Stimulation Of Cyclic GMP Research Articles

Malignant glioma progression and nitric oxide

Glioblastoma multiforme, the most common of the malignant gliomas, carries a dismal prognosis in spite of the most aggressive therapy and recent advances in molecular pathways of glioma progression. Although it has received relatively little attention in the setting of malignant gliomas, nitric oxide metabolism may be intimately associated with the disease process. Interestingly, nitric oxide has both physiological roles (e.g., neurotransmitter-like activity, stimulation of cyclic GMP), and pathophysiological roles (e.g., neoplastic transformation, tumor neovascularization, induction of apoptosis, free radical damage). Moreover, whether nitric oxide is neuroprotective or neurotoxic in a given disease state, or whether it enhances or diminishes chemotherapeutic efficacy in malignant neoplasia, is unresolved. This review discusses the multifaceted activity of nitric oxide with particular reference to malignant gliomas.

Signal transduction underlying carbachol-induced PGE 2 generation and cox-1 mRNA expression of rat brain

In this paper we have determined the different signal pathways involved in M 1 and M 3 muscarinic acetylcholine receptor (mAChR) dependent stimulation of cyclo-oxygenase 1 (cox-1) mRNA gene expression and PGE 2 production on rat cerebral frontal cortex. Carbachol stimulation of M 1 and M 3 mAChR exerts an increase in cox-1 mRNA gene expression without affecting cox-2 mRNA expression and increased PGE 2 generation. Besides, increased phosphoinositide (PI) turnover and stimulation of nitric oxide synthase (NOS) and cyclic GMP (cGMP) production. Inhibitors of phospholipase A 2 (PLA 2), COX and phospholipase C (PLC), calcium/calmodulin (CaM), NOS and soluble guanylate cyclase prevent the carbachol effect. These results suggest that carbachol-activation of M 1 and M 3 mAChR increased PGE 2 release associated with an increased expression of cox-1 and NO–cGMP production. The mechanism appears to occur directly to PLC stimulation and indirectly to PLA 2 activation. These results may contribute to understand the effects and side effect of non-steroidal anti-inflammatory drugs in patients with cerebral degenerative diseases.

H(1)-Receptor activation triggers the endogenous nitric oxide signalling system in the rat submandibular gland.

BACKGROUND: Histamine is released from mast cells by immunologic and non-immunologic stimuli during salivary gland inflammation, regulating salivary secretion. The receptor-secretory mechanism has not been studied in detail. AIMS: The studies reported were directed toward elucidating signal transduction/second messenger pathways within the rat submandibular gland associated with 2-thiazolylethylamine (ThEA)-induced H(1)-receptor responses. MATERIALS AND METHODS: To assess the H(1) receptor subtype expression in the rat submandibular gland, a radioligand binding assay was performed. The study also included inositolphosphates and cyclic GMP accumulation, protein kinase C and nitric oxide synthase activities, and amylase release. RESULTS: The histamine H(1) receptor subtype is expressed on the rat submandibular gland with high-affinity binding sites. The ThEA effect was associated with activation of phosphoinositide-specific phospholipase C, translocation of protein kinase C, stimulation of nitric oxide synthase activity and increased production of cyclic GMP. ThEA stimulation of nitric oxide synthase and cyclic GMP was blunted by agents able to interfere with calcium movilization, while a protein kinase C inhibitor was able to stimulate ThEA action. On the other hand, ThEA stimulation evoked amylase release via the H1 receptor but was not followed by the L-arginine/nitric oxide pathway activation. CONCLUSIONS: These results suggest that, apart from the effect of ThEA on amylase release, it also appears to be a vasoactive chemical mediator that triggers vasodilatation, modulating the course of inflammation.

Exogenous nitric oxide inhibits apoptosis in guinea pig gastric mucous cells

BACKGROUNDIncreased nitric oxide (NO) synthase activity and enhanced apoptosis are features of gastric mucosa infected with Helicobacter pylori and a causative relation has been suggested. However, although NO can promote...

Endothelin stimulates nitric oxide-dependent cyclic GMP formation in rat cerebellar astroglia.

Endothelins (ETs) elicit a diversity of cellular responses in cultured astrocytes that suggest an important role of these peptides in glial cell function. Stimulation of astroglial ET receptors induces phosphoinositide (PI) hydrolysis and intracellular calcium mobilization, but little is known about the signalling events that occur downstream of this system. Here we show that in rat cerebellar astroglia in culture ETs produce a receptor-mediated stimulation of cyclic GMP (cGMP) formation that is rapid and totally dependent on nitric oxide synthase (NOS) activity. The effect is blocked by an inhibitor of PI phospholipase C, compound U73122, and by depletion of intracellular calcium stores with thapsygargin. These results indicate that calcium released by inositol trisphosphate is responsible for NOS activation and subsequent cGMP formation.

Role of K+ channel opening and stimulation of cyclic GMP in the vasorelaxant effects of nicorandil in isolated piglet pulmonary and mesenteric arteries: relative efficacy and interactions between both pathways.

1. The effects of the K+ channel opener levcromakalim, the guanylate cyclase stimulant nitroprusside and the dual drug nicorandil (K+ channel opener and guanylate cyclase stimulant) were analysed in piglet isolated endothelium-denuded pulmonary (PA) and mesenteric (MA) arteries stimulated by noradrenaline (NA) or by the thromboxane A2 mimetic U46619. 2. Nicorandil, levcromakalim and verapamil were less potent in PA than in MA, the efficacy of levcromakalim was also reduced in PA. The effects of nicorandil and levcromakalim were similar in arteries pre-contracted by NA and U46619, whereas verapamil was more potent in arteries pre-contracted by NA. Nitroprusside was equipotent in MA pre-contracted by either NA or U46619 and in PA pre-contracted by NA whereas in PA pre-contracted by U46619, nitroprusside showed lower potency and efficacy. 3. The relaxant effects of levcromakalim and nitroprusside were inhibited by 10(-5) M glibenclamide and 10(-6) M ODQ, respectively. Nicorandil-induced relaxation was inhibited by ODQ in all experimental conditions, whereas glibenclamide had inhibitory effects in PA and MA pre-contracted by U46619, had no effect in PA pre-contracted by NA and in MA pre-contracted by NA it was only inhibitory in the presence of ODQ. 4. No apparent interactions were found between nitroprusside and levcromakalim as indicated by the lack of effects of pretreatment with one of them (producing 20-35% relaxation) on the potency of the relaxant response to the other. However, in PA pre-contracted by U46619, where nitroprusside or levcromakalim induced only partial relaxation, the combination of both mechanisms (either by combining nitroprusside plus levcromakalim or by nicorandil) was able to induce full vasodilatation. 5. In conclusion, K+ channel opening and guanylate cyclase stimulation are independent pathways that induce additive vasorelaxation in piglet PA and MA. The mechanism of action of nicorandil is dependent on the artery and on the nature of the agonist employed to precontract the artery. The relative efficacy of K+ channel opening vs guanylate cyclase stimulation may partially explain the preferential contribution of each mechanism to the relaxant effects of nicorandil.

Reexamination of opioid stimulation of cGMP formation in cell lines of neuronal origin.

1. The present study reexamines a previous notion on opioid stimulation of cyclic GMP (cGMP) formation and the retraction of the original findings. 2. The effect of opioid agonists on cGMP accumulation in two cell lines of neuronal origin was measured. The proportion of cGMP stimulation in NG108-15 neuroblastoma x glioma hybrid cells resembled the proportion of [Ca2+]in elevation by opioids in this culture. The failure of opioids to stimulate cGMP formation in SK-N-SH human neuroblastoma coincided with the lack of cGMP stimulation by other Ca2+ mobilizing agents in these cells. The nitric oxide donor nitroprusside elevated cGMP in both cell lines. 3. The implication of the opioid-Ca(2+)-NO-cGMP cellular pathway for opioid activity in vivo is discussed.

Tricyclic Antidepressants Inhibit Human Natural Killer Cells

The commonly used antidepressants imipramine, amitriptyline, and nortriptyline were found to significantly inhibit human natural killer (NK) cell-mediated cytolysisin vitroand suppress the stimulation of NK cells by IFN-γ. This is a previously unrecognized biologic property of these drugs with psychotropic activity. Tricyclic antidepressants did not decrease effector–target cell conjugation formation, nor did they induce target cell resistance to NK lysis, indicating that the drugs might interfere with the killing mechanism of the effector cells. Kinetic data reveal that the drug interference is related to an early postbinding event in the activation of NK cells. Results also showed that the inhibitory effect of tricyclic antidepressants on human NK cell activity occurred in parallel to an increase in intracellular cyclic GMP concentration. However, the attenuation in the cyclic GMP formation by methylene blue, a selective inhibitor of soluble guanylate cyclase, was not accompanied by a corresponding increase in NK cell cytolytic activity. It is suggested that the stimulation of cyclic GMP was not directly involved in the inhibitory effect of antidepressants on NK cells and perhaps was a secondary phenomenon. This immune cell modulatory property of tricyclic antidepressants seems to indirectly provide evidence for the concept that human brain neurons and NK cells might share regulatory system(s).

Agonist properties of a stable hexapeptide analog of neurotensin, NαMeArg-Lys-Pro-Trp-tLeu-Leu (NT1)

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca 2+ ([Ca 2+] i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8–13), N α MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca 2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca 2+-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities ( B max) of 130.2 and 87.5 fmol/mg protein, respectively. The respective K D values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present ( K I > 10,000) in either membrane preparation. Although the correlation of binding betwen HT-29 cell membranes and mouse brain membranes was quite significant ( r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8–13), xenopsin, neuromedin N, NT(9–13), kinetensin and ( d-Trp 11)-NT] increased intracellular Ca 2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca 2+] i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Segmental differences in the effects of guanylin and Escherichia coli heat‐stable enterotoxin on Cl‐ secretion in human gut.

1. Mucosally added synthetic guanylin and Escherichia coli heat-stable enterotoxin (STa) increased short-circuit current (ISC) across isolated muscle-stripped human intestine in vitro. 2. Serosal bumetanide inhibited ISC responses indicating that guanylin and STa stimulate electrogenic chloride secretion. 3. ISC responses were markedly greater in the colon than in the jejunum. 4. Pretreatment with indomethacin did not significantly alter the effects of guanylin and STa. 5. Both peptides induced concentration-dependent increases in the cyclic GMP content of human intestinal mucosa in vitro; cyclic AMP levels remained unchanged. 6. In contrast to ISC responses, increases in cyclic GMP content induced by guanylin and STa were markedly greater in the jejunum than in the colon. 7. Sodium nitroprusside (SNP) but not human alpha-atrial natriuratic peptide (CDD/ANP(99-126)) increased chloride secretion in human intestine; both agents induced small increases in intestinal cyclic GMP content. 8. Guanylin, STa and the nitric oxide (NO) donor SNP increased electrogenic chloride secretion across human intestinal mucosa in vitro by stimulation of cyclic GMP. The discrepancy between the effects on chloride secretion and intracellular cyclic GMP content suggest different cellular action sites of guanylin and STa in human small and large intestine.

Muscarinic cholinergic stimulation of the nitric oxide-cyclic GMP signaling system in cultured rat sensory neurons

Acetylcholine or carbachol stimulated cyclic GMP production in neuronal cultures from embryonic rat dorsal root ganglia but not in non-neuronal dorsal root ganglia cultures. Acetylcholine stimulation of cyclic GMP production was mediated by muscarinic receptors and required extracellular Ca 2+. Basal cyclic GMP production and acetylcholine-evoked cyclic GMP production were attenuated by Methylene Blue, suggesting the involvement of soluble guanylate cyclase and nitric oxide synthase. l- N G-Monomethyl arginine attenuated basal, acetylcholine or carbachol-stimulated cyclic GMP production; this inhibition of acetylcholine and carbachol stimulation of cyclic GMP was reversed by l-arginine. These results suggest that a nitrosyl factor mediates basal, as well as acetylcholine- and carbachol-stimulated, cyclic GMP production. Selective destruction of small diameter neurons by capsaicin pretreatment of dorsal root ganglion neuronal cultures abolished acetylcholine and capsaicin stimulation of cyclic GMP, but did not affect sodium nitroprusside stimulation of cyclic GMP. These results suggest that acetylcholine evoked production of a nitrosyl factor in capsaicin-sensitive (small diameter) sensory neurons, which subsequently stimulated a soluble guanylate cyclase and cyclic GMP production in adjacent neuronal and/or non-neuronal cells. These results demonstrate that muscarinic agonists stimulate the nitric oxide-cyclic GMP signalling system in capsaicin-sensitive sensory neurons. Thus, the noxious character of acetylcholine when administered peripherally may be mediated by nitric oxide-cyclic GMP.

Regulation of natriuretic peptide receptor A and B expression by transforming growth factor-beta 1 in cultured aortic smooth muscle cells.

Two types of natriuretic peptide receptors (NPR-A and NPR-B) are membrane guanylate cyclases whose relative expression varies in different tissues. Because natriuretic peptides have been shown to inhibit aortic smooth muscle proliferation, we investigated the regulation of NPR-A and NPR-B in these cells under different proliferative conditions. NPR subtype mRNA levels were measured by our newly developed quantitative reverse transcription-polymerase chain reaction assay using mutated NPR-A and NPR-B cRNA as internal standards. The functional impact of their expression was determined by atrial natriuretic peptide (ANP)- and C-type natriuretic peptide (CNP)-induced stimulation of cyclic GMP production. In the intact aorta, NPR-B mRNA levels were found to be 10-fold higher than those of NPR-A. This dominance was further amplified (1000-fold) in long-term cultures (10 to 15 passages) of aortic smooth muscle cells (ASMC). Higher cyclic GMP production with CNP than with ANP was observed in cultured ASMC from Wistar-Kyoto (WKY) rats. Similar stimulation by the two agonists was noted in spontaneously hypertensive rat (SHR) cells, paralleled by a 10-fold increase in NPR-A mRNA levels and ANP stimulation of cyclic GMP in hypertensive cells. The present study also evaluated NPR-A and NPR-B mRNA control by transforming growth factor-beta 1 (TGF-beta 1), an important regulator of cell proliferation that is overexpressed in SHR ASMC. TGF-beta 1 decreased both NPR-A and NPR-B mRNA levels with a predominant effect in SHR cells at high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct inhibition of platelet function by organic nitrates via nitric oxide formation

This study investigates the mechanisms of platelet inhibition by the nitrate esters isosorbide dinitrate, isoidide dinitrate, isomannide dinitrate, isosorbide 2-mononitrate and isosorbide 5-mononitrate as compared to the spontaneous nitric oxide (NO)-donor linsidomine, the active metabolite of molsidomine. Nitrates and linsidomine dose-dependently inhibited aggregation, ATP secretion and thromboxane formation of washed human platelets at a rank order of potency, identical with that for stimulation of cyclic GMP in cultured rat lung fibroblasts. While linsidomine (0.1 mM) caused a 3-fold platelet cGMP elevation, there was a weak (≦ 30%) but significant cGMP stimulation by organic nitroesters, which was tightly correlated with inhibition of platelet aggregation ( r = 0.926, P = 0.008). Zaprinast (2 μM) potentiated, while methylene blue (1 μM) and oxyhemoglobin (10 μM) reversed the antiaggregatory effects. Linsidomine (0.5 μM−0.1 mM) dose-dependently released NO in a cell-free system. No spontaneous NO release was detected with organic nitroesters (0.1 mM). These data suggest that, to some extent, bioactivation of organic nitroesters occurs in platelets resulting in platelet inhibition via the NO/cGMP system.

Heterogeneous regulation of renal atrial natriuretic factor receptor subtypes in one-kidney, one clip hypertensive rats.

To investigate the regulation of papillary atrial natriuretic factor (ANF) receptors and the differential regulation of glomerular ANF receptor subtypes in one-kidney, one clip (1-K,1C) hypertension. Plasma immunoreactive ANF levels and atrial and ventricular ANF content were measured in 1-K,1C rats and their normotensive uninephrectomized controls. Glomerular and papillary ANF receptor subtypes were characterized by competitive radioligand binding assay. Stimulation of cyclic GMP (cGMP) production, by increasing ANF doses, was examined in isolated glomeruli. Plasma amino-terminal ANF levels were higher in 1-K,1C than in control rats. ANF concentrations in the left atrium were lower and ventricular ANF concentrations were higher in the 1-K,1C group. Competition binding curves for glomerular membranes revealed the presence of two binding sites (ANF-R1 and ANF-R2) in both groups. Total glomerular ANF receptor density was lower in 1-K,1C rats than in their normotensive controls. Both receptor subtypes were downregulated in 1-K,1C rats and their relative proportion was similar to that in glomerular preparations from the controls. ANF-stimulated cGMP production by isolated glomeruli was lower in 1-K,1C than in normotensive rats. In papillary membranes, ANF was bound to a homogeneous population of high-affinity binding sites in both groups. A modest augmentation in the density of papillary ANF receptors was observed in 1-K,1C rats. Glomerular and papillary ANF receptors may be regulated differently in the presence of elevated plasma ANF levels. The present results indicate that the downregulation of glomerular ANF-R1 and the reduced cGMP response may lead to altered sodium and water handling by the kidney of 1-K,1C hypertensive rats.

The pineal adrenergic → cyclic GMP response develops two weeks after the adrenergic → cyclic AMP response

Pineal metabolism is regulated primarily by noradrenergic innervation. Stimulation of the adult gland with norepinephrine elevates both cyclic AMP and cyclic GMP production, through remarkably similar mechanisms requiring activation of both β- and α 1-adrenergic receptors. As described here, however, the adrenergic stimulation of cyclic GMP is first detectable about 2 weeks after the cyclic AMP response can be detected. This indicates there is a profound difference in when cyclic AMP- and cyclic GMP-regulated processes can be adrenergically regulated.

Pertussis toxin does not inhibit α 1-adrenergic potentiation of β-adrenergic stimulation of cyclic AMP accumulation in rat pinealocytes

The hypothesis that G i might be involved in the α 1-adrenergic, protein kinase C (PKC)-mediated amplification of β-adrenergic cyclic AMP stimulation in rat pinealocytes was investigated. Treatment of pinealocytes with a high concentration of pertussis toxin (500 ng/ml, 18 h) almost completely (approximately 95%) inactivated two cell membrane G-proteins (kDa 40.7 and 39.8) judged by back ADP-ribosylation of pinealocyte membrane proteins. However, this treatment failed to inhibit either the β-adrenergic (isoprepaline, ISO 10 −6 M), α 1-plus β-adrenergic (noradrenaline, NA 10 −5 M) or β -adrenergic plus 12-O-tetradecanoylphorbol 13-acetate (TPA 10 −7 M) induced stimulation of cyclic AMP or cyclic GMP. These results suggest that α 1-adrenergic potentiation of β-adrenergic stimulation of cyclic AMP and cyclic GMP does not involve a pertussis toxin-sensitive G-protein.

β-Adrenergic regulation of cyclic GMP in rat pinealocytes

Stimulation of rat pinealocyte cyclic GMP by β-adrenergic agonists (in the presence of prazosin to block α 1-adrenoceptors) showed an identical potency order (isoproterenol> epinephrine = norepinephrine>> salbutamol> terbutaline) to the stimulation of pinealocyte cyclic AMP. The increase in cyclic AMP and cyclic GMP produced by norepinephrine was inhibited by various subtype-selective β-adrenergic antagonists with the same potency order (propranolol> atenolol = ICI 118,551>> practolol). These results indicate that pinealocytes have a single β-adrenoceptor, of the β 1-subtype. Activation of this β 1-adrenoceptor mediates adrenergic stimulation of not only cyclic AMP but also cyclic GMP.

Characterization of the atrial natriuretic peptide clearance receptor using a vaccinia virus expression vector.

A recombinant vaccinia virus has been used to direct the expression of the atrial natriuretic peptide clearance receptor (ANP C-receptor) in mammalian cell lines normally deficient in this protein. The recombinant receptor binds 125I-ANP-(102-126) in a specific and saturable manner and carboxyl-terminal truncated and internal-deleted ANP analogs bind to this site with high affinity. Following the covalent attachment of 125I-ANP-(102-126) to the recombinant ANP C-receptor, the protein exhibits an electrophoretic mobility identical to that of the native ANP C-receptor of cultured vascular cells. Expression of the ANP C-receptor in heterologous cells does not affect ANP-stimulated cyclic GMP accumulation, confirming previous reports that this novel ANP receptor subpopulation is not coupled to cyclic GMP metabolism. Furthermore, specific antisera, generated by inoculating rabbits with living recombinant virus, block 125I-ANP binding to the ANP C-receptor but do not inhibit ANP stimulation of cyclic GMP, supporting the existence of two receptor subpopulations that are functionally and immunologically distinct.

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.

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.