Pharmacology and Receptor Binding of Atrial Natriuretic Factor in Vascular Smooth Muscle

Characterization of the vascular pharmacology and receptor binding of atrial natriuretic factor (ANF) has been achieved utilizing a synthetic peptide which contains the sequence and biological activity of ANF. The synthetic ANF (sANF) relaxes aortic segments contracted by agonists or by low (15 to 20 mM) but not high (40 mM) concentrations of K⁺. The relaxation to sANF is well maintained, reversible, independent of the vascular endothelium and correlated with increases in cyclic GMP (with no change in cyclic AMP). Plasma membranes prepared from rabbit aorta arid kidney possess high affinity (K^d= 100 pM) specific binding sites for sANF. An excellent correlation exists between the receptor binding and pharmacology for several synthetic analogs of ANF. The presence of these receptors appears consistent with the activation of particulate (but not soluble) guanylate cyclase by sANF. sANF does exhibit a profound regional vasodilator selectivity which can be explained, in part, by changes in receptor density.     

Cytosolic Ca2+ attenuates ANF-induced cyclic GMP response in vascular smooth muscle cells

Despite a high density of atrial natriuretic factor (ANF) receptors, cultured vascular smooth muscle cells of the spontaneously hypertensive rat (SHR) manifest a blunted cyclic GMP (cGMP) response to ANF. We explored the role of cytosolic free Ca2+ ([Ca2+]i) in the ANF-induced cGMP response of cultured aortic vascular smooth muscle cells from SHR and two normotensive rat strains: Wistar-Kyoto (WKY) and American Wistar. Exposure to 500 nmol/l A23187 in Ca2+-containing but not in Ca2+-deficient medium resulted in a decline in the ANF-induced cGMP response at maximal ANF concentration (500 nmol/l; SHR from 1004 +/- 98 to 423 +/- 67, P less than 0.001; WKY from 1791 +/- 209 to 625 +/- 90, P less than 0.001; American Wistar from 1496 +/- 125 to 559 +/- 96 fmol/10(6) cells/4 min, P less than 0.001). The same phenomenon was observed by depolarization with 50 mmol/l KCl in Ca2+-containing medium. There were no significant differences among the rat strains in basal levels of [Ca2+]i. If Ca2+ plays a role in the blunted cGMP response to ANF in vascular smooth muscle cells of the SHR, this effect may be exerted by a distinct pool of the ion in the submembrane domain which is associated with the particulate guanylate cyclase system.     

Cyclic GMP phosphodiesterase and guanylate cyclase activities in rabbit ovaries and the effect of in-vivo stimulation with LH

The activities of guanylate cyclase and cyclic GMP (cGMP) phosphodiesterase, enzymes that are responsible for maintaining tissue levels of cGMP, were determined in the ovaries of rabbits killed without treatment or 4 h after administration of LH. Ovarian activities of the two enzymes were determined in the 100 000 g supernatant fraction (cytosol) and the resulting pellet (particulate fraction). Significant phosphodiesterase and cyclase activities were detected in both the cytosol and particulate fractions. Administration of LH had no significant effect on phosphodiesterase activity in either of the tissue fractions. On the other hand, LH caused a significant drop in guanylate cyclase activity in the cytosol and particulate fractions. This drop in the cyclase activity may be the cause of the decreased rabbit ovarian concentrations of cGMP that we have previously observed after LH stimulation.     

Atrial natriuretic peptide, oxytocin, and vasopressin increase guanosine 3',5'-monophosphate in LLC-PK1 kidney epithelial cells

The effect of atrial natriuretic peptide (ANP), arginine vasopressin (AVP), and oxytocin (OT) on cAMP and cGMP accumulation was investigated in LLC-PK1 kidney epithelial cells. The addition of ANP, AVP, and OT to intact cells produced a time- and concentration-dependent increase in cGMP accumulation. ANP produced a 1.7-fold increase in cGMP at 10 pM and a maximal 28-fold increase in cGMP at 1 microM. ANP had no effect on basal or AVP-induced stimulation of cAMP accumulation. OT was 10-fold more potent than AVP at increasing cGMP levels, producing a 2.1-fold increase in cGMP at 0.1 nM, whereas AVP was 100-fold more potent at increasing cAMP levels. At a concentration of 1 microM, AVP and OT produced a maximal 12 to 14-fold increase in cGMP, while OT and AVP produced 50- and 90-fold increase in cAMP, respectively. The selective OT agonist [Thr4, Gly7]oxytocin was very effective at increasing cGMP, but not at increasing cAMP levels. The V2-vasopressin agonist [deamino-Pen1,Val4, D-Arg8]vasopressin did not increase cGMP levels, but produced a 20-fold increase in cAMP levels. The addition of ANP together with either AVP or OT produced an additive increase in cGMP content. Simultaneous addition of AVP and OT did not lead to a greater increase in cAMP or cGMP levels. These results suggest that the AVP- and OT-induced increase in cGMP is mediated by OT receptors, whereas the increase in cAMP is probably mediated by vasopressin receptors. ANP increased the activity of particulate guanylate cyclase by 6-fold, while AVP and OT has no effect on particulate guanylate cyclase activity. The relatively selective inhibitor of soluble guanylate cyclase, methylene blue, had no effect on the ANP-induced increase in cGMP content in intact cells, but produced a 50% inhibition of the increase in cGMP by AVP and OT. Methylene blue did not alter the stimulation of cAMP by AVP or OT. These results demonstrate that ANP, AVP, and OT increase cGMP in LLC-PK1 kidney epithelial cells. The increase in cGMP by ANP is mediated by particulate guanylate cyclase, whereas AVP and OT probably increase cGMP by interacting with OT receptors coupled to soluble guanylate cyclase.     

Receptor regulation of atrial natriuretic factor

Two atrial natriuretic factor (ANF) receptor subtypes are present in vascular smooth muscle cells: the B receptors (or biologically active) coupled to a guanylate cyclase and the C receptors (clearance) representing 95% of the total number of ANF binding sites but noncoupled to a guanylate cyclase. Using binding experiments with 125I-ANF and measurement of cGMP production stimulated by ANF, we were able to demonstrate that ANF receptors are sensitive to homologous (induced by ANF) and heterologous regulation (induced by angiotensin II, AII) in rat cultured vascular smooth muscle cells. The effect of the two hormones showed marked differences, in their time course, their reversibility and their consequence on guanylate cyclase activity. Although both ANF and AII reduced the total number of ANF binding sites after 18 h, ANF induced a desensitization of the guanylate cyclase whereas AII elicited a potentialization of this system. From these results, we have concluded that in vascular cells B receptors are sensitive to homologous regulation and C receptors are sensitive to heterologous regulation by AII. This also highlights a specific interaction between ANF and AII at the receptor level.     

Epidermal growth factor enhances guanylate cyclase activity in vivo and in vitro

Epidermal growth factor (EGF) increases DNA synthesis and cell division both in vivo and in vitro. The mechanism by which EGF increases growth and DNA synthesis is unknown. Since the intracellular messenger cGMP stimulates DNA synthesis, the present investigation was designed to determine if EGF might have part of its mechanism of action through activating guanylate cyclase [EC 4.6.1.2], the enzyme that catalyzes the formation of cGMP. EGF enhanced soluble and particulate guanylate cyclase activities as well as cGMP levels 2- to 3-fold in hypophysectomized and nonhypophysectomized tissues both in vivo and in vitro. EGF increased guanylate cyclase activity 0.5 h after ip injection in mice, and this increased activity was still present 12 h later. Guanylate cyclase activity was increased to a greater extent secondary to EGF in hypophysectomized cecum compared to nonhypophysectomized cecum. Dose-response curves revealed that maximal stimulation of guanylate cyclase by EGF occurred at 1 nM. There was no augmented guanylate cyclase activity when the concentration of EGF was decreased to 0.01 nM. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of EGF.     

Correlation of nitric oxide and atrial natriuretic peptide changes with altered cGMP homeostasis in liver cirrhosis.

BACKGROUND: Cyclic GMP (cGMP) concentration is increased in plasma of patients with liver cirrhosis. Three possible mechanisms may contribute: increased cGMP synthesis by soluble (activated by nitric oxide), or particulate (activated by atrial natriuretic peptide (ANP)) guanylate cyclase or increased release from cells. AIM: The aim of this work was to analyze the possible contributors to increased plasma cGMP and to assess whether changes in the parameters of the system vary with the degree of liver disease (Child Pugh score) or by the presence of ascites. METHODS: We measured cGMP in plasma and lymphocytes, soluble guanylate cyclase activation by nitric oxide in lymphocytes, nitrates and nitrites and ANPs (activator of particulate guanylate cyclase) in plasma. We analyzed the correlation between changes in different parameters to discern which parameters contribute to increased plasma cGMP. RESULTS: The plasma content of nitrates+nitrites, ANP and cGMP are increased. Activation of soluble guanylate cyclase by nitric oxide is increased in patients while basal cGMP in lymphocytes is decreased. CONCLUSIONS: Both increased ANP and increased activation of soluble guanylate cyclase by nitric oxide contribute to increased plasma cGMP in patients. The concentrations of ANP and cGMP in plasma increase with the degree of disease and are higher in patients with ascites.     

Regulation of hepatic nuclear guanylate cyclase.

In immunohistochemical studies of rat liver tissue slices and purified nuclei, adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) immunofluorescence on the nuclear membrane are sequentially increased after glucagon administration. An explanation for the increased cGMP immunofluorescence was sought in experiments in which guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2]activity of hepatic subcellular fractions was determined. The results showed that a nuclear guanylate cyclase exists which can be distinguished from the soluble and crude particulate guanylate cyclases. The activity of the nuclear enzyme was increased by 35% in nuclei isolated from rats 30 min after glucagon injection, the time at which maximal nuclear membrane cGMP immunofluorescence is observed. Because glucagon altered both cAMP location and levels prior to the observed changes in nuclear cGMP metabolism, the hypothesis that cAMP acted as the second messenger was tested. In vitro incubation of nuclei isolated from control rats with 10(-5) M cAMP produced a 25% increase in nuclear guanylate cyclase activity. With nuclei isolated from glucagon-treated rats, no significant increase in enzyme activity was observed; this indicates that maximal stimulation of nuclear guanylate cyclase by cAMP occurred at levels that are obtained in vivo after glucagon administration. These findings suggest that hepatic nuclear cGMP content may be regulated by a specific organelle guanylate cyclase and that cAMP may be one of the determinants of this enzyme's activity.     

Effects of atrial natriuretic factor and vasopressin on cyclic nucleotides in cultured kidney cells

The cellular mechanism of the action of atrial natriuretic factor (ANF) is thought to involve activation of guanylate cyclase. Increasing evidence shows a direct tubular effect of ANF. Part of the ANF-induced diuresis has been suggested to be due to inhibition of the action of arginine vasopressin (AVP) in the cortical collecting tubule. In this study we investigated the effect of ANF on cyclic nucleotide production in primary cultures of cortical collecting tubule cells immunodissected with a monoclonal antibody. ANF caused a dose-dependent stimulation in cyclic guanosine 3',5'-monophosphate (cGMP) production; the half-maximal stimulation was observed at approximately 1 nM of ANF. ANF (0.01-100 nM) had no effect on cyclic adenosine 3',5'-monophosphate (cAMP) accumulation in cortical collecting tubule cultures. AVP caused a dose-dependent increase in cAMP production, and this effect was not altered by the simultaneous addition of ANF (100 nM). Similarly, ANF-induced cGMP stimulation was not influenced by AVP (10 nM). We conclude that 1) ANF has a direct stimulatory action on cGMP production by cultured cortical collecting tubule cells and 2) any interaction between ANF and AVP is likely to occur at steps distal to cyclic nucleotide formation.     

Ovarian cyclic GMP concentration and guanylate cyclase activity in immature rats after treatment with pregnant mare serum gonadotrophin

We have previously reported that the LH-induced decrease in the concentration of ovarian cyclic GMP (cGMP) in the rabbit was accompanied by a drop in ovarian guanylate cyclase activity. The present experiments were carried out to see if the increase in cGMP concentration that occurs in immature rat ovaries after stimulation with pregnant mare serum gonadotrophin (PMSG) is also accompanied by changes in guanylate cyclase activity. Total ovarian cGMP, along with ovarian weight, was found to be increased at 16 h after PMSG treatment. Ovarian concentrations of cGMP, however, increased only after that period (at 20, 24 and 48 h) and the increase was progressive. Guanylate cyclase activity was found in both the cytosol and 100 000 g particulate fractions of the immature rat ovaries. Forty-three hours after PMSG treatment, activity in the particulate fraction was found to be significantly increased. This increase in guanylate cyclase activity was also found at 20 h but not at 16 h. Thus, the increase in ovarian cGMP concentration in immature rats after PMSG treatment was accompanied by increased guanylate cyclase activity.     

Cell biology of atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) exhibits a wide spectrum of cardiovascular, endocrine, metabolic and renal actions. cGMP is the major mediator of ANP at the cellular level and only tissues possessing particulate guanylate cyclase appear to present ANP-induced actions. Three types of ANP receptors have recently been cloned. Two of them (A and B receptors) are homologous and contain guanylate cyclase catalytic domains. The C receptor could possibly regulate the metabolic fate of ANP. Data obtained by the radiation inactivation method suggest the presence of an inter- or intramolecular inhibitory component of nearly 90 kilodaltons that represses the catalytic activity of guanylate cyclase within its membrane environment. The mechanism of guanylate cyclase stimulation by ANP could involve this inhibitory component. Preliminary data suggest that the hyperresponsiveness of the particulate guanylate cyclase/cGMP system in hypertension occurs through modulation of the inhibitory component.     

Sodium nitroprusside inhibition of parathyroid hormone release is not mediated through cyclic GMP

Sodium nitroprusside effected a rapid, dose-dependent increase in intracellular cGMP accumulation in freshly dispersed bovine parathyroid cells. The effect was half-maximal between 10(-4) and 3 X 10(-4)M, maximal at 3 X 10(-3)M nitroprusside and could be amplified (approximately 50%) by the addition of methylisobutylxanthine (4 X 10(-4)M). The dose-response characteristics were similar to those previously described for the inhibition of cAMP accumulation and PTH release by this agent. Neither dibutyryl cGMP (10(-3)M) nor 8'-bromo-cGMP (10(-3)M) mimicked the inhibitory effect of nitroprusside on cAMP accumulation or PTH release. Dose-dependent stimulation of guanylate cyclase was found in a particulate preparation of parathyroid cells; activity was maximal at 10(-4)M nitroprusside while higher concentrations appeared to inhibit the enzyme. Nitroprusside significantly reduced both (-)isoproterenol and guanine nucleotide-stimulated adenylate cyclase activity in the particulate preparation with maximum inhibition between 10(-3)-10(-2)M. cGMP concentrations as high as 10(-4)M did not affect agonist-stimulated cAMP synthesis. Thus, although the kinetic and dose-response characteristics of the nitroprusside effect on cGMP suggest a linkage to its previously described effects on cAMP and PTH secretion, no direct evidence was found to indicate a causal relationship between the two. Rather it would appear that the effects on the adenylate and guanylate cyclase enzymes occur in parallel, possibly the result of some common primary perturbation of cellular physiology.     

Atrial natriuretic peptide and sodium nitroprusside stimulate cyclic GMP accumulation by avian skin fibroblasts and epiphyseal growth-plate chondroprogenitor cells

Chondroprogenitor cells derived from avian tibia epiphyseal growth plate, and skin fibroblasts were cultured in vitro. In the fibroblasts, human (1-28) and rat (5-28) atrial natriuretic peptide (ANP) stimulated cyclic GMP (cGMP) production in a dose-dependent manner without affecting cAMP. Sodium nitroprusside also stimulated cGMP accumulation by chondroprogenitor cells and fibroblasts, but the maximum cGMP accumulation elicited by sodium nitroprusside was much lower than that obtained with ANP. The effects of ANP and sodium nitroprusside on chondroprogenitor cells and skin fibroblasts were additive. Human ANP increased cGMP production by the particulate fraction prepared either from chondroprogenitor cells or fibroblasts. Sodium nitroprusside, at concentrations of up to 1 mmol/l, did not affect cGMP production by the particulate fraction prepared from either cell type. The present study provides additional evidence that avian growth-plate chondroprogenitor cells and skin fibroblasts are targets for ANP. ANP and nitroprusside activate different guanylate cyclase isoenzymes--the particulate and soluble forms of the enzyme respectively. The data suggest that most of the guanylate cyclase activity in these cells is localized in the particulate fraction.     

Effects of atrial natriuretic factor on cyclic nucleotides, bone resorption, collagen and deoxyribonucleic acid synthesis, and prostaglandin E2 production in fetal rat bone cultures

We examined the effects of synthetic human atrial natriuretic factor (human ANF 99-126) on adenylate cyclase activity, cAMP and cyclic GMP (cGMP) levels, bone resorption, collagen and DNA synthesis, and prostaglandin E2 (PGE2) production in fetal rat bone organ cultures. ANF (100 nM) inhibited PTH- and PGE2-stimulated cAMP production but had no effect on basal cAMP production in 21-day fetal rat calvaria. ANF increased cGMP levels, and this was not affected by PTH. ANF (10 nM) partially inhibited bone resorption stimulated by PGE2 but had no effect on control or PTH-stimulated resorption in 19-day fetal rat long bones. ANF had no effect on collagen and DNA synthesis or PGE2 production and did not alter responses to PTH or PGE2 in the fetal rat calvaria. Thus, ANF has no major direct effect on bone resorption or formation, but it is possible that ANF modulates the local regulatory function of PGE2 in bone.     

The role of Ca2+ and calmodulin in the regulation of atrial natriuretic peptide-stimulated guanosine 3',5'-cyclic monophosphate accumulation by isolated mouse Leydig cells

We have investigated the role of Ca2+ and calmodulin in the stimulation of cGMP formation by mouse Leydig cells in response to rat atriopeptin-II (rAP-II). Removal of extracellular Ca2+ had no influence on the levels of cGMP accumulated by the cells stimulated with rAP-II. The amounts of testosterone produced by unstimulated and rAP-II-stimulated cells were, however, reduced by 50% in the absence of Ca2+ from the incubation medium. Addition of ionomycin to the Leydig cells led to a dose-related inhibition of rAP-II-stimulated cGMP formation, but the basal cGMP level was not affected. These experiments were carried out in the presence of a phosphodiesterase inhibitor. The inhibitory effect of ionomycin was absolutely dependent upon the presence of Ca2+ in the medium. The guanylate cyclase activity required the presence of a cation, and Mn2+, Mg2+, or Ca2+ could function as the required cation. There was no direct inhibition of the cyclase activity by Ca2+ up to as high a concentration as 8 mM. Furthermore, three structurally unrelated calmodulin antagonists, W7, trifluoperazine, and calmidazolium, but not W5, caused a dose-related inhibition of rAP-II-stimulated cGMP accumulation by the cells. The inhibitory effect of calmodulin antagonists was not exerted directly at the level of guanylate cyclase activity, since the particulate enzyme was not inhibited by any of these drugs. We conclude, therefore, that extracellular Ca2+ is not essential for rAP-II-mediated stimulation of cGMP formation by mouse Leydig cells, at least under the short term incubation conditions used. An excessive ionophoretic influx of Ca2+ into the cells impairs the ability of rAP-II to stimulate cGMP formation. Therefore, it appears that a finely regulated level of intracellular Ca2+ is required for optimal activation of atrial natriuretic peptide-responsive guanylate cyclase in mouse Leydig cells, and calmodulin plays an important role in this process.     

Nitric oxide as a signal in thyroid.

It is now well established that agonist activation of the PIP2/calcium cascade in the thyroid results in the enhancement of cGMP accumulation presumably by activation of the soluble guanylate cyclase. In many tissues the physiological signal controlling soluble guanylate cyclase is nitric oxide (NO) and its synthesis from arginine is controlled by the intracellular Ca2+. In this report we show results that suggest that NO may be the intermediate of the cGMP response to the activation of the PIP2/calcium cascade. In dog thyroid slices, incubation with carbamylcholine or A23187 increases significantly free intracellular Ca2+ levels and the cGMP content of the slices. NG-Monomethyl-L-arginine (NMMA), a competitive inhibitor of arginine for nitric oxide synthase, inhibited these cGMP responses but not the action of sodium nitroprusside which activates soluble guanylate cyclase directly. The inhibition was relieved by arginine. Methylene blue, which blocks the activation of soluble guanylate cyclase by NO, also decreased the three stimulatory effects. NMMA and methylene blue also decreased the basal levels of cGMP. NO may therefore be an important autocrine and paracrine factor in thyroid.     

Papillary collecting tubule responsiveness to atrial natriuretic factor in Dahl rats

There is evidence that atrial natriuretic factor (ANF) has an action in the inner medullary collecting duct. In addition, the prehypertensive Dahl salt-sensitive (S) rat has an intrinsic tendency toward less natriuresis than the Dahl salt-resistant (R) rat has when challenged with ANF. To test the hypothesis that renal papillary collecting tubule cells from prehypertensive S rats might be genetically less responsive to ANF, S and R cells were grown in culture and studied for responsiveness to ANF by measurement of cyclic nucleotide responses. There was a concentration-dependent effect of ANF on renal papillary collecting tubule cell synthesis of intracellular cyclic guanosine 3',5'-monophosphate (cGMP) in both strains. However, the S cells were hyporesponsive compared with the R cells (p less than 0.002, by analysis of variance). Likewise, in response to Na nitroprusside, the S cells were hyporesponsive compared with the R cells as measured by intracellular cGMP accumulation (p less than 0.03, by analysis of variance). Arginine vasopressin stimulated intracellular cAMP equally in both strains. Also, ANF equally enhanced intracellular cGMP in glomerular mesangial cells from S and R rats, indicating possible specificity of the reduced responsiveness to ANF to the distal nephron of S rats. Plasma ANF levels had a slight tendency to be higher in prehypertensive S rats than in R rats (p = 0.088, by t test). These results suggest that the papillary collecting duct of Dahl S and R rats may differ in guanylate cyclase activity. This difference may partially explain the impaired natriuretic responses of S rats and could represent a factor contributing to the development of salt-sensitive hypertension.     

Atrial natriuretic factor receptor heterogeneity and stimulation of particulate guanylate cyclase and cyclic GMP accumulation

Since the seminal discovery by deBold that atria contain factors that produce diuresis and natriuresis, the biologic effects attributed to ANF have expanded to the point where the name "atrial natriuretic factor" seems inappropriate. In addition to promoting diuresis and natriuresis, ANF has been shown to produce vascular smooth muscle relaxation and to inhibit the secretion of aldosterone from the adrenal cortex, renin from the juxtaglomerlular apparatus, vasopressin from the hypothalamus, and salt and water intake after central administration. ANF also promotes intestinal secretion and stimulates testosterone synthesis in Leydig cells. However, the cellular mechanisms whereby ANF elicits these diverse effects are poorly understood. ANF has been reported to inhibit adenylate cyclase in a number of tissues. However, the significance of ANF inhibition of adenylate cyclase is unknown. This effect cannot be associated with vascular relaxation since decreased cyclic AMP would be expected to promote contraction rather than relaxation. ANF inhibition of adenylate cyclase may mediate the inhibitory effects of ANF on hormone secretion from the anterior pituitary gland. The inhibition of adenylate cyclase could also explain the inhibitory effect of ANF on aldosterone synthesis, since agents that stimulate cyclic AMP increase aldosterone synthesis. However, ANF also inhibits the dibutyryl-cyclic AMP-induced stimulation of aldosterone secretion, suggesting that an inhibition of adenylate cyclase cannot account fully for the inhibitory effects of ANF on aldosterone synthesis. There is no evidence to support a role for cyclic AMP in the diuretic and natriuretic action of ANF. An inhibition of membrane phosphoinositide breakdown by ANF and the subsequent formation of IP3 and intracellular calcium release could explain the inhibitory effects of ANF on vascular contraction and steroid synthesis. However, there is very little evidence to suggest that ANF regulates phosphoinositide metabolism, while some recent studies suggest that ANF may regulate calcium fluxes in vascular tissue. Clearly, cyclic GMP has emerged as the most likely intracellular mediator of ANF effects. ANF increases cyclic GMP in a wide range of tissues by selectively activating particulate guanylate cyclase. However, it is not known which effects of ANF are mediated by cyclic GMP. The discovery that ANF increases cyclic GMP in vascular tissue clearly suggests that cyclic GMP mediates the vascular relaxation effect of ANF, since other classes of vasodilators also increase cyclic GMP. There is preliminary evidence that cyclic GMP may inhibit renin secretion and sodium transport in kidney cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Homologous and heterologous regulation of atrial natriuretic factor receptors in smooth muscle cells

Two subtypes of atrial natriuretic factor (ANF) receptors are present in vascular smooth muscle cells: B(biologically active) receptors coupled to a guanylate cyclase and C (clearance) receptors (95 per cent of the total number of ANF binding sites) non coupled to any identified second messenger system. We compared the homologous receptor regulation induced by ANF to the heterologous one elicited by angiotensin II (Ag II). Binding of (3-[125I]iodotyrosyl) rat ANF and cGMP production stimulated by ANF were measured after 18 hours preincubation of rat cultured vascular smooth muscle cells (10(6) cells/dish) at 37 degrees C with ANF or Ag II. The hormones (10 nM) decreased to the same extent the total apparent number of ANF binding sites (control: 208 +/- 25 fmol/10(6); ANF : 82 +/- 20 fmol/10(6) cells; Ag II : 90 +/- 9 fmol/10(6) cells) The diminution of the number of ANF binding sites induced by ANF exposure was reversed by 85 per cent following 10 minutes treatment of the cells with 10 mM AcOH. Moreover, treatment with ANF (10 nM) led to a diminution of cGMP stimulation induced by ANF, this effect being still present after washing the cells with 10 mM AcOH. In contrast, diminution of ANF building sites consecutive to Ag II exposure was not affected by AcOH treatment and a potentiation of cGMP production elicited by ANF was observed. These results suggest that, in rat vascular smooth muscle cells, B receptors are sensitive to homologous down regulation and C receptors are sensitive to heterologous regulation by Ag II.     

Vasopressin-mediated inhibition of atrial natriuretic factor-stimulated cGMP accumulation in an established smooth muscle cell line.

Rat thoracic aortic smooth muscle cells (line A10, ATCC CRL 1476) display a high density of atrial natriuretic factor (ANF) receptors. ANF stimulated the accumulation of cGMP in these cells in a time- and dose-dependent fashion. These cells are known to display a high density of vasopressin receptors of the vascular V1 subtype. These vasopressin receptors mediate inhibition of isoproterenol-stimulated cAMP accumulation and stimulation of inositol phosphate accumulation and calcium fluxes. Addition of [8-arginine]vasopressin ([Arg8]VP) to these cells inhibited ANF-stimulated cGMP accumulation. Inhibition of cGMP accumulation was dependent on the concentration of [Arg8]VP, with half-maximal and maximal effects occurring at 0.4 and 10 nM, respectively. [Arg8]VP did not have significant effects on basal cGMP levels. The inhibition by [Arg8]VP appears to be mediated by V1 receptors, since the V2 renal receptor agonist [1-desaminocysteine,8-D-arginine]vasopressin was ineffective. Also, the selective V1 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-methyltyrosine),8-arginine]vasopressin and the mixed V1/V2 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-ethyl-D-tyrosine),4-valine,8-arginine]vasopressin blocked the [Arg8]VP-mediated effect, whereas the selective V2 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-D-isoleucine,4-valine,8-arginine]vasopressin was minimally effective. These data show that in rat aortic smooth muscle cells, V1 receptors are negatively coupled to guanylate cyclase. These data also suggest that the vasoconstrictor activity of [Arg8]VP might involve inhibition of ANF-receptor-mediated vascular relaxation through inhibition of cGMP accumulation in addition to its effects on isoproterenol-mediated cAMP accumulation and inositol phosphate accumulation and calcium fluxes.