Interaction of atrial natriuretic peptide-stimulated guanylate cyclase and vasopressin-stimulated calcium signaling pathways in the glomerular mesangial cell

Receptors for atrial natriuretic peptide (ANP) have been demonstrated in renal mesangial cells as well as other cell types in the glomerulus. The biochemical basis for the effects of ANP on glomerular hemodynamics remains undefined. Using cultured rat glomerular mesangial cells, we demonstrated a concentration-dependent stimulation of cGMP production in intact cells, and of guanylate cyclase in membranes. Despite the presence of a guanylate cyclase response, ANP had no inhibitory effect on basal inositol trisphosphate production nor on basal cytosolic calcium. Arginine vasopressin stimulated IP3 production, caused a rise in cytosolic calcium as measured using the calcium-sensitive fluorescent probe Indo-1, and caused mesangial cell contraction. ANP caused a slight but significant enhancement of vasopressin-stimulated IP3 production, but had no effect on the cytosolic calcium response nor on the contractile response. 8-Bromo-cGMP likewise had no effect on the generation of the calcium signal. These results indicate that the effects of ANP on glomerular hemodynamics are not mediated by an alteration in the generation of the calcium signal in mesangial cells. In contrast, addition of calcium inhibited ANP stimulated guanylate cyclase activity.     

Adenine nucleotide regulation of particulate guanylate cyclase from rat lung.

Adenine nucleotides activate basal particulate guanylate cyclase in rat lung membranes. Activation is specific for adenine and not guanine, cytidine or uridine nucleotides. The concentration of adenine nucleotides yielding half-maximum activation of particulate guanylate cyclase is 0.1 mM and this nucleotide activates the enzyme by increasing maximum velocity 11-fold without altering affinity for substrate. Activation is specific for particulate guanylate cyclase, since soluble enzyme is inhibited by adenine nucleotides. Similarly, activation is specific for magnesium as the enzyme substrate cation cofactor, since adenine nucleotides inhibit particulate guanylate cyclase when manganese is used. Adenine nucleotide regulation of particulate guanylate cyclase may occur by a different molecular mechanism compared to other activators, since the effects of these nucleotides are synergistic with those of detergent, hemin and atrial natriuretic peptides. Cystamine inhibits adenine nucleotide activation of particulate guanylate cyclase at concentrations having minimal effects on basal enzyme activity suggesting a role for critical sulfhydryls in mechanisms underlying nucleotide regulation of particulate guanylate cyclase. Purification and quantitative recovery of particulate guanylate cyclase by substrate affinity chromatography results in the loss of adenine nucleotide regulation. These data suggest that adenine nucleotides may be important in the regulation of basal and activated particulate guanylate cyclase and may be mediated by an adenine nucleotide-binding protein which is separate from that enzyme.     

Detection of guanylate cyclases A and B stimulated by natriuretic peptides in gastrointestinal tract of rat

Summary The ultracytochemical localization of membrane-bound guanylate cyclases A and B has been studied after stimulation with atrial natriuretic peptide, C-type natriuretic peptide and brain natriuretic peptide in the gastrointestinal tract of rat. The two isoforms are stimulated differently by the three peptides. The results showed that the atrial and C-type natriuretic peptides stimulated guanylate cyclase activity, whereas the brain peptide seemed not to activate enough of the enzyme to detect. The guanylate cyclase activity had a wider distribution in stomach and small intestine than in large intestine; nevertheless, the reaction product of guanylate cyclase A activity had a wider localization in the stomach, whereas the reaction product of guanylate cyclase B activity had a wider distribution in the small intestine. In the small and large intestine, we detected mostly similar localizations of guanylate cyclase activity irrespective of the peptide used; in the stomach the reaction products of guanylate cyclase A and B were detected in different cell types or in different sites of the same cell. In all the gastrointestinal tract, guanylate cyclase activity was detected mainly in three types of cells: exocrine and endocrine cells; undifferentiated and mature epithelial cells; and smooth muscle cells. These localizations of guanylate cyclase activity suggest its role in regulating glandular secretion, cellular proliferation and muscular activity. This revised version was published online in November 2006 with corrections to the Cover Date.     

Differential stimulation of rat lung particulate guanylate cyclase activity by atrial natriuretic peptide and sodium nitroprusside

The effects of alpha-rat atrial natriuretic peptide (alpha-rANP) and sodium nitroprusside on the activity of rat lung particulate guanylate cyclase were examined. The particulate guanylate cyclase in partially purified rat lung membranes was stimulated by both alpha-rANP and nitroprusside. The effects of alpha-rANP and nitroprusside were, however, not additive. Diamide and N-ethylmaleimide almost completely abolished the nitroprusside-mediated stimulation, while they had only moderate effects on the alpha-rANP-mediated stimulation of the enzyme activity. ATP potentiated the enzyme stimulation by alpha-rANP, whereas it had no effect on the nitroprusside-mediated stimulation. These findings suggest that the stimulation of lung particulate guanylate cyclase activity by alpha-rANP and nitroprusside is mediated by different mechanisms.     

Calcium and calmodulin regulate atrial natriuretic factor stimulation of cyclic GMP in a human renal cell line.

We examined calcium and calmodulin regulation of atrial natriuretic factor stimulation of particulate-membrane guanylate cyclase (ANF-s-GC) in SK-NEP-1 cells. W7 and trifluoropiperazine, but not W5, inhibited whole cellular ANF-stimulated cyclic GMP accumulation (ANF-s-cGMP). EGTA and LaCl3 decreased ANF-s-GC and calmodulin reversed this inhibition. A23187-induced inhibition of ANF-s-cGMP was only partly reversible by IBMX. H7 or staurosporine counteracted the inhibitory effect of A23187. Calcium inhibited basal and ANF-s-GC. These data suggest that at low concentrations of calcium, ANF-s-GC was calcium-calmodulin dependent but high concentrations of calcium inhibited ANF-s-GC through phosphodiesterase, through inhibition of GC, and probably through protein kinase C.     

Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate

In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of guanylate cyclase by atrial natriuretic factor in isolated human glomeruli

A 23 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) stimulated guanylate cyclase activity in isolated human glomeruli in a concentration- and time-dependent manner. ANF activated particulate guanylate cyclase whereas it had no effect on soluble guanylate cyclase. These results demonstrate that the glomerulus is a target structure for ANF in humans. They also suggest that ANF-induced increase in glomerular filtration rate is due to a direct effect of this peptide on the glomerular cells mediated by activation of glomerular guanylate cyclase.     

Characterization and regulation by protein kinase C of renal glomerular atrial natriuretic peptide receptor-coupled guanylate cyclase

The nature and regulation of atrial natriuretic peptide (ANP)-sensitive guanylate cyclase in rat renal glomerular membranes was examined. By affinity crosslinking techniques, three bands with apparent molecular masses of 180, 130 and 64 kDa were specifically labeled with [125I]ANP. A specific antibody to the 180 kDa membrane guanylate cyclase of rat adrenocortical carcinoma recognized a 180 kDa band on Western blot analysis of solubilized, GTP-affinity purified glomerular membrane proteins. The same antibody completely inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. Partially purified protein kinase C inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. It is concluded that a 180 kDa ANP-sensitive guanylate cyclase is present in glomerular membranes, and that this enzyme is inhibited directly by protein kinase C.     

Purification and characterization of the 180-kDa membrane guanylate cyclase containing atrial natriuretic factor receptor from rat adrenal gland and its regulation by protein kinase C

The original concept that cyclic GMP is one of the mediators of the hormone-dependent process of steroidogenesis has been strengthened by the characterization of a 180-kDa protein from rat adrenocortical carcinoma and rat and mouse testes. This protein appears to have an unusual characteristic of containing both the atrial natriuretic factor (ANF)-binding and guanylate cyclase activities, and appears to be intimately involved in the ANF-dependent steroidogenic signal transduction. In rat adrenal glands we now demonstrate: 1) the direct presence of a 180-kDa ANF-binding protein in GTP-affinity purified membrane fraction as evidenced by affinity cross-linking technique and by the Western blot analysis of the partially purified enzyme; 2) that the enzyme is biochemically and immunologically different from the soluble guanylate cyclase as there is no antigenic cross-reactivity of 180-kDa guanylate cyclase antibody with soluble guanylate cyclase; 3) in contrast to the soluble guanylate cyclase, the particulate enzyme is not stimulated by nitrite-generating compounds and hemin; and 4) protein kinase C inhibits both the basal and ANF-dependent guanylate cyclase activity and phosphorylates the 180-kDa guanylate cyclase. These results reveal the presence of a 180-kDa protein in rat adrenal glands and support the contention that: (a) this protein contains both the guanylate cyclase and ANF receptor; (b) the 180-kDa enzyme is coupled with the ANF-dependent cyclic GMP production; (c) the 180-kDa enzyme is biochemically distinct from the nonspecific soluble guanylate cyclase; and (d) there is a protein kinase C-dependent negative regulatory loop for the operation of ANF-dependent cyclic GMP signal pathway which acts via the phosphorylation of 180-kDa guanylate cyclase.     

Soluble guanylate cyclase in molecular mechanisms underlying the therapeutic action of drugs

The influence of ambroxol (a mucolytic agent) on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside (SNP) and Sin-1) were investigated. Ambroxol in the range of concentrations from 0.1 to 10 ??M had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the SNP-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC₅₀ values of 3.9 and 2.1 ??M, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin (an antimalarial agent) on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1?100 ??M) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the SNP-induced activation of human platelet guanylate cyclase with the IC₅₀ value of 5.6 ??M. Artemisinin (10 ??M) also inhibited (by 71 ± 4.0%) the activation of the enzyme by a thiol-dependent NO-donor, the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 ??M), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the signaling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.     

Ultracytochemical localization of particulate guanylate cyclase after stimulation with natriuretic peptides in lamb olfactory mucosa

Summary The ultracytochemical localization of particulate guanylate cyclase has been studied in lamb olfactory mucosa after activation with rat atrial natriuretic factor (rANF), porcine brain natriuretic peptide (pBNP), porcine C-type natriuretic peptide (pCNP) or rat brain natriuretic peptide (rBNP). Particulate guanylate cyclase is the receptor for these peptides and recently two subtypes of the cyclase have been identified. These isoforms are stimulated differently by ANF, BNP and CNP. Under our experimental conditions, rANF, pCNP and pBNP were strong activators of particulate guanylate cyclase in lamb olfactory mucosa, as demonstrated by the presence of reaction product. Samples incubated in basal conditions without rANF, pCNP or pBNP, or samples incubated in presence of rBNP did not reveal any cyclase activity. The rANF-stimulated cyclase activity was localized in the apical portion of olfactory epithelium. pCNP-stimulated guanylate cyclase was detected to the lamina propria in association with secretory cells of Bowman's glands and with cells in close relation with Bowman's glands (elongated cells and myoepithelial cells). The cyclase activity stimulated by pBNP was limited to cells of Bowman's glands. The present data indicate that ANF and CNP are recognized by different receptors and that BNP and CNP bind to the same receptor.     

Effects of thiol inhibitors on hepatic guanylate cylase activity.

Several thiol blocking agents inhibit basal guanylate cyclase activity of 100 000 X g hepatic supernatant fractions and the stimulation of enzyme activity by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), NaN3, NaNO2 and nitroprusside. The relative potency of the thiol blockers as inhibitors was CdCl2 greater than p-hydroxymercuribenzoate greater than N-ethylmaleimide greater than arsenite greater than iodoacetamide. Inhibition of basal and MNNG-responsive soluble guanylate cyclase activities by arsenite was markedly potentiated by an equimolar concentration of 2,3-dimercaprol, but not by mercaptoethanol. Inhibition of soluble guanylate cyclase by either arsenite or CdCl2 was completely reversed by excess 2,3-dimercaprol. Qualitatively similar effects were observed with DE-52 cellulose purified soluble hepatic guanylate cyclase, and suggested an involvement of closely juxtaposed thiol groups in the regulation of enzyme activity. For several reasons inhibition by thiol blockers appeared to be mediated through multiple mechanisms and/or sites of interaction: (1) Concentrations of the thiol inhibitors which had no effect on basal activity strikingly inhibited the responsiveness of the enzyme to a submaximal concentration of MNNG. (2) CdCl2 abolished the action of excess MnCl2 to stimulate purified guanylate cyclase, but was a relatively ineffective inhibitor when MnCl2 and GTP were present in equimolar concentrations. By contrast, arsenite-2,3-dimercaprol was uniformly effective in inhibiting guanylate cyclase activity in the presence or absence of excess MnCl2. (3) Arsenite-2,3-dimercaprol increased the Km for MnGTP (control, 0.13 +/- 0.02 mM; 0.2 mM arsenite-2,3-dimercaprol, 0.31 +/- 0.03 mM), whereas CdCl2 had no effect on this parameter. (4) Hepatic particulate guanylate cyclase activity was significantly inhibited by arsenite 2,3-dimercaprol but not by CdCl2. Thus, the data not only indicate that vicinal dithiol groups are required for expression of basal guanylate cyclase activity and enzyme responses to agonists, but strongly suggest the involvement of more than one interacting site containing free thiol residues.     

Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 microM and 36 microM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (less than 100 microM) and inhibited by high concentrations (greater than 100 microM) of calcium, half-maximal effects were obtained with 8 microM and 500 microM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2 inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 microM, it was kinetically a mixed-type inhibition. La3+ and CA2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EDTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium-potassium action potential of Paramecium.     

Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA

The potent diuretic and natriuretic peptide hormone atrial natriuretic factor (ANF), with vasodilatory activity also stimulates steroidogenic responsiveness in Leydig cells. The actions of ANF are mediated by its interaction with specific cell surface receptors and the membrane-bound form of guanylate cyclase represents an atrial natriuretic factor receptor (ANF-R). To understand the mechanism of ANF action in testicular steroidogenesis and to identify guanylate cyclase/ANF-R that is expressed in the Leydig cells, the primary structure of murine guanylate cyclase/ANF-R has been deduced from its cDNA sequence. A cDNA library constructed from poly(A+) RNA of murine Leydig tumor (MA-10) cell line was screened for the membrane-bound form of ANF-R/guanylate cyclase sequences by hybridization with a rat brain guanylate cyclase/ANF-R cDNA probe. The amino acid sequence deduced from the cDNA shows that murine guanylate cyclase/ANF-R cDNA consists of 1057 amino acids with 21 amino acids comprising the transmembrane domain which separates an extracellular ligand-binding domain (469 amino acid residues) and an intracellular guanylate cyclase domain (567 amino acid residues). Upon transfection of the murine guanylate cyclase/ANF-R cDNA in COS-7 cells, the expressed protein showed specific binding to 125I-ANF, stimulation of guanylate cyclase activity and production of intracellular cGMP in response to ANF. The expression of guanylate cyclase/ANF-R cDNA transfected in rat Leydig tumor cells stimulated the production of testosterone and intracellular cGMP after treatment with ANF. The results presented herein directly show that ANF can regulate the testicular steroidogenic responsiveness in addition to its known regulatory role in the control of cardiovascular homeostasis.     

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.     

Physical and functional association of the atrial natriuretic peptide receptor with particulate guanylate cyclase as demonstrated using detergent extracts of bovine lung membranes

Coupling of the atrial natriuretic peptide (ANP) receptor to particulate guanylate cyclase has been demonstrated kinetically and chromatographically using bovine lung plasma membranes and their detergent extracts. Addition of ANP to the membrane suspension stimulated guanylate cyclase activity 2-5-fold indicating the presence of ANP-sensitive particulate guanylate cyclase. The enzyme retained the ability to respond to ANP even after solubilization with digitonin. Characterization of the solubilized enzyme by gel filtration and affinity chromatography revealed that the ANP receptor and particulate guanylate cyclase exist as a functionally but not covalently linked stable complex.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues

The effects on guanylate cyclase and cyclic GMP accumulation of a synthetic peptide containing the amino acid sequence and biological activity of atrial natriuretic factor (ANF) were studied. ANF activated particulate guanylate cyclase in a concentration- and time- dependent fashion in crude membranes obtained from homogenates of rat kidney. Activation of particulate guanylate cyclase by ANF was also observed in particulate fractions from homogenates of rat aorta, testes, intestine, lung, and liver, but not from heart or brain. Soluble guanylate cyclase obtained from these tissues was not activated by ANF. Trypsin treatment of ANF prevented the activation of guanylate cyclase, while heat treatment had no effect. Accumulation of cyclic GMP in kidney minces and aorta was stimulated by ANF activation of guanylate cyclase. These data suggest a role for particulate guanylate cyclase in the molecular mechanisms underlying the physiological effects of ANF such as vascular relaxation, natriuresis, and diuresis.     

Inhibitory effect of Ca(2+) on ATP-mediated stimulation of NPR-A-coupled guanylyl cyclase in renal glomeruli from spontaneously hypertensive and normotensive rats.

Atrial natriuretic peptide (ANP) regulates blood pressure mainly through the occupation of the guanylyl cyclase-coupled receptor NPR-A, which requires ATP interaction for maximal activation. This study investigates the effect of extracellular Ca(2+) on ATP-mediated regulation of NPR-A-coupled guanylyl cyclase activity in glomerular membranes from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). ATP induced a significant increase in basal and ANP(1-28)-stimulated guanylyl cyclase activity that was greater in SHR than in WKY. Extracellular Ca(2+) inhibited ATP-stimulated guanylyl cyclase activity in a concentration-dependent manner, but did not modify basal and ANP(1-28)-stimulated guanylyl cyclase activity. In the presence of ATP, NPR-A showed higher affinity for ANP(1-28) and lower Bmax. Ca(2+) did not modify NPR-A-ANP(1-28) binding properties. The different effects of extracellular Ca(2+) on ANP(1-28)- or ATP-mediated guanylyl cyclase activation suggest that these events are differentially regulated. Addition of extracellular Ca(2+) induced similar effects in hypertensive and normotensive rats, suggesting that it is not responsible for the elevated cGMP production observed in SHR.     

Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose-response manner, with an IC50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.