Effect of atrial natriuretic factor on plasma vasopressin in conscious rats

An intravenous (IV) bolus injection (10 μg) of synthetic rat atrial natriuretic factor [ANF (Arg 101-Tyr 126)] into normal conscious Sprague-Dawley rats produced a significant decrease of plasma arginine vasopressin (AVP) while 1-, 2-, and 5-μg doses exerted no such effect. Mean arterial blood pressure (MAP) was lowered about 15 mmHg by an IV 10 μg bolus injection of ANF. When plasma AVP rose significantly in rats exposed to such osmotic stimuli as 600 mM NaCl and 900 mM mannitol intraperitoneally (IP), subsequent IV injection of ANF (10 μg) markedly depressed this parameter. Lower doses of ANF were ineffective against 600 mM NaCl IP. The significant elevation of plasma AVP levels by hypertonic sucrose 900 mM IP was not modified by ANF (10 μg). Blood pressure remained unchanged after IP administration of various osmotic stimuli, except mannitol, and in all these experiments an IV bolus of ANF exerted a lowering effect on MAP. Seventy-two hr water deprivation (mixed osmotic and volume stimulus) resulted in elevated plasma AVP levels which were unaffected by an IV bolus injection of ANF at doses of 0.06–10 μg. Immunoreactive ANF (IR-ANF) rose in plasma to 39.3±13 ng/ml 1 min after an IV bolus injection of 10 μg ANF, dropping to 1.01±0.2 ng/ml after 5 min and to 0.32±0.01 ng/ml after 10 min (when ANF and AVP interactions were studied), but still remained approximately six times higher than in control rats. These results suggest that, in the conscious rat, only pharmacological levels of ANF observed after an IV bolus infusion may influence both resting and osmotically-stimulated AVP levels.     

B and C Types Natriuretic Peptides Modify Norepinephrine Uptake and Release in the Rat Adrenal Medulla

Vatta, M. S., M. F. Presas, L. G. Bianciotti, M. Rodriguez–fermepin, R. Ambros and B. E. Fernandez. B and C types natriuretic peptides modify norepinephrine uptake and release in the rat adrenal medulla. Peptides 18(10) 1483–1489, 1997.—We have previously reported that atrial natriuretic factor (ANF) modulates adrenomedullar norepinephrine (NE) metabolism. On this basis, the aim of the present work was to study the effects of B and C types natriuretic peptides (BNP and CNP) on the uptake, intracellular distribution and release of ³H-NE. Experiments were carried out in rat adrenal medulla slices incubated “in vitro.” Results showed that 100 nM of both, CNP and BNP, enhanced total and neuronal NE uptake. Both peptides (100 nM) caused a rapid increase in NE uptake during the first minute, which was sustained for 60 min. NE intracellular distribution was only modified by CNP (100 nM), which increased the granular fraction and decreased the cytosolic pool. On the other hand, spontaneous as well as evoked (KCl) NE release, was decreased by BNP and CNP (50 and 100 nM for spontaneous release and 1, 10, 50 and 100 nM for evoked output). The present results suggest that BNP and CNP may regulate catecholamine secretion and modulate adrenomedullary biological actions mediated by catecholamines, such as blood arterial pressure, smooth muscle tone, and metabolic activities.     

Bile secretion is centrally regulated by C-type natriuretic peptide

1. Current evidence supports that C-type natriuretic peptide (CNP) is the brain natriuretic peptide. Natriuretic peptide receptors and mRNA CNP have been reported in the liver and in discrete areas and nucleus of the central nervous system involved in the regulation of gastrointestinal physiology. In the present work, we sought to establish the role of CNP in the central regulation of bile secretion in the rat and to delineate the possible pathways and mechanisms involved.2. To examine the role of CNP on bile secretion, the peptide was applied in the brain lateral ventricle (1, 10, and 100 ng/L) and bile samples were collected every 15 min for 60 min. The role of the autonomic nervous system in CNP response was assessed by atropine or combined phentolamine and propranolol administration.3. Centrally applied CNP diminished basal as well as bile salt-evoked bile flow in a dose-dependent manner. CNP reduced bile acid output as well as sodium and potassium excretion, supporting CNP effect on bile acid-dependent flow. CNP also decreased chloride excretion and increased bile pH. The excretion of total glutathione was not affected by centrally applied CNP suggesting that this peptide does not alter bile acid-independent flow. Neither parasympathetic nor sympathetic blockade abolished CNP inhibitory response on bile secretion. Mean arterial pressure and portal venous pressure were not modified by CNP.4. Present findings show that centrally applied CNP modulates bile secretion in a dose-dependent fashion. CNP alkalinized bile and reduced bile acid-dependent flow without affecting bile acid-independent flow. The inhibitory response of CNP on bile secretion was not mediated by the autonomic nervous system. Present findings give further support to the role of CNP as the brain natriuretic peptide.     

Increases in heart rate and blood pressure produced by microinjections of atrial natriuretic factor into the AV3V region of rat brain

Recent studies have provided evidence for the dense localization of atrial natriuretic factor (ANF) in the anteroventral third ventricle (AV3V) region of the rat brain. This area is currently thought to be involved in the regulation of blood pressure and fluid and electrolyte balance. To investigate whether ANF may play a role in central cardiovascular regulation, the effects of microinjection of ANF into the preoptic suprachiasmatic nucleus (POSC), which is located in the AV3V region of the brain, were examined in the present study. Low doses of ANF (2–4 pmol) produced modest elevations in systolic and diastolic pressures, approximately 10–14%, and a small rise in HR of roughly 7%. Higher doses of ANF (20–40 pmol) produced significant increases in systolic (15–19%), mean arterial (12–14%) and pulse (25–36%) pressures. In addition, much larger increases in HR, approximately 20%, were produced by these higher doses of ANF. The onset of effects produced by ANF on BP and HR was seen 15–45 min after injection. Peak effects were usually observed approximately 60–150 min after onset, and the duration of the effect was 2–4 hours, after which time values usually returned to baseline. These studies indicate that ANF produces significant increases in BP and HR when injected at pmol doses into the POSC, and lends support to the idea that this peptide may play an important role in central cardiovascular regulatory mechanisms.     

Centrally applied atrial natriuretic factor diminishes bile secretion in the rat.

Little is known about the role of centrally applied peptides in the regulation of bile secretion. We previously reported that the intravenous injection of atrial natriuretic factor (ANF) reduces bile acid dependent flow without affecting portal venous pressure in the rat. In the present work, we studied the effects of centrally applied ANF on bile secretion and the possible pathways involved. Rats were cannulated in the brain lateral ventricle for the administration of 1, 10 and 100 ng/microl ANF. After 1 week, the common bile duct was cannulated and bile samples were collected every 15 min for 60 min after the administration of ANF. The excretion rate of various biliary components was assessed. Bile secretion experiments were also performed after bilateral truncal vagotomy or atropine administration to evaluate the participation of a vagal pathway. In addition, the role of the sympathetic system was addressed by combined administration of propranolol and phentolamine. Centrally applied ANF did not modify blood pressure but diminished bile flow and bile acid output. It also reduced sodium and potassium secretion but did not modify protein or phospholipid excretion. Neither bilateral truncal vagotomy nor atropine administration abolished ANF response. Furthermore, combined administration of adrenergic antagonists did not alter ANF inhibitory effect on bile flow. In conclusion, centrally applied ANF reduced bile acid dependent flow not through a vagal or adrenergic pathway in the rat, suggesting the involvement of a peptidergic pathway.     

High salt intake-induced changes in atrial natriuretic factor kinetics are mediated by clearance receptors.

We have reported a paradoxical plasma atrial natriuretic factor (ANF) decline following prolonged high salt intake that was attributed to an increased tissue uptake of circulating ANF, leading to its augmented distribution volume (Vas) and metabolic clearance rate (MCR) as compared with control rats on a standard diet. To explore this phenomenon further, we evaluated possible chronic salt-loading-induced changes in ANF clearance (C-ANF) receptors, which appear to play a major role in ANF removal from the circulation. We studied changes in plasma [125I]ANF(1-28) and its pharmacokinetics after preoccupation of C-ANF receptors by its specific ligand, C-ANF(4-23), in high-salt-treated rats and their controls. Following C-ANF(4-23) administration, we detected significantly higher circulating [125I]ANF levels throughout the study period (8 min) in high-salt-fed rats compared with the controls (280-470% vs 100-215% increase of basal values, P less than 0.05). C-ANF(4-23) infusion caused a significantly greater decrease of the metabolic clearance rate and distribution volume of [125I]ANF in high-salt-fed rats than in control animals (74 +/- 6% vs 41 +/- 6% and 75 +/- 4% vs 50 +/- 5% of basal values, respectively; P less than 0.05). These data suggest that a prolonged high salt diet may increase the availability of C-ANF receptors and, through this mechanism, may negatively modulate plasma ANF concentrations. C-ANF receptors may thus fulfill a regulatory function on circulating ANF during prolonged salt loading in rats.     

Atrial natriuretic factor inhibits the CRH-stimulated secretion of ACTH and cortisol in man.

Corticotrophic secretion of ACTH is stimulated by corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and suppressed by glucocorticoids. In vitro and preclinical studies suggest that atrial natriuretic factor (ANF) may be a peptidergic inhibitor of pituitary-adrenocortical activity. The aim of this study was to elucidate a possible role of ANF as a modulator of ACTH release in humans. A bolus injection of 100 micrograms human CRH (hCRH) during a 30 min intravenous infusion of 5 micrograms/min human alpha atrial natriuretic factor (h alpha ANF) was administered at 19:00 to six healthy male volunteers. In comparison to saline, a blunted CRH-stimulated secretion of ACTH (mean maximum plasma level +/- SD 45 min after hCRH: saline 46.2 +/- 14.2 pg/ml, h alpha ANF 34.6 +/- 13.8 pg/ml, p-value = 0.007) and a delayed rise (10 min) in cortisol were detected. The maximum plasma cortisol levels remained nearly unchanged between saline and h alpha ANF administration (mean maximum plasma level +/- SD 60 min after hCRH: saline 182 +/- 26 ng/ml, h alpha ANF 166 +/- 54 ng/ml). No effects of h alpha ANF on basal cortisol levels were observed; in contrast, basal ACTH plasma levels were slightly reduced. Basal blood pressure and heart rate remained unaffected. In the control experiment, infusion of 3 IU AVP in the same experimental paradigm increased basal and stimulated ACTH and cortisol levels significantly in comparison to saline. These observations suggest that intravenously administered haANF inhibits the CRH-stimulated release of ACTH in man.     

The effect of hypovolemia on the renal and cardiovascular responses to atrial natriuretic factor (ANF) infusion.

The renal and cardiovascular effects of ANF infusion have been examined in separate series of experiments; in conscious instrumented sheep following either hemorrhage (10 mL/kg body weight) or removal of 500 mL of plasma by ultrafiltration. Renal arterial infusion of hANF (99-126) at 50 micrograms/h increased sodium excretion from 99 +/- 30 to 334 +/- 102 (p less than 0.05) in normal animals, and from 77 +/- 31 to 354 +/- 118 mumol/min in hemorrhaged animals. Similarly in sheep following ultrafiltration, cardiac output and stroke volume were reduced by intravenous infusion of ANF (100 micrograms/h), although these effects were less marked than those observed in normal animals. The rapid modulation of natriuretic responses to ANF observed in volume expanded animals is not seen in this model of acute volume depletion suggesting that the mechanism through which the renal response to ANF is modulated in low sodium or volume states is not simply the reverse of that which produces rapid enhancement of response following blood volume expansion.     

Atrial natriuretic factor is a circulating hormone

The radioimmunoassay of atrial natriuretic factor (ANF) has been applied for determination of immunoreactive ANF (IR-ANF) in rat plasma. Immunoreactive ANF has been extracted from rat plasma by immunoaffinity column on Sepharose-4B anti-ANF or by Vycor glass. The mean concentrations of IR-ANF in ether anesthetized rats were found to be 1.61 +/- 0.14 ng/ml in female and 1.25 +/- 0.21 ng/ml in male rats when extracted on Sepharose-4B anti-ANF, and 1.21 +/- 0.10 ng/ml in females and 1.02 +/- 0.11 ng/ml in males when extracted by Vycor glass. A close linear correlation has been observed between the plasma IR-ANF concentrations in aorta and jugular vein. The described results indicate that atrial cardiocytes secrete atrial natriuretic factor into plasma. The heart is, therefore, an endocrine organ.     

Effects of synthetic atrial natriuretic factor (ANF) on renin-aldosterone axis in the conscious newborn calf

The effects of synthetic atrial natriuretic factor (ANF) on the renin-aldosterone axis were studied in fifteen 4-7 day-old male milk-fed calves divided into 3 groups of 5 animals each. Synthetic ANF intravenous (i.v.) administration (1.6 micrograms/kg body wt over 30 min) induced a transient significant fall in plasma renin activity (from 2.5 +/- 0.3 to 1.7 +/- 0.3 ng angiotensin l/ml/h; P less than 0.05) but failed to reduce basal plasma aldosterone levels in the first group of animals. Administration (i.v.) of angiotensin II (AII) (0.8 micrograms/kg body wt for 75 min) was accompanied by a progressive fall in plasma renin activity (from 2.2 +/- 0.3 to 0.8 +/- 0.1 ng angiotensin l/ml/h; P less than 0.01) and by an increase in plasma aldosterone levels (from 55 +/- 3 to 86 +/- 5 pg/ml; P less than 0.01) both in the second and the third groups; addition of ANF to AII infusion (AII: 0.5 mu/kg body wt for 45 min; AII: 0.3 micrograms/kg body wt and ANF 1.6 micrograms/kg body wt during 30 min) in the third group did not modify plasma renin activity or AII-stimulated plasma aldosterone levels when compared to the AII-treated group. These findings show that in the newborn calf ANF is able to reduce plasma renin activity but fails to affect basal and AII-stimulated plasma aldosterone levels, suggesting that the zona glomerulosa of the newborn adrenal cortex is insensitive to a diuretic, natriuretic and hypotensive dose of the atrial peptide.     

The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.     

Atrial natriuretic factor is unlikely to be involved in the reduced aldosterone production in the Brattleboro rat

We have previously reported that basal and stimulated aldosterone production in Brattleboro rat (DI) lacking hypothalamic arginine vasopressin is lower than that observed in control Long-Evans rat (LE). In the present study, we investigated the secretion under various experimental conditions, adrenal binding sites, and the aldosterone-inhibiting effect of atrial natriuretic factor (ANF). In the conscious resting state, the plasma ANF concentration was similar between LE and DI rats. Pentobarbital anaesthesia (5 mg/100 g body wt.) reduced the plasma ANF concentration equally in both groups, with or without captopril pretreatment. Morphine (10 mg/100 g body wt.) increased ANF secretion dramatically and equally in the two groups of pentobarbital anaesthetized (2 mg/100 g body wt.) rats. In dexamethasone pretreated-pentobarbital anaesthetized rats, a concurrent i.v. ANF infusion (50 ng/min) did not change significantly the corticosterone response to ACTH (1-24) (1 mI.U./100 g body wt.) but steeply depressed ACTH-induced aldosterone production to a similar extent between DI and LE rats. A single class of adrenal ANF receptor sites was found with a similarity in high affinity and maximum binding capacity between the two groups of rats. Taken together, these results suggest that the reduced aldosterone production by Brattleboro rat adrenals is unlikely to be related to the inhibitory effect of ANF.     

Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus

We have previously reported that atrial natriuretic factor (ANF) increased neuronal norepinephrine (NE) uptake and reduced basal and evoked neuronal NE release. Changes in NE uptake and release are generally associated to modifications in the synthesis and/or turnover of the amine. On this basis, the aim of the present work was to study ANF effects in the rat hypothalamus on the following processes: endogenous content, utilization and turn-over of NE; tyrosine hydroxylase (TH) activity; cAMP and cGMP accumulation and phosphatidylinositol hydrolysis. Results showed that centrally applied ANF (100 ng/microl/min) increased the endogenous content of NE (45%) and diminished NE utilization. Ten nM ANF reduced the turnover of NE (53%). In addition, ANF (10 nM) inhibited basal and evoked (with 25 mM KCl) TH activity (30 and 64%, respectively). Cyclic GMP levels were increased by 10 nM ANF (100%). However, neither cAMP accumulation nor phosphatidylinositol breakdown were affected in the presence of 10 nM ANF. The results further support the role of ANF in the regulation of NE metabolism in the rat hypothalamus. ANF is likely to act as a negative putative neuromodulator inhibiting noradrenergic neurotransmission by signaling through the activation of guanylate cyclase. Thus, ANF may be involved in the regulation of several central as well as peripheral physiological processes such as cardiovascular function, electrolyte and fluid homeostasis, endocrine and neuroendocrine synthesis and secretion, behavior, thirst, appetite and anxiety that are mediated by central noradrenergic activity.     

Atrial natriuretic factor and renal sodium excretion during ventilation with PEEP in hypervolemic dogs.

Controlled mandatory ventilation with positive end-expiratory pressure (PEEP) reduces renal sodium excretion. To examine whether atrial natriuretic factor (ANF) is involved in the renal response to alterations in end-expiratory pressure in hypervolemic dogs, experiments were performed on anesthetized dogs with increased blood volume. Changing from PEEP to zero end-expiratory pressure (ZEEP) increased sodium excretion by 145 +/- 61 from 310 +/- 61 mumol/min and increased plasma immunoreactive (ir) ANF by 104 +/- 27 from 136 +/- 21 pg/ml. Changing from ZEEP to PEEP reduced sodium excretion by 136 +/- 36 mumol/min and reduced plasma irANF by 98 +/- 22 pg/ml. To examine a possible causal relationship, ANF (6 ng.min-1.kg body wt-1) was infused intravenously during PEEP to raise plasma irANF to the same level as during ZEEP. Sodium excretion increased by 80 +/- 36 from 290 +/- 78 mumol/min as plasma irANF increased by 96 +/- 28 from 148 +/- 28 pg/ml. We conclude that alterations in end-expiratory pressure lead to great changes in plasma irANF and sodium excretion in dogs with increased blood volume. Comparison of the effects of altering end-expiratory pressure and infusing ANF indicates that a substantial part of the changes in sodium excretion during variations in end-expiratory pressure can be attributed to changes in plasma irANF.     

Microvascular effects of atrial natriuretic peptide in rat cremaster

Experiments utilized the open cremaster preparation to test the hypothesis that atrial natriuretic peptide (ANP)-induced volume changes result from microvascular resistance alterations. Atrial natriuretic peptide (25, 100, and 500 ng/kg/min, IV) or vehicle was infused into anesthetized rats. At the two highest ANP infusion rates, mean arterial pressure was significantly reduced from 104 ± 3 (control) to 87 ± 2 and 77 ± 2 mmHg, respectively. Hematocrit was 41.0 ± 0.8 and 45.6 ± 0.9% (p < 0.05) at the end of vehicle and ANP infusions, respectively. Despite these effects of ANP, there were no significant arteriolar or venular diameter alterations. Thirty μM nitroprusside significantly dilated all vessel segments except large venules. These observations suggest that resistance alterations in the skeletal muscle microvasculature are not the cause of ANP-induced fluid movement.     

Role of spleen in ANF-induced reduction in plasma volume.

Atrial natriuretic factor (ANF) causes an increase in hematocrit that cannot be accounted for by urinary losses. The mechanism behind this phenomenon was studied in intact and splenectomized rats. Rat ANF 99-126 was infused i.v. for 30 min into conscious rats at rates of 0 (saline control), 0.05, or 0.1 microgram/min. Plasma volume was then determined by dilution of the dye, Evan's Blue. In one group of rats, red cell volume was determined using 51Cr-labelled erythrocytes. ANF infusion was continued uninterrupted throughout the experiments. In the intact rats, ANF (0.10 microgram/min) caused hematocrit to increase from 38.9 +/- 0.5 to 41.2 +/- 0.4% (p < 0.005). Splenectomy so attenuated this response to ANF that it failed to reach significance. Similarly, ANF (0.10 microgram/min) caused plasma volume to fall from 5.1 +/- 0.1 to 4.5 +/- 0.1 mL/100 g body wt. (p < 0.005) in the intact rats, but did not affect plasma volume in the splenectomized rats. As a result, blood volume was significantly reduced by ANF in the intact rats, but remained unchanged in the splenectomized rats. Red cell volume did not change in response to infusion of ANF, nor did ANF affect the rate of clearance of Evan's Blue out of the plasma. It is concluded that the spleen is an important site of movement of protein-poor fluid out of the vasculature, and that this exchange is influenced by ANF.     

Cardiac natriuretic peptides and pancreatic islet blood flow in anesthetized rats.

The aim of the study was to evaluate effects of cardiac natriuretic peptides on splanchnic circulation, especially to the pancreatic islets. Pentobarbital-anesthetized rats were infused intravenously (0.01 ml/min for 20 min) with saline, atrial natriuretic peptide (ANP; 0.25 or 0.5 microg/kg BW/min), brain natriuretic peptide (BNP; 0.5 microg/kg BW/min) or C-type natriuretic peptide (CNP; 0.5 or 2.0 microg/kg BW/min). Splanchnic blood perfusion was then measured with a microsphere technique. Mean arterial blood pressure was decreased by ANP and BNP, but not by CNP. The animals given the highest dose of ANP became markedly hypoglycemic, whilst no such effects were seen in any of the other groups of animals. Total pancreatic blood flow was decreased by the highest dose of CNP, whereas no change was seen after administration of the other peptides. Islet blood flow was increased by the highest dose of ANP. Neither BNP nor CNP affected islet blood flow. None of the natriuretic peptides influenced duodenal, colonic or arterial hepatic blood flow. It is concluded that cardiac natriuretic peptides exert only minor effects on splanchnic blood perfusion in anesthetized rats. However, islet blood perfusion may be influenced by ANP.     

Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis

Although recent major advances have developed a much better understanding of the pathophysiological pathways, tubulointerstitial fibrosis (TIF) is still currently incurable. Therefore, early detection may mean that the condition is more manageable than it was in the past. C-type natriuretic peptide (CNP) has been found to be a potent vasodilator but a weak natriuretic factor. In addition, CNP has also been believed to be produced in tubular cells and presented as a local modulator with anti-inflammatory and anti-proliferative effects. Elimination of CNP occurs by three main mechanisms, neutral endopeptidase, natriuretic peptide receptor-C and urinary excretion. Among them, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. In the present study, subgroups of rats were subjected to unilateral ureteral obstruction (UUO) or sham operation and observed for 24 h to 3 months. Urinary CNP excretion was significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to sham-operated rats. Urinary CNP excretion was also markedly higher than CNP concentrations both in abdominal aorta and in renal vein, and almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin. Urinary CNP excretion was negatively correlated with urinary protein concentration, blood urea nitrogen and creatinine, while positively correlated with albumin. In conclusion, the increased urinary CNP excretion is strongly associated with TIF progression, and may serve as an early marker of TIF.     

Cloning and functional expression of the bovine natriuretic peptide receptor-B (natriuretic factor R1c subtype)

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED₅₀ of 12±2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a K_d of 13±1 pM, and natriuretic peptides compete for [¹²⁵I]-CNP binding with a rank order of pCNP>pBNP>rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.Abbreviations BSA bovine serum albumin - dNTP deoxynucleotide triphosphate - SDS sodium dodecyl sulfate - DEAE-dextran diethylaminoethyl-dextran - EDTA ethylenediamine tetraacetic acid - Tris Tris(hydroxymethyl)aminomethane - DMSO dimethyl sulfoxide - RP-HPLC reverse phase-high performance liquid chromatography - AMV avian myeloblastosis virus - Arg arginine - Lys lysine     

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R_1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R_1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-t20 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R_1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R_1A and ANF-R_1C subtypes. A10 and NIH cells which express high density of ANF-R₂ receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R_1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R_1A and ANF-R_1C subtypes are modulated in the same manner. In the presence of Mn²⁺, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg²⁺ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R_1A, the ANF-R_1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R_1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R_1A subtype.Abbreviation ANF atrial natriuretic factor - BNP brain natriuretic peptide - CNP C-type natriuretic peptide - ATP adenosine-5-triphosphate - IBMX 3-isobutyl-1-methylxanthine - TPA 12-O-tetradecanoyl-phorbol-13-acetate - FKL forskolin - PKC calcium-phospholipid-dependent protein kinase - PKA cAMP-dependent protein kinase - PKG cGMP-dependent protein kinase - C-ANF [Cys¹¹⁶]-ANF-(102-116)-NH₂ - CC chromaffin cells