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.     

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.     

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.     

Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase

Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G(s)α to C2 and the ensuing 7° rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.     

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.     

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.     

Negative regulation of atrial natriuretic factor receptor coupled membrane guanylate cyclase by phorbol ester. Potential protein kinase C regulation of cyclic GMP signal in isolated adrenocortical carcinoma cells of rat

Rat adrenocortical carcinoma cells possess a high density of atrial natriuretic factor (ANF) receptors which are coupled with membrane guanylate cyclase and corticosterone production. Herein we show that pretreatment of these cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, attenuates the ANF-stimulated cyclic GMP accumulation in a dose-dependent manner. The half maximum inhibitory concentration of PMA was 10(-10) M. When these cells were incubated with PMA in the presence of 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine, a protein kinase C inhibitor, the PMA-mediated attenuation of ANF-stimulated cyclic GMP formation is blocked. These results suggest that protein kinase C negatively regulates the ANF-receptor coupled membrane guanylate cyclase system in these cells.     

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).     

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.     

A ring-reversed analog of atrial natriuretic peptide retains receptor binding, guanylate cyclase stimulation.

We have prepared an atrial natriuretic peptide analog, ANP[13-27][1-12], in which the connectivity of the disulfide-linked ring has been reversed by formally cleaving the ring and cyclizing the N- and C-terminal tails. This analog, which retains many of the spatial relationships of the native molecule, binds to both ANP-A and ANP-C receptor subtypes, and triggers the production of cyclic-GMP by ANP-A. ANP-C binding of ANP[13-27][1- 12] is roughly equipotent to that of ANP itself, although the ring cleavage falls within the putative ANP-C binding domain. ANP[13-27][1-8], a truncated analog in which much of this binding domain has been removed, surprisingly maintains a high affinity for ANP-C; however, this peptide has lost the ability to activate the ANP-A-linked guanylate cyclase.     

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.     

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.     

Characterization of the functional domains of the natriuretic peptide receptor/guanylate cyclase by radiation inactivation.

Radiation inactivation has been used to evaluate the molecular size of domains responsible for atrial natriuretic peptide (ANP)-binding and cyclase functions of the ANP receptor/guanylate cyclase. Two types of inactivation curves were observed for cyclase function in both adrenal cortex and aortic smooth muscle cells: 1) biphasic with enhanced guanylate cyclase activity after exposure to low radiation doses and 2) linear after preincubation of membrane proteins with 0.5 microM ANP or solubilization with Triton X-100. The existence of an inhibitory component was the simplest model that best explained the types of radiation curves obtained. Activation of guanylate cyclase by ANP or Triton X-100 could occur via the dissociation of this inhibitory component from the catalytic domain. On the other hand, the loss of ANP-binding activity was linear with increasing radiation exposures under basal, ANP treatment, and Triton X-100 solubilization conditions. Radiation inactivation sizes of about 30 kDa for cyclase function, 20 kDa for ANP-binding function, and 90 kDa for inhibitory function were calculated. These studies suggest that the ANP receptor/guanylate cyclase behaves as a multidomain protein. The results obtained by radiation inactivation of the various biological functions of this receptor are compatible with the hypothesis of an intramolecular inhibitory domain repressing the guanylate cyclase catalytic domain within its membrane environment.     

Expression of mRNA for atrial natriuretic peptide receptor guanylate cyclase (ANPRA) in human retina

Summary 1. Guanylate cyclase plays an important role in the visual cycle. Here we report the mRNA expression for the atrial natriuretic peptide receptor type A form of guanylate cyclase (ANPRA) in human retina.2. Polymerase chain reaction using two sets of primers on the cDNAs reverse-transcribed from human retinal poly(A)⁺ RNA amplified two products under two different reaction conditions. The primers used in the reaction were designed from the reported sequence of human placental ANPRA cDNA.3. Sequencing of the amplified products showed 100% sequence homology to the human placental ANPRA gene. Northern blot analysis indicated the presence of a 4.4-kb ANPRA mRNA in human retina, similar to that present in human brain.     

C-type natriuretic peptide decreases soluble guanylate cyclase levels by activating the proteasome pathway

Natriuretic peptides (NP) activate particulate guanylate cyclase (pGC) and nitric oxide (NO) activates soluble guanylate cyclase (sGC). Both guanylate cyclases catalyse the formation of the same second messenger, cyclic guanosine 3',5'-monophosphate (cGMP), which activates the cGMP-dependent protein kinases (PKG). PKG then starts a signalling cascade that mediates many cardiovascular and renal effects, such as smooth muscle relaxation and diuresis. Many cell types possess both sGC and pGC. Because both GC-cGMP systems play complementary roles, an interaction between the two pathways might represent an important physiological control mechanism. In this report we demonstrate an interaction between the two pathways. C-type natriuretic peptide (CNP) decreased the beta-subunit of sGC (sGC-beta) steady-state protein levels and enzymatic activity in cultured human mesangial cells (HMC) in a time- and dose-dependent manner. This down-regulation was not dependent on changes in sGC-beta mRNA levels. Treatment of the cells with the stable cGMP analogue 8-Br-cGMP or the phosphodiesterase type-5 inhibitor Zaprinast produced the same down-regulatory effect. Inhibition of PKG or proteasome activity prevented the CNP-induced reduction of sGC-beta protein levels and activity. Taken together, these results demonstrate that pGC activation induces a post-transductional down-regulation of sGC by a mechanism involving PKG and the proteasome pathway.     

Phosphorylation of high- and low-molecular-mass atrial natriuretic peptide analogs by cyclic AMP-dependent protein kinase

Synthetic high- and low-molecular-mass atrial peptides were phosphorylated in vitro by cyclic AMP-dependent protein kinase and [32P]ATP. From a series of atrial peptide analogs, it was deduced that the amino acid sequence, Arg101-Ser104 of atriopeptin was required for optimal phosphorylation. Phosphorylated AP(99-126) was less potent than the parent atriopeptin in vasorelaxant activity and receptor-binding properties. These results indicate that the presence of a phosphate group at the N-terminus of AP(99-126) decreases the interaction of the peptide with its receptor and, as a consequence, decreases bioactivity. These observations are in contrast to those of Rittenhouse et al. [(1986) J. Biol. Chem. 261, 7607-7610] who reported that phosphorylation of AP(101-126) enhanced the stimulation of Na/K/Cl cotransport in cultured vascular smooth muscle cells.     

Atrial natriuretic peptide and guanylin-activated guanylate cyclase isoforms in human sweat glands

The ultracytochemical localization of membrane-bound guanylate cyclases A and C, stimulated by atrial natriuretic peptide and guanylin respectively, has been studied in human sweat glands. The results showed that the peptides stimulated guanylate cyclases A and C in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was present on the plasma membranes and on intracellular membranes involved in the secretory mechanism. In eccrine glands, the cells of the excretory duct also presented enzymatic activity on the plasma membranes. In both glands, myoepithelial cells, surrounding the secretory cells, exhibited only guanylate cyclase A activity. These localizations of enzymatic activity suggest a role for both atrial natriuretic peptide and guanylin in regulating glandular secretion.     

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.     

Atrial natriuretic peptide receptors and activation of guanylate cyclase in rat cardiac sarcolemma

Two classes of atrial natriuretic peptide (ANP) receptors are present in purified sarcolemmal membrane fractions isolated from rat ventricle. Scatchard analysis using [125I]-ANP reveals high affinity (Kd approximately 10(-11) M) and low affinity (Kd approximately 10(-9) M) binding sites. Basal guanylate cyclase activities associated with these membrane fractions range from 3.2 +/- 1.3 pmol/min/mg protein in the presence of Mg2+ to 129 +/- 17 pmol/min/mg protein in the presence of Mn2+. Millimolar concentrations of adenosine triphosphate (ATP) potentiates Mg2+- but not Mn2+-supported activity. Binding of ANP to the low affinity site but not the high affinity site results in a maximum 2-fold activation of Mn2+- and up to 6-fold activation of Mg2+/ATP supported guanylate cyclase activities.     

Guanylyl cyclase / atrial natriuretic peptide receptor-A: role in the pathophysiology of cardiovascular regulation

Atrial natriuretic factor (ANF), also known as atrial natriuretic peptide (ANP), is an endogenous and potent hypotensive hormone that elicits natriuretic, diuretic, vasorelaxant, and anti-proliferative effects, which are important in the control of blood pressure and cardiovascular events. One principal locus involved in the regulatory action of ANP and brain natriuretic peptide (BNP) is guanylyl cyclase / natriuretic peptide receptor-A (GC-A/NPRA). Studies on ANP, BNP, and their receptor, GC-A/NPRA, have greatly increased our knowledge of the control of hypertension and cardiovascular disorders. Cellular, biochemical, and molecular studies have helped to delineate the receptor function and signaling mechanisms of NPRA. Gene-targeted and transgenic mouse models have advanced our understanding of the importance of ANP, BNP, and GC-A/NPRA in disease states at the molecular level. Importantly, ANP and BNP are used as critical markers of cardiac events; however, their therapeutic potentials for the diagnosis and treatment of hypertension, heart failure, and stroke have just begun to be realized. We are now just at the initial stage of molecular therapeutics and pharmacogenomic advancement of the natriuretic peptides. More investigations should be undertaken and ongoing ones be extended in this important field.