Amiloride increases the sensitivity of particulate guanylate cyclase to atrial natriuretic factor

The natriuretic agent amiloride induces a shift of the dose-response curve of particulate guanylate cyclase to atrial natriuretic factor (ANF) to the left. The ANF concentration for half-maximal activation of guanylate cyclase is shifted from 20 to 3 nM in the presence of 100 microM amiloride. This effect is observed with GTP*Mn2+, but not with GTP*Mg2+ as substrate. Amiloride derivatives, which inhibit a specific Na+-channel, also shift the dose-response curve to the left. These data suggest that some of the effects of amiloride may be mediated by an increased sensitivity of particulate guanylate cyclase to ANF.     

Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung

An atrial natriuretic factor (ANF) receptor from rat lung was solubilized with Lubrol-PX and purified by sequential chromatographic steps on GTP-agarose, DEAE-Sephacel, phenyl-agarose, and wheat germ agglutinin-agarose. The ANF receptor was enriched 19,000-fold. The purified receptor has a binding profile and properties that correspond to the affinity and specificity found in membranes and crude detergent extracts. Polyacrylamide gel electrophoresis of the purified preparation in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of one major protein band with a molecular mass of 120,000 daltons. When purified preparations were incubated with 125I-ANF, then cross-linked with disuccinimidyl suberate, the 120,000-dalton protein was specifically radiolabeled. This high affinity binding site for ANF co-purified with particulate guanylate cyclase. Particulate guanylate cyclase was purified to a specific activity of 19 mumol cyclic GMP produced/min/mg of protein utilizing Mn-GTP as substrate. This represented a 15,000-fold purification compared to the initial lung membrane preparation with Lubrol-PX. Gel permeation high performance liquid chromatography and glycerol density gradient sedimentation studies of the purified preparation also resulted in co-migration of specific ANF binding and guanylate cyclase activities. The co-purification of these activities suggests that both ANF binding and guanylate cyclase activities reside in the same macromolecular complex. Presumably ANF binding occurs at the external membrane surface and cyclic GMP synthesis at the internal membrane surface of this transmembrane glycoprotein.     

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.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

Regulation of guanylate cyclase activity by atrial natriuretic factor and protein kinase C

Summary The putative second messenger of certain atrial natriuretic factor (ANF) signal transductions is cyclic GMP. Recently, we purified a 180-kDa protein, apparently containing both ANF receptor and guanylate cyclase activities, and hypothesized that this is one of the cyclic GMP transmembrane signal transducers. The enzyme is ubiquitous and appears to be conserved. Utilizing the 180-kDa membrane guanylate cyclase, we now show that the 180-kDa guanylate cyclase is regulated in opposing fashions by two receptor signals—ANF stimulating it and protein kinase C inhibiting it. Furthermore, protein kinase C phosphorylates the 180-kDa enzyme. This suggests a novel switch on and switch off mechanism of the cyclic GMP signal transduction. Switch off represents the phosphorylation while switch on the dephosphorylation of the enzyme.     

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.     

Comparison of particulate guanylate cyclase in cells with and without atrial natriuretic peptide receptor binding activity

Summary A line of kidney cells (PK,) which does not possess measurable ANP binding but has an active particulate guanylate cyclase has been identified. The physical characteristics of this enzyme were compared with those of particulate guanylate cyclase and ANP receptors isolated from rat lung. Although receptor and enzyme appear to reside on the same protein in the lung while the cyclase from PK₁ cells does not possess ANP binding activity, these proteins exhibit identical physical characteristics. Guanylate cyclase from PK₁ cells and rat lung and ANP receptor from lung co-eluted during gel filtration chromatography, with a Stokes radius of 6.1 nm. Also, these activities co-migrated through sucrose density gradients with S_20,w values of 10.4 to 10.9. Using these parameters, a molecular weight of about 270 kD was estimated for all three activities. Furthermore, these enzyme activities exhibited similar mobilities in isoelectric focusing gels, with a pI of 6.1. Thus, although particulate guanylate cyclase from lung presumably possesses receptor binding activity, it is physically identical to a form of this enzyme associated with no measurable binding activity. Possible explanations for these observations are discussed.     

Association of the atrial natriuretic factor receptor with guanylate cyclase in solubilized rat glomerular membranes

The elution profile of solubilized rat glomerular membranes from a gel filtration column showed two peaks of 125I-ANF (atrial natriuretic factor) binding (367 +/- 21, 156 +/- 12 KDa). Over 85% of the total binding for the extract was in the 367 KDa peak. Guanylate cyclase activity was correlated with 125I-ANF specific binding. ANF activation of guanylate cyclase was also observed. As observed previously with particulate membrane, Scatchard-analysis of ANF binding data with the solubilized extract was consistent with a two-site model. Both affinities (Kd's), 4 pM and 1 nM, are within the range of blood concentrations reported for ANF. These observations suggest that most rat glomerular ANF receptors are large molecular complexes coupled with guanylate cyclase in the 300-350 KDa size range.     

ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase

ATP is an obligatory agent for the atrial natriuretic factor (ANF) and the type C natriuretic peptide (CNP) signaling of their respective receptor guanylate cyclases, ANF-RGC and CNP-RGC. Through a common mechanism, it binds to a defined ARM domain of the cyclase, activates the cyclase and transduces the signal into generation of the second messenger cyclic GMP. In this presentation, the authors review the ATP-regulated transduction mechanism and refine the previously simulated three-dimensional ARM model (Duda T, Yadav P, Jankowska A, Venkataraman V, Sharma RK. Three dimensional atomic model and experimental validation for the ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase. Mol Cell Biochem 2000;214:7-14; reviewed in: Sharma RK, Yadav P, Duda T. Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism. Can J Physiol Pharmacol 2001;79: 682-91; Sharma RK. Evolution of the membrane guanylate cyclase transduction system. Mol Cell Biochem 2002;230:3-30). The model depicts the ATP-binding dependent configurational changes in the ARM and supports the concept that in the first step, ATP partially activates the cyclase and primes it for the subsequent transduction steps, resulting in full activation of the cyclase.     

Mechanism of activation of particulate guanylate cyclase by atrial natriuretic peptide as deduced from radiation inactivation analysis

The interaction between the receptor (Rc) for atrial natriuretic peptide (ANP) and the effector enzyme particulate guanylate cyclase (GC) has been studied by radiation inactivation. Irradiation of bovine lung membranes produced an increase in GC activity at low radiation doses followed by a dose-dependent reduction at higher doses. This deviation from linearity in the inactivation curve disappeared when lung membranes were pretreated with ANP. Essentially identical results were also obtained with adrenal membranes. Based on these radiation inactivation data, the following dissociative mechanism of activation of particulate guanylate cyclase by ANP has been proposed: Rc.GC(inactive) + ANP----Rc.ANP + GC(active).     

Atrial natriuretic factor stimulates luteal guanylate cyclase

Effect of a synthetic atrial natriuretic peptide, rat atriopeptin II (rAP-II) on the formation of cyclic nucleotides and progesterone production in Percoll-purified rat luteal cells was investigated. Incubation of luteal cells with varying concentrations of rAP-II resulted in a dose-related stimulation of intracellular cyclic GMP content; maximum stimulation being achieved with 10 nM rAP-II. The increase in cyclic GMP formation was extremely rapid and a 12-fold increase in the cyclic GMP content over basal level was attained within 5 min of incubation of the cells with 10 nM rAP-II. In the presence of phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine, both basal and rAP-II-stimulated levels of cyclic GMP were increased approximately 10 times, but the magnitude of stimulation remained similar in the presence or absence of the inhibitor. The atrial peptide at the concentration of 1-100 nM, however, had no effect on either basal or gonadotropin-stimulated progesterone production and cyclic AMP formation by the luteal cells. Furthermore, the increase in the level of cellular cyclic GMP content of rAP-II was demonstrated to result from a selective activation of particulate guanylate cyclase.     

ATP signaling site in the ARM domain of atrial natriuretic factor receptor guanylate cyclase

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single transmembrane spanning modular protein. It binds ANF to its extracellular module and activates its intracellular catalytic module located at its carboxyl end. This results in the accelerated production of cyclic GMP, which acts as a critical second messenger in decreasing blood pressure. Two mechanistic models have been proposed for the ANF signaling of ANF-RGC. One is ATP-dependent and the other ATP-independent. In the former, ATP works through the ARM (ATP-regulated transduction module) of ANF-RGC. This model has recently been challenged [Antos et al. (2005) J Biol Chem 280:26928-26932] in support of the ATP-independent model. The present in-depth study analyzes the major principles of this challenge and concludes that the challenge lacks merit. The study then moves on to dissect the ATP mechanism of ANF signaling of ANF-RGC. It shows that the ATP photoaffinity probe, [gamma(32)P]-8-azido-ATP, reacts with Cys(634) residue in the ATP-binding pocket of ARM, and also signals the ANF-dependent activation of ANF-RGC. The target site of the 8-azido (nitrene) group is between the Cys(634) and Val(635) bond of the ATP-binding pocket. Thus, the study experimentally validates the ARM model-predicted role of Val(635) in the folding pattern of the ATP-binding pocket. And, it also identifies another residue Cys(634) that along with eight already identified residues is a part of the fold around the adenine ring of the ATP pocket. This information establishes the direct role of ATP in ANF signal transduction model of ANF-RGC, and provides a significant advancement on the mechanism by which the ATP-dependent transduction model operates.     

Participation of adenosine 5'-triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor

The addition of ANF to Percoll-purified liver plasma membranes produced a slight activation of guanylate cyclase; the ANF-stimulated cyclase activity was further increased upon the addition of ATP to the enzyme assay mixture. The effect of ATP to potentiate the cyclase activation was concentration-dependent, required Mg2+ as a divalent cation, and was seen with membranes from various tissues and cells. ATP increased the maximal velocity of the cyclase without a change in the affinity for GTP or ANF. Phosphorylation by ATP might not be involved since ANF-stimulated guanylate cyclase was enhanced by non-phosphorylating ATP analogues as well. Thus, an allosteric ATP binding site is suggested to participate in ANF-induced regulation of membrane-bound guanylate cyclase.     

HeLa cells contain the atrial natriuretic peptide receptor with guanylate cyclase activity

Receptors for atrial natriuretic peptide (ANP) are heterogeneous: an approximately 140-kDa receptor exhibits ANP-stimulated guanylate cyclase activity whereas an approximately 65-kDa receptor is thought to act only as a clearance-storage protein. We have used photoaffinity labeling techniques to show that the human cell line, HeLa, contains predominantly the approximately 140-kDa ANP receptor. In contrast, several other cell lines contain primarily the approximately 65-kDa receptor. In HeLa cells, ANP bound specifically to high affinity binding sites (Kd approximately 2 nM) and stimulated a rapid, dose-dependent accumulation of cGMP. These cell lines can thus provide useful models to study the multiple mechanisms of ANP action.     

Allosteric modification, the primary ATP activation mechanism of atrial natriuretic factor receptor guanylate cyclase

ANF-RGC is the prototype receptor membrane guanylate cyclase being both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single transmembrane-spanning protein. After binding these hormones at the extracellular domain it at its intracellular domain signals activation of the C-terminal catalytic module and accelerates the production of its second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. ATP is obligatory for the posttransmembrane dynamic events leading to ANF-RGC activation. It functions through the ATP-regulated module, ARM (KHD) domain, of ANF-RGC. In the current over a decade held model "phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A" [Potter, L. R., and Hunter, T. (1998) Mol. Cell. Biol. 18, 2164-2172]. The presented study challenges this concept. It demonstrates that, instead, ATP allosteric modification of ARM is the primary signaling step of ANF-GC activation. In this two-step new dynamic model, ATP in the first step binds ARM. This triggers in it a chain of transduction events, which cause its allosteric modification. The modification partially activates (about 50%) ANF-RGC and, concomitantly, also prepares the ARM for the second successive step. In this second step, ARM is phosphorylated and ANF-RGC achieves additional (～50%) full catalytic activation. The study defines a new paradigm of the ANF-RGC signaling mechanism.     

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.     

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.     

Inhibition of pituitary adenylate cyclase by atrial natriuretic factor

The effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in rat anterior and posterior pituitary homogenates. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in anterior and posterior pituitary homogenates in a concentration dependent manner. The maximum inhibitions observed were 42% in anterior pituitary with an apparent Ki of 10(-10) M, and 25% with an apparent Ki of 10(-11) M in posterior pituitary. Corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and prostaglandins (PGE1) stimulated adenylate cyclase to various degrees in anterior pituitary homogenates and ANF inhibited the stimulatory effect of all these hormones. In addition ANF was also able to inhibit the stimulation exerted by NaF and forskolin which activate adenylate cyclase by receptor independent mechanism. Similarly, the stimulatory effects of N-Ethylcarboxamide adenosine (NECA), NaF and forskolin on adenylate cyclase in posterior pituitary homogenates were also inhibited by ANF. This is the first study demonstrating the inhibitory effect of ANF on pituitary adenylate cyclase.     

Atrial natriuretic factor activates membrane-bound guanylate cyclase of chief cells

The present study examined the effect of atrial natriuretic factor (ANF) on cGMP generation by dispersed chief cells from guinea pig stomach. ANF caused a rapid dose-dependent increase in cGMP, a 7-fold increase in cGMP caused by 1 microM ANF, with or without 3-isobutyl-1-methylxanthine present. Methylene blue reduced cGMP in response to nitroprusside but not ANF. Guanylate cyclase activity of a chief cell membrane fraction doubled in response to ANF, but was not affected by nitroprusside. ANF had no effect on guanylate cyclase activity of the soluble fraction of lysed chief cells. Dose-response curves for whole cell cGMP production and membrane guanylate cyclase activity in response to ANF were closely related. These data indicate that ANF increases chief cell cGMP production by activating particulate guanylate cyclase, providing functional evidence that chief cells possess surface membrane receptors for ANF.     

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.