atria and released by atrial stretch. The hormone promotes sodium and water excretion by the kidney, inhibits the renin-angiotensin-aldosterone system, and reduces systemic arterial pressure. Specific receptors for ANF are present in the kidney, adrenal glands, vascular smooth muscle, platelets and central nervous system. Congrestive heart failure is characterized by increased circulating levels of ANF; however, there appears to be an attenuation in the renal response to the hormone.Recent investigations have reported the effect of systemic administration of synthetic ANF to normal individuals and those with congestive heart failure. The hormone may promote a significant natriuresis and diuresis in addition to reducing arterial pressure and inhibiting renin and aldosterone secretion. Substantial questions remain as to the full physiologic significance and therapeutic potential of this hormone.     

Interaction of atrial natriuretic factor and osmoregulatory hormones in the Pekin duck

This study examined the influence of plasma atrial natriuretic factor (ANF) on the osmoregulatory hormones arginine vasotocin (AVT), angiotensin II (AII), aldosterone, and corticosterone in the blood of conscious Pekin ducks. Synthetic chicken ANF was iv infused at a nonhypotensive, natriuretic, and diuretic dose of 100 ng.kg-1.min-1 in normally hydrated ducks and birds in which the circulating levels of AVT, AII, aldosterone, and corticosterone were elevated by 24-hr dehydration. In normally hydrated animals the effect of ANF was limited to a reduction (P less than 0.05) in the basal concentration of aldosterone; in dehydrated birds both AII and aldosterone levels were reduced (P less than 0.05). ANF infusion was consistently without effect on plasma AVT or corticosterone concentrations. The iv infusions of AII with or without simultaneous infusion of ANF showed that ANF inhibited (P less than 0.05) the aldosterone response to AII. Autoradiographic and membrane binding techniques indicated that there were both AII and ANF receptors in the subcapsular zone of the duck adrenal gland and that ANF had no affinity for the AII binding sites. The results demonstrate that ANF is capable of modulating the duck angiotensin-aldosterone system and suggest a role for this peptide in avian salt and fluid homeostasis.     

Cyclic guanosine monophosphate responses to atrial natriuretic factor, brain natriuretic peptide, but not C-type natriuretic peptide, and the characterization of their receptors in rat medullary thick ascending limb

The effects of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on renal medullary thick ascending limb (mTAL) have not been fully understood. The aim of this study is to examine the second-messenger responses of rat mTAL to ANF, BNP, and CNP. Characterizations of the ANF, BNP, and CNP receptors in mTAL were also performed by radioligand studies. Results showed that ANF and BNP were both capable of eliciting cyclic guanosine monophosphate (cGMP) responses in mTAL. Conversely, no cGMP response was observed upon stimulation by CNP in mTAL. The presence of ANF receptors was demonstrated by radioligand studies. One receptor site was found, and the K_d and maximum binding capacity were 4.0 ± 0.45 nmol/L and 277.8 ± 47.7 fmol/mg protein, respectively. BNP receptors were also found in mTAL, and ANF and BNP were sharing the same receptor. On the contrary, no CNP receptor could be shown by radioligand studies. These results suggest that guanylyl cyclase-coupled receptors (atrial natriuretic peptide receptor-A [ANPR-A]) specific for ANF and BNP are present in rat mTAL, while those for CNP (ANPR-B) are absent. ANF and BNP but not CNP act on mTAL to control water excretion.     

Localization and quantification of atrial natriuretic factor binding sites in the kidney of Xenopus laevis.

Atrial natriuretic factor (ANF) binding sites in the skin, the bladder, and the kidney of the anuran amphibian Xenopus laevis were localized and quantified using quantitative in vitro autoradiography. Specific binding of 125I-rANF occurred only in the glomeruli and in the adrenal tissue of the kidney. The association of 125I-rANF binding was much higher than the dissociation and there was no steady state between the ligand and the binding sites. Scatchard and Hill's analyses of saturation experiments showed 125I-rANF to bind to heterogeneous sites with positive cooperativity. The effective concentrations, where 50% of maximal binding occurs (EC50), in glomeruli and in adrenal tissue were 75.7 +/- 8.5 and 74.7 +/- 12.1 pM (n = 8), respectively. The corresponding maximum binding capacities (Bmax) were 0.847 +/- 0.131 fM/mm2 in glomeruli and 1.161 +/- 0.179 fM/mm2 in adrenal tissue. Displacement studies have demonstrated the same affinity of these 125I-rANF binding sites to unlabeled rANF, hANF, and rAtriopeptin II, while 125I-labeled rANF had a much higher affinity. The N-terminal ANF fragment (99-109) and the C-terminal rANF fragment (116-126) had only weak displacing effects, whereas unrelated peptides did not alter the binding of 125I-rANF. The osmotic stress of acclimation to 1.5% salt water increased renal but not adrenal ANF binding.     

The heart, an endocrine gland

Two independent series of biomedical investigations have led to the discovery that the atria are a peptide-secreting endocrine gland. The first is mainly morphological and starts with the finding that mammalian atrial but not ventricular cardiocytes contain "dense bodies". These "dense bodies" later called "specific granules" were found to be different from lysosomes, to be made up of proteins and to incorporate both 3H-leucine and 3-H-fucose in a pattern typical of peptide-secreting endocrine cells. The finding that rat atrial granulation varied with the sodium and water balance led to the crucial observation that atrial extracts have natriuretic and diuretic effects. In less than 4 years, this new natriuretic hormone has been purified, sequenced and synthetized, and its cDNA and gene have been cloned. The ANF gene has been assigned to the distal short arm of chromosome 1 in band 1P36 while the mouse gene is localized in chromosome 4. The native and synthetic hormones exert identical wide ranging effects (possibly through particulate guanylate cyclase stimulation and adenylate cyclase inhibition) on the kidney, blood vessels, adrenal cortex and pituitary. Physiopathologic implications of the hormone in experimental hypertension, congestive heart failure and expansion of blood volume are already beginning to emerge. On the other hand, the search for natriuretic hormones or factors by studies of negative pressure breathing, atrial distention experiments, head-out water immersion, expansion of blood volume, Na+/K-ATPase inhibition and parabiosis experiments in Dahl rats has provided a general framework within which to interpret this new cardiac function.     

Central administration of atrial natriuretic factor inhibits saline preference in the rat

Atrial natriuretic factors (ANFs), produced in myocytes of mammalian atria, exert potent natriuretic and diuretic actions in the kidney as well as a variety of other actions coordinated to normalize extracellular fluid volume. Recently, ANF-like immunoreactivity has been detected in the hypothalamus of the rat, and central administration of ANF has been shown to block dehydration-induced water intake. We describe here the ability of 0.2 and 2.0 nmol atriopeptin III to inhibit saline intake when infused into the third ventricle of conscious, salt-depleted rats; an effect that was dose-related and long-lasting (24 h). These studies provide further evidence for a central nervous system action of ANF, which, together with its established renal and adrenal actions, might be an important feature of the coordinated physiological control of fluid volume.     

Evolution of lactogen receptors in selected rabbit tissues during pregnancy

The ontogeny of lactogen receptors in brain, adipose, liver, kidney, adrenal gland, mammary gland, ovarian and uterine tissues of pregnant rabbits was evaluated in this study using 125I bovine prolactin as tracer. Brain and adipose tissues were found to have very low receptor numbers throughout pregnancy (less than 20 fmol/mg of protein), while liver and kidney had higher but constant levels of receptor through the same period (200 and 100 fmol/mg of protein, respectively). Mammary gland and adrenal gland tissues exhibited sharp increases in prolactin binding between 15 and 17 days with both having peak receptor binding at 17 days of around 200 fmol/mg of protein. Ovarian and uterine receptor binding increased slowly after day five of pregnancy and reached peak levels of approximately 225 fmol/mg of protein at day 20. Scatchard analysis of the binding of protein in the tissues having increased binding during the course of pregnancy, revealed that its affinity for sites in these tissues was the same at 5 and 20 days of gestation, indicating the rise in binding to be a result of increased numbers of available receptors. Sub-organ localization studies found the binding of prolactin to adrenal gland, ovary and uterus to be essentially located in adrenal cortex, nonluteal ovary and endometrium. Incubation of membranes from each of the tissues showing significant change during pregnancy, from several time points of pregnancy, with 5.0 M MgCl2 produced little change in apparent receptor numbers; suggesting that receptor occupancy levels of endogenous prolactin was low.     

Specific membrane receptors for atrial natriuretic factor in renal and vascular tissues.

Membranes from rabbit aorta and from rabbit and rat kidney cortex possess high-affinity (Kd = 10(-10) M) specific binding sites for atrial natriuretic factor (ANF). Similar high-affinity sites are present in an established cell line from pig kidney, LLC-PK1. Results of fractionation studies indicate that the receptors are localized in the plasma membrane of these tissues. The binding is time-dependent and saturable. An excellent quantitative correlation was found between the affinity of synthetic ANF and analogs of intermediate activity to aorta membranes and the half-maximal concentration needed for relaxation of rabbit aorta rings contracted by addition of serotonin. Furthermore, the binding affinity of the receptor in kidney membranes is consistent with the concentration required for in vivo natriuresis in the rat. Biologically inactive synthetic ANF fragments and other peptide hormones such as angiotensin II and vasopressin do not significantly inhibit binding. These data suggest that the receptors for ANF in vascular and renal tissues are responsible for mediating the physiological actions of this peptide in these target tissues.     

Specific binding of atrial natriuretic factor in brain microvessels

Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. We examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using 125I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity (dissociation constant, approximately 10(-10) M) and with a binding capacity of 58 fmol/mg of protein. The binding of 125I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function.     

Atrial natriuretic factor: specific binding to renal glomeruli during the development of two-kidney, one clip hypertension in the rat

Blood pressure (BP), atrial and plasma concentrations of atrial natriuretic factor (ANF), hematocrit and renal glomerular ANF receptors were studied during the development of hypertension in two-kidney, one clip (2-K, 1C) rats and were compared with normotensive controls. Plasma ANF was elevated in the 2-K, 1C group in all stages of hypertension, even after 3 weeks when BP, although higher than in sham-operated animals, had not yet reached arbitrarily-set hypertensive levels. At this time, hematocrit was higher in the hypertensive rats than in the controls, but the difference later disappeared. Lower atrial ANF concentrations were observed in the 2-K, 1C group at week 3, but only in the right atrium. No difference in ANF levels was noted in either the left or right atrium between hypertensive and normotensive animals 5 and 7 weeks after clipping. The glomerular ANF receptor population was markedly smaller in the clipped left kidney of 2-K, 1C rats during all stages of hypertension, and in the untouched right kidney at 5 and 7 weeks after surgery, but was larger in the non-clipped right kidney in the pre-hypertensive phase (3 weeks). It is concluded that receptor density changes during the evolution of high BP in the 2-K, 1C Goldblatt model of experimental hypertension. Our data suggest that the increases and decreases in density of renal glomerular ANF receptors may play a role in the differential handling of sodium by the clipped and non-clipped kidney during the various stages of development of 2-K, 1C hypertension in the rat.     

Tyrosine Hydroxylase Activity in the Brain and Adrenal Gland of Rats Following Chronic Administration of Ethanol

The effects of chronic administration and ethanol withdrawal on the activity of tyrosine hydroxylase were examined in the adrenal gland and six brain regions, including the frontal cortex, hippocampus, locus coeruleus, striatum, substantia nigra, and hypothalamus. Ethanol was administered to rats by oral intubation every 8 hr for 48 hr in amounts based upon their state of intoxication. One hr after the last intubation, tyrosine hydroxylase activity was not significantly different from control values in either the adrenal gland or brain. Forty hr following the last ethanol intubation, tyrosine hydroxylase activity was significantly increased above control values in both the adrenal gland and locus coeruleus. Tyrosine hydroxylase activity in the remaining five brain areas was unaffected by ethanol treatment. Immunotitration studies indicate that the increases in tyrosine hydroxylase activity found in the adrenal gland and locus coeruleus 40 hr after ethanol administration were due to an increase in enzyme protein. These data indicate that high blood ethanol concentrations maintained over a period of time (48 hr) do not affect adrenal gland or brain tyrosine hydroxylase activity. However, withdrawal from ethanol following 48 hr of treatment does produce an increase in tyrosine hydroxylase activity in the adrenal gland and locus coeruleus, similar to that seen following other stresses.     

Atrial natriuretic factor inhibits the stimulation of aldosterone secretion by angiotensin II, ACTH and potassium in vitro and angiotensin II-induced steroidogenesis in vivo

Since atrial natriuretic factor (ANF) blocks the contractile effect of angiotensin II on vascular strips, we investigated the action of the synthetic 48-73 ANF (previously called 8-33 ANF) on another target tissue of angiotensin II, the adrenal glomerulosa. ANF did not affect basal aldosterone output by isolated rat adrenal glomerulosa cells. ANF inhibited aldosterone secretion stimulated by 10(-8)M angiotensin II with an IC50 of 1.3 X 10(-9)M. Aldosterone secretion stimulated by 2.9 X 10(-10)M ACTH and by 15 mM potassium was similarly inhibited by ANF. In vivo, ANF blocked the effect of angiotensin II infused iv on aldosterone secretion in conscious unrestrained rats. We conclude that ANF is a non-selective inhibitor of stimulated aldosterone output.     

The genetic contribution of the natriuretic peptide system to cardiovascular diseases

Three types of natriuretic peptides (NP) have been isolated: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The NP family elicits a number of vascular, renal and endocrine effects that help to maintain blood pressure and extracellular fluid volume. These effects are mediated by the specific binding of NP to cell surface receptors that have been characterized, purified and cloned from cells of the vasculature, kidney, adrenal gland and brain. There are 3 subtypes of NP receptors: type A natriuretic peptide receptor (NPRA), type B natriuretic peptide receptor (NPRB), and type C natriuretic peptide receptor (NPRC). All 3 subtypes affect cellular second messenger activity. NPRA and NPRB are guanylyl cyclase receptors, and their activation increases cGMP levels. Activation of NPRC results in inhibition of adenylyl cyclase activity. Human NPRA has a high structural homology with human NPRB, and contains a highly-conserved guanylyl cyclase domain. ANP and BNP bind primarily to NPRA, which is found in the vasculature, causing vasodilation and inhibition of vascular smooth muscle cell proliferation. The present paper contains a review of NPs and their receptors and the genetic contribution of the NP system to cardiovascular diseases such as essential hypertension and myocardial infarction.     

Distribution and characterization of natriuretic peptide receptors in the kidney of the toad, Bufo marinus.

The location and characteristics of atrial natriuretic peptide binding sites in the kidney of the toad, Bufo marinus, were determined. Specific (125)I-rANP binding sites were observed on glomeruli and blood vessels, but little if any binding was observed over regions corresponding to the renal tubules. (125)I-rANP binding in tissue sections and/or isolated membranes was completely displaced in the presence of 1 microM rat ANP, frog ANP, and porcine C-type natriuretic peptide (membranes only); however, residual binding remained after incubation with 1 microM of the NPR-C ligand, C-ANF, indicating the presence of two distinct binding sites. Electrophoresis of kidney membranes cross-linked to (125)I-rANP identified specific bands at approximately 70 and 140 kDa which correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP production rates in membrane preparations, while C-ANF had no stimulatory effect. Two partial cDNA clones generated using primers based on conserved regions of vertebrate natriuretic peptide receptors showed high homology to an NPR-C and the natriuretic peptide guanylate cyclase receptors (NPR-GC), respectively. This study provides evidence that the kidney of B. marinus contains both NPR-C and NPR-GC and that the glomerulus is potentially the principal site of ANP regulation in the kidneys.     

Dehydration-induced alterations in rat brain vasopressin and atrial natriuretic factor immunoreactivity

Potent natriuretic and spasmolytic peptides present in cardiac extracts recently have been identified. These atrial natriuretic factors (ANF) exert vascular and renal actions quite contrary to those of vasopressin (AVP). The ability of ANF to inhibit AVP secretion suggested a role for the peptides in the control of AVP release. The present studies report the measurement of ANF-like immunoreactivity within brain regions associated with the hypothalamo-neurohypophyseal tract and demonstrate significant water deprivation-induced reductions in ANF content of several structures (neural lobe, organum vasculosum lamina terminalis, suprachiasmatic and supraoptic nuclei) but not in others (median eminence, paraventricular nucleus, cortex and pituitary). The data suggest the production of ANF-like peptides within the brain and, further, the involvement of central ANF in extracellular fluid volume regulation.     

Renal response to acute volume overload in conscious rats with atrial appendectomy

The presence of volume receptors and a potent natriuretic factor (ANF) in mammalian cardiac atria strongly suggests a central role of the atria in extracellular fluid volume regulation. ANF is stored within granules in atrial appendages, and their removal could alter the response to volume overload. We tested this hypothesis in conscious Wistar rats two weeks after sham operative or atrial appendectomy. The results indicate that removal of the atrial appendages significantly reduced their urinary excretion of water, sodium and potassium during the first hour following acute volume overload. It is concluded that atrial appendectomy alters the ability of rats to handle acute volume overload possibly through a reduction in the ANF available for release.     

Vascular atrial natriuretic factor receptors in spontaneously hypertensive rats.

OBJECTIVE: The aim was to investigate vascular receptors for atrial natriuretic factor (ANF) in spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Wistar rats (WR) at different ages. METHODS: Relaxation and guanylate cyclase responses of blood vessels to atrial natriuretic factor were investigated, as was the binding of 125I-ANF to vascular membranes and ANF receptor subtypes, using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in reducing conditions, after solubilisation and irreversible binding of 125I-ANF. RESULTS: Vascular relaxation responses of aorta showed an increased sensitivity to ANF in four week old SHR [pD2 = 8.9 (SEM 0.1) v 8.5(0.1) in WKY rats, p < 0.05] while sensitivity was similar for the three strains at older ages. Production of cyclic GMP in mesenteric arteries in response to 100 nmol.litre-1 ANF was greater (p < 0.05) in SHR than in WKY rats at four weeks of age, but was similar in older rats. The density of binding sites for ANF in mesenteric arteries, however, was lower in SHR at four weeks (p < 0.01), and increased in older rats, becoming similar to that of normotensive rats at 12 weeks of age. Affinity of ANF sites was similar in all strains. The proportion of high and low molecular weight ANF binding peptides in solubilised blood vessel membranes on SDS-PAGE was similar in all strains except in four week old SHR, in which binding to the high molecular weight band (presumably the guanylate cyclase containing receptor) was increased relative to the low molecular weight band (non-cyclase-coupled receptor) in comparison to other strains and ages. CONCLUSIONS: Activity of guanylate cyclase in response to occupancy of ANF receptors may be increased in young SHR. Normal relaxation of blood vessels in response to ANF in older SHR could result in failure to counteract the increased vasoconstrictor activity present in these rats, which could play a role in the increase in blood pressure.