Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase.

We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37 degrees C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 +/- 0.077 nM and the Bmax. was 216 +/- 33 fmol bound/10(6) cells, which corresponds to 130,000 +/- 20,000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 microM-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increased the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demonstrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.     

Inhibition of atrial natriuretic peptide-induced cyclic GMP accumulation in the bovine endothelial cells with anti-atrial natriuretic peptide receptor antiserum

Using an antiserum raised against the purified atrial natriuretic peptide (ANP) receptor that has a disulfide-linked homodimeric structure and represents one subtype of the multiple ANP receptors, we showed that the receptor is coupled to the guanylate cyclase activation; formerly, this type of ANP receptor is not considered to be coupled to the cyclase. The specificity of the antiserum was determined by immunoblot analysis and immunoprecipitation. The anti-receptor antiserum did not compete with 125I-ANP for binding to the receptor but it lowered the affinity of the receptor. When added to bovine endothelial cell cultures, the antiserum blocked the cyclic GMP response of the cells triggered by ANP. These results indicate that the subtype of the ANP receptor recognized by the antiserum is responsible for the activation of particulate guanylate cyclase as well as the double function type receptor that has been assumed to contain both the receptor domain and the catalytic domain for cGMP synthesis on the same molecule. The presence of dissociative complexes of ANP receptor and particulate guanylate cyclase was also demonstrated by radiation inactivation analysis.     

Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM. Stimulation was also present but less substantial (2-fold at 5 microM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin.     

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.     

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.     

Atrial Natriuretic Factors Stimulate Accumulation and Efflux of Cyclic GMP in C6–2B Rat Glioma and PC12 Rat Pheochromocytoma Cell Cultures

Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of platelet aggregation by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.     

An oxidized analog of alpha-human atrial natriuretic polypeptide is a selective agonist for the atrial-natriuretic-polypeptide clearance receptor which lacks a guanylate cyclase.

The differences in biological functions between alpha-human atrial natriuretic polypeptide (alpha-hANP) and its oxidized analog, MetSO-alpha-hANP, have been investigated. Analysis of the ANP receptor subtypes by affinity labeling has shown that a bovine pulmonary aortic endothelial cell line (CPAE cells) primarily expresses ANP-R1 (R, receptor) coupled to particulate guanylate cyclase, while Hela cells from human cervical carcinoma predominantly express ANP-R2, which lacks a guanylate cyclase. alpha-hANP could bind to both ANP receptor subtypes with high affinity, while MetSO-alpha-hANP showed more selective binding to ANP-R2 than to ANP-R1. The activity of MetSO-alpha-hANP for stimulation of guanylate cyclase coupled to ANP-R1 was about 520-fold less than that of alpha-hANP (median effective dose = 2.5 nM for alpha-hANP, 1.3 microM for MetSO-alpha-hANP), indicating that MetSO-alpha-hANP was a partial agonist for this receptor. While this oxidized analog could inhibit the cAMP production through ANP-R2, with 0.15 times the activity of alpha-hANP (median concentration = 0.31 nM for alpha-hANP, 2.0 nM for MetSO-alpha-hANP). In in vivo studies, the diuretic activity of MetSO-alpha-hANP was 25-100-fold less than that of alpha-hANP. In addition, MetSO-alpha-hANP could potentiate the diuretic activity of alpha-hANP that was also caused by C-ANF4-23, a specific agonist for ANP-R2. These results demonstrate that MetSO-alpha-hANP can act as an agonist more selective for ANP-R2 than for ANP-R1, both in vivo and in vitro. The relationship between receptor selectivities and the conformation of alpha-hANP or MetSO-alpha-hANP was also discussed.     

A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps).

This paper describes the purification, sequence, and biological properties of a 38-amino acid residue peptide from the venom of Dendroaspis angusticeps which shared important sequence homologies with natriuretic peptides. Dendroaspis natriuretic peptide (DNP) relaxed aortic strips that had been contracted by 40 mM KCl with a potency (K0.5 = 20 nM) similar to that of atrial natriuretic peptide (ANP) and larger than that of C type natriuretic peptide (CNP). The relaxing actions of ANP and DNP (both at 100 nM) were mutually exclusive. Bovine aortic endothelial cells responded to ANP (K0.5 = 3 nM) and DNP (K0.5 = 3 nM) but not to CNP by a large activation of guanylate cyclase. Rat aortic myocytes showed larger cGMP responses to CNP (K0.5 = 10 nM) than to ANP or DNP (K0.5 = 100 nM). Finally, DNP completely prevented the specific 125I-ANP binding to clearance receptors in cultured aortic myocytes with a potency (Kd = 10 nM) that was less than that of ANP (Kd = 0.3 nM). It is concluded that DNP is a new member of the family of natriuretic peptides and that it recognizes ANPA receptors and clearance, ANPc receptors, but not CNP-specific ANPB receptors.     

Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells

In a previous study, we found that atriopeptin I was much weaker (EC50 greater than 500 nM) than atrial natriuretic factor (ANF-(8-33)) (EC50 = 0.3 nM) at increasing cyclic GMP in cultured endothelial cells. In this study, we used the cross-linking reagent disuccinimidyl suberate to investigate whether the differences in activity were due to the presence of multiple ANF receptors. When 98% of the ANF-binding sites on endothelial cells were occupied by tyrosine-atriopeptin I after cross-linking, there was no difference in the concentration-response curve to ANF-(8-33) with regard to cyclic GMP accumulation. In contrast, when 96% of the binding sites were occupied by cross-linked ANF-(8-33), a 60% decrease in the maximal cyclic GMP response was observed after the readdition of ANF-(8-33). These results suggest that ANF-(8-33) is binding to an additional site that atriopeptin I does not effectively bind. Affinity cross-linking of 125I-ANF to intact endothelial cells resulted in the labeling of two sites of Mr approximately 66,000 and approximately 130,000. Approximately 94% of the 125I-ANF binding sites had an Mr approximately 66,000. Labeling of this site was inhibited by both tyrosine-atriopeptin I (KI = 0.9 nM) and ANF-(8-33) (KI = 0.09 nM). Although 0.1 microM tyrosine-atriopeptin (AP I) inhibited labeling of the 66,000-dalton site to nearly the same degree as ANF-(8-33), it produced only a 4-fold increase in cyclic GMP compared to a 400-fold increase with ANF-(8-33). These results suggest that the 66,000-dalton site is not coupled to guanylate cyclase and cyclic GMP formation. Tyrosine-AP I (KI greater than 10 nM) was much weaker at competing for the 130,000-dalton site than ANF-(8-33) (KI = 0.075 nM). Because the EC50 for cyclic GMP stimulation for tyrosine-AP I (greater than 100 nM) and ANF-(8-33) (0.4 nM) is closer to the KI values for the 130,000-dalton protein, this site probably mediates the marked stimulation of cyclic GMP. Our results demonstrate that endothelial cells contain two binding sites for ANF-(8-33) and suggest that only the less abundant site (Mr approximately 130,000) is the receptor coupled to the activation of guanylate cyclase.     

Biochemical studies of soluble atrial natriuretic peptide (ANP) receptors from rat olfactory bulb and vascular smooth muscle cells

1. Aim. The biochemical characteristics of atrial natriuretic peptide receptors (ANP-R) derived from rat vascular smooth muscle (A-10 cell line) and central nervous system (CNS; olfactory bulb) tissue were compared. 2. Method and Results. ANP-Rs from each source were solubilized with 40 to 65% efficiency utilizing the nonionic detergent Lubrol-PX. Upon solubilization, the ANP-R from each source maintained the ability to bind 125I-ANP (99-126) with a high affinity; Scatchard analysis indicated that the VSMC ANP-R displayed a Kd for the radioligand of approximately 10 pM, whereas the olfactory receptor possessed a Kd of about 165 pM. The Bmax values for the soluble VSMC and olfactory ANP-Rs were 285 and 30 fmol/mg protein, respectively. Competition binding studies indicated that the VSMC ANP-R bound ANP(99-126), ANP(103-126), and ANP(103-123) with similar affinities, whereas the olfactory ANP-R was much more sensitive to changes in the COOH-terminal structure of the competing peptide. The soluble ANP-Rs from VSMC and olfactory were chromatographically indistinguishable on phenyl-, DEAE-, and wheat germ agglutinin-agarose columns. However, the ANP-Rs could be distinguished using GTP-agarose; the olfactory ANP-R was capable of binding to the resin, whereas the VSMC ANP-R was not. 3. Conclusions. Coupled with other studies, these data suggest that the A10 VSMC ANP-R observed in this study may not be coupled to guanylate cyclase and may represent a receptor serving a clearance function, whereas a significant proportion of the olfactory CNS ANP-R appears to be associated with GTP-binding proteins, likely particulate guanylate cyclase, and probably represents a coupled form of the receptor.     

Identification and characterization of parathyroid hormone receptors on dog kidney, human kidney, chick bone and human dermal fibroblast. A comparative study of functional and structural properties.

To evaluate the functional and structural characteristics of the parathyroid hormone (PTH) receptors on different tissues and the possible heterogeneity in structure and function, PTH receptors on dog kidney membrane, human kidney membrane, chick bone cell membrane and human dermal fibroblast membrane were evaluated. The results showed that human kidney plasma membrane, canine kidney plasma membrane and chick bone cell membrane possess one single class of PTH receptor with a Kd (dissociation constant) of 1-5 nM and an IC50 also of 1-5 nM. The number of binding sites was 800 fmol per mg of protein for chick bone cell particulate membrane, 1-5 pmol per mg of protein for human kidney plasma membrane and 2.2 pmol per mg of protein for dog kidney plasma membrane. Photoaffinity labelling identified a major binding component with a molecular mass of 70 kDa in all three types of membrane. The plasma membrane fraction from human dermal fibroblast contained two different binding sites for PTH with high (Kd = 2 nM) and low (Kd = 580 nM) affinities respectively. The IC50 for the adenylate cyclase is about 2 nM, which is similar to the Kd of the high-affinity site. Photoaffinity labelling also demonstrated a major binding component with a molecular weight of 70 kDa. We conclude that structural and functional similarity exists among the PTH receptors present on chick bone cell membrane, dog kidney membrane and human kidney membrane. The human dermal fibroblast possesses two different binding sites, one of which is coupled to adenylate cyclase.     

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.     

Natriuretic peptide receptors in cultured rat diencephalon

To characterize the type of cell expressing natriuretic peptide receptors in the brain and the nature of these receptors, we conducted studies in primary cultured glial and neuronal cells derived from fetal rat diencephalon. The glial predominant cultures (95% of total cells and glial fibrillary acidic protein positive) expressed nearly a 10-fold greater specific binding of the natriuretic peptides to cell surface receptors compared with the neuron-predominant cultures. Scatchard analysis of binding studies with 125I-atrial natriuretic peptide (ANP) and 125I-brain natriuretic peptide (BNP) revealed a single class of receptors with dissimilar affinities (0.25 +/- 0.09 and 0.74 +/- 0.07 nM, respectively, n = 3 experiments p less than 0.01) but similar numbers of binding sites for both peptides (93 and 88 fmol/mg of protein, respectively). Cross-linking of 125I-ANP and BNP to cultured glia followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography identified distinct bands at either approximate Mr 130,000, or 102,000 and 66,000, corresponding to two high molecular weight (B) receptors and one low molecular weight (C) receptor described in other tissues. Different subtypes of astrocytes appeared to express different B receptors. Binding and cross-linking of radiolabeled ANP or BNP were competitively inhibited equally by unlabeled ANP or BNP, indicating that ANP and BNP probably bind the same receptors. The glial cultures functionally expressed a receptor(s) with guanylate cyclase activity; BNP was less potent than ANP in stimulating cGMP at lower concentrations. These results indicate that both high and low molecular weight natriuretic peptide receptors are expressed in astrocyte-predominant cultures from the fetal diencephalon and suggest that glia participate in several actions of ANP which are probably mediated through this area of the brain.     

Effects of atriopeptin on particulate guanylate cyclase from rat adrenal

Atriopeptin II activated particulate guanylate cyclase 5-10-fold in a concentration- and time-dependent fashion in crude membranes obtained from homogenates of rat adrenal cortex or medulla. Similar effects were observed with other atriopeptin analogs. Soluble guanylate cyclase and adenylate cyclase in these preparations were not activated. Accumulation of cyclic GMP in minces of adrenal cortex or medulla was increased 6-8-fold due to atriopeptin II activation of particulate guanylate cyclase. Several thiol-reactive agents blocked the activation of particulate guanylate cyclase, suggesting that free thiol groups on membrane proteins may be important in atriopeptin receptor-guanylate cyclase coupling.     

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.     

Human prepro atrial natriuretic factors 26-55, 56-92, and 104-123 increase renal guanylate cyclase activity

Human prepro atrial natriuretic factors 26-55, 56-92, and 104-123 as well as human atrial natriuretic factor (4-28) in the present investigation increased renal cortical and medullary cyclic GMP levels and maximally enhanced particulate guanylate cyclase activity [E.C. 4.6.1.2] two-fold in whole kidney homogenates, renal cortical and medullary membranes, and in isolated distal nephrons at their 1 microM concentrations. Dose-response relationships revealed that the half maximal [ED50] activation of guanylate cyclase was at their 10 nM concentrations in rat, rabbit, and dog kidneys. Both human atrial natriuretic factor and the prepro factors decreased adenylate cyclase activity. These results suggest that prepro factors 26-55, 56-92, 104-123 may also be functionally active.     

Atrial natriuretic peptide induces breakdown of phosphatidylinositol phosphates in cultured vascular smooth-muscle cells

Discrepancies exist between extent of guanylate cyclase activation by atrial natriuretic peptide (ANP) in cell-free systems and ANP-stimulated levels of cyclic GMP in whole cells, and also between receptor affinity and dose effectiveness of ANP. Therefore, we have investigated whether, in addition to receptor-coupled guanylate cyclase activation, other second-messenger cascade systems may be involved in mediating both an increase in cyclic GMP and the physiological response to ANP. Equilibrium 125I-ANP binding studies on cultured thoracic aorta smooth muscle cells revealed the existence of low-affinity (approximately 10(-8) M, 84.5 fmol/10(5) cells) and high-affinity (approximately 10(-10) M, 12.5 fmol/10(5) cells) binding sites. We confirm that ANP elevates intracellular cyclic GMP (EC50 approximately 10(-8) M) and inhibits agonist-(isoproterenol and forskolin)-induced increases in intracellular cyclic AMP (IC50 approximately 10(-9) M). ANP also stimulated breakdown of phosphatidylinositol phosphates and generation of inositol phosphates with a half-maximally effective concentration of approximately 10(-10) M. The extent of phosphatidylinositol polyphosphate hydrolysis was small (120%) in comparison to that of phosphatidylinositol (Ptd-Ins) (200%). Ptd-Ins hydrolysis was paralleled by the appearance of glycerophosphoinositol, and there was also a close temporal relationship between these processes and the accumulation of intracellular cyclic GMP. Smooth muscle cells released [3H]arachidonic acid label in response to ANP (EC50 approximately 10(-10) M). Taken together, the data suggest that the vasorelaxant hormone ANP has stimulatory effects on phosphoinositol lipid metabolism via both phospholipase C (generation of inositol phosphates) and phospholipase A2 (generation of releasable [3H]arachidonic acid and indirectly glycerophosphoinositol). In contrast, stimulation of phosphatidylinositol phosphate breakdown by the vasoconstrictive hormone angiotensin II is not associated with glycerophosphoinositol formation, and neither cyclic GMP nor cyclic AMP levels were influenced by this hormone.     

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.