Formycin triphosphate as a probe for the ATP binding site involved in the activation of guanylate cyclase.

Formycin A triphosphate (FTP), a fluorescent analog of ATP, slightly increased basal guanylate cyclase activity, but significantly potentiated guanylate cyclase activity stimulated by atrial natriuretic factor (ANF) in rat lung membranes. FTP potentiated ANF-stimulated guanylate cyclase activity with an EC50 at about 90 microM and inhibited ATP-stimulated guanylate cyclase activity with an IC50 at about 100 microM. These results indicate that FTP binds more tightly than ATP for the same binding site. Therefore, FTP would be an excellent tool for studying the ATP binding site.     

Differences in the nitric oxide/soluble guanylyl cyclase signalling pathway in the myocardium of neonatal and adult rats

The effects of a nitric oxide-donor, S-nitroso-N-acetylpenicillamine, and a direct activator of soluble guanylyl cyclase, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), on force of contraction (F(c)) and L-type Ca(2+) currents (I(Ca(L))) were investigated in myocardial preparations from neonatal and adult rats. Since hearts from adult and neonatal animals contained 160 and 47 mg/100 g wet weight myoglobin, respectively, its possible interaction with both drugs was also investigated. Both S-nitroso-N-acetylpenicillamine (100 microM) and YC-1 (30 microM) were ineffective in myocardial preparations from adult rats but reduced the magnitude of I(Ca(L)) and F(c) in preparations from neonatal rats. The latter effects were antagonised by 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 50 microM) and attenuated by myoglobin (30-300 microM), which also attenuated the effects of both drugs on pre-contracted aortic rings. The differential effects of S-nitroso-N-acetylpenicillamine and YC-1 in the myocardium from adult and neonatal rats may result from developmental changes in the content of myoglobin and/or in the NO/soluble guanylyl cyclase signal pathway.     

HS-142-1, a novel nonpeptide atrial natriuretic peptide (ANP) antagonist, blocks ANP-induced renal responses through a specific interaction with guanylyl cyclase-linked receptors.

HS-142-1, a novel microbial product, blocked 125I-labeled rat atrial natriuretic peptide (rANP) (= ANF(99-126)) binding to bovine adrenocortical membranes, where guanylyl cyclase-containing receptors are predominantly expressed. However, HS-142-1 only slightly inhibited [125I]rANP binding to bovine lung membranes where only a small portion of binding sites are coupled to guanylyl cyclase. Further, HS-142-1 only recognized the 135 kDa ANP receptor, which is considered to be the guanylyl cyclase-containing receptor based on the results obtained in affinity cross-linking studies with bovine adrenocortical and lung membranes. Under identical conditions, Atriopeptin I selectively recognized guanylyl cyclase-free receptors both in binding and affinity cross-linking experiments. When injected intravenously (1 mg/kg) to anesthetized rats, HS-142-1 abolished ANP-induced diuresis and natriuresis. These results suggest that HS-142-1 works in vivo through a specific interaction with the ANP functional receptor, and that HS-142-1 will be a powerful tool for understanding the physiological roles of ANP in distinction from its pharmacological effects.     

HS-142-1, a novel nonpeptide atrial natriuretic peptide (ANP) antagonist,blocks ANP-induced renal responses through a specific interaction with guanylyl cyclase-linked receptors

HS-142-1, a novel microbial product, blocked ¹²⁵I-labeled rat atrial natriuretic peptide (rANP) (= AN(99–126)) binding to bovine adrenocortical membranes, where guanylyl cyclase-containing receptors are predominantly expressed. However, HS-142-1 only slightly inhibited [¹²⁵I]rANP binding to bovine lung membranes where only a small portion of binding sites are coupled to guanylyl cyclase. Further, HS-142-1 only recognized the 135 kDa ANP receptor, which is considered to be the guanylyl cyclase-containing receptor based on the results obtained in affinity cross-linking studies with bovine adrenocortical and lung membranes. Under identical conditions. Atriopeptin I selectively recognized guanylyl cyclase-free receptors both in binding and affinity cross-linking experiments. When injected intravenously (1 mg/kg) to anesthetized rats, HS-142-1 abolished ANP-induced diuresis and natriuresis. These results suggest that HS-142-1 works in vivo through a specific interaction with the ANP functional receptor, and that HS-142-1 will be a powerful tool for understanding the physiological roles of ANP in distinction from its pharmacological effects.     

Cyclosporine impairs the guanylyl cyclase activity of the natriuretic peptide receptor in the glomerulus.

In order to elucidate the involvement of the atrial natriuretic peptide (ANP) and its receptor (natriuretic peptide receptor; NPR) system in cyclosporine-induced nephrotoxicity, we investigated the cyclosporine A (CsA)-induced changes in characteristics of the NPR/guanylyl cyclase system in the glomerulus and inner medulla of the rat kidney. CsA was administered intramuscularly to rats for 2 weeks (CsA group). Particulate guanylyl cyclase activity was measured in glomerular and inner medullary membranes. For receptor characteristics, quantitative in vitro receptor autoradiography was performed. The guanylyl cyclase activity in the glomerulus from the CsA group was attenuated compared with that from the control. However, the activity in the inner medulla was not affected by CsA treatment. Direct application of CsA to normal glomerular membrane completely abolished the ANP-induced guanylyl cyclase activation. Binding studies, using(125)I-ANP, revealed that B(max)was decreased in the CsA group, while K(d)was not affected in the glomerulus. However, in the inner medulla, neither B(max)nor K(d)was affected by CsA treatment. CsA did not displace the(125)I-ANP bindings to NPRs in the normal rat kidney. Local tissue ANP as well as plasma ANP concentration in both groups was not significantly different. These results indicate that CsA impairs the guanylyl cyclase activity mainly in the glomerulus by the decrease in NPR population and/or by direct inhibition, suggesting that the ANP/NPR system might be involved in CsA-induced nephrotoxicity.     

GABA and benzodiazepine receptors in the offspring of dams receiving diazepam: ontogenetic studies

Ontogenesis of the regulation of ³H-GABA and ³H-diazepam binding to rat brain plasma membranes treated with 0.05% Triton X-100 has been studied. The density of ³H-diazepam and ³H-GABA binding in cortex, cerebellum and corpus striatum at birth was approximately one third of the adult values. They increased at the same rate and reached the adult values between 14–21 days after birth. Study of the binding characteristics showed that the K_D for high and low affinity for ³H-GABA, and for ³H-diazepam did not change during ontogenesis and the increase reflects only an increase of Bmax. The number of Triton X-100 treatments of crude synaptic membrane (CSM) required to maximize ³H-GABA for the high affinity component were different at various postnatal days: only one treatment was required in 1-day old rats, two in 7- and 14-day old rats and three in adult animals. In addition, the capability of muscimol to stimulate ³H-diazepam binding in both frozen-thawed and Triton X-100 treated membrane preparations decreased with increasing age. Binding of ³H-GABA and ³H-diazepam to brain of newborn rats whose dams received diazepam throughout pregnancy (100 mg/kg, × os, bid) was also studied. No significant differences were observed in the ontogenetic development of both bindings. However, in the cortex of these newborn rats the capability of muscimol to stimulate ³H-diazepam binding was greatly reduced in Triton X-100-treated membranes.     

Indomethacin decreases particulate guanylyl cyclase activity in rat kidney

1. Effects of non-steroidal anti-inflammatory drugs on the local atrial natriuretic peptide (ANP) and nitric oxide (NO) systems in the kidney were investigated. 2. Male Sprague-Dawley rats were treated with indomethacin (5 mg/kg, every 12 h, i.p.) for 2 days. The expression of ANP and natriuretic peptide receptor-A (NPR-A) mRNA was determined in the kidney, as was that of endothelial NO synthase (NOS) proteins. Particulate and soluble guanylyl cyclase activities were determined separately. 3. Following treatment with indomethacin, urinary sodium excretion decreased significantly. Although the renal expression of ANP was not changed significantly, that of NPR-A decreased significantly. The expression of NOS increased significantly. Particulate guanylyl cyclase activity was decreased, whereas the activity of soluble guanylyl cyclase was increased. The catalytic activity of Na(+)/K(+)-ATPase was increased, with no significant changes in its expression. The expression of the type 3 Na/H exchanger and Na-K-2CL cotransporters increased significantly. 4. The indomethacin-induced decrease in urinary sodium excretion may be attributed, at least in part, to decreased activity of the local ANP/cGMP system. The increased activity of the NO/cGMP system may be a compensatory response to the diminished activity of the prostaglandin system.     

Structural requirements of ATP for activation of basal and atrial natriuretic factor-stimulated guanylate cyclase in rat lung membranes

ATP has been reported to increase basal and atrial natriuretic factor (ANF)-stimulated guanylate cyclase activity. The structural features of ATP involved in the activation of guanylate cyclase were examined by employing a variety of ATP analogs with modification either at the phosphate chain or at the ribose moiety. Among the natural adenine nucleotides, ATP and ADP were able to increase both basal and ANF-stimulated guanylate cyclase activities in rat lung membranes. AMP had no effect. ATP was more effective than AMPPCP (the non-hydrolyzable analog of ATP), and ADP was more effective than ADP beta S and AMPCP (the hydrolysis-resistant analogs of ADP) to increase basal and ANF-stimulated guanylate cyclase activities. Removal of the oxygen atom from the ribose moiety of ATP or ADP significantly reduced their potency. Thus, the length of the phosphate chain and the hydroxyl groups at the ribose moiety are both determinants for nucleotide mediated guanylate cyclase activation.     

Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.     

Influence of elastase-induced emphysema and the inhalation of an irritant aerosol on deposition and retention of an inhaled insoluble aerosol in Fischer-344 rats

The purpose of this study was to assess the effects of elastase-induced pulmonary emphysema and the inhalation of an irritant aerosol (Triton X-100, a nonionic surfactant similar to those used in a number of pressurized consumer products) on pulmonary deposition and retention of an insoluble test aerosol, 59Fe-labeled Fe2O3. Untreated rats or rats pretreated by intratracheal instillation with elastase were exposed to an aerosol of 59Fe-labeled Fe2O3 either 18 hr or 7 days after exposure to aerosolized Triton X-100 which was administered in doses of 20, 100, or 200 micrograms/g of lung. Rats pretreated with elastase had significantly lower pulmonary deposition of 59Fe than the untreated controls (p less than 0.005). Pulmonary deposition of Fe2O3 was unaffected by pretreatment with Triton X-100. Elastase treatment alone had no effect on retention of Fe2O3. Triton X-100 administered 18 hr prior to exposure of rats to Fe2O3 aerosol resulted in dose-related increases in whole-body retention of 59Fe. When rats were exposed to Triton X-100 7 days before exposure to Fe2O3, increased retention of 59Fe was noted only in those treated at the highest Triton X-100 dose level (200 micrograms/g).     

Action of detergents on 3H-dihydroergokriptine binding and localization of alpha-adrenoceptors in synaptosomal membranes

3H-dihydroergokriptine (3H-DHE) binding was carried out in synaptosomal membranes from basal ganglia of the cat. A single type of binding site with Kd 3.7 nM, Hill number = 0.95 and Bmax = 1000 pmol/g protein was found. 3H-DHE bound to alpha-adrenoceptors and not to serotonin or dopamine receptors. At very low concentrations, some detergents enhanced binding, but at higher concentrations of those used (Triton X-100, Nonidet P-40, deoxycholate and digitonin), inhibited the binding of 3H-DHE. After binding to the membrane protein, the 3H-DHE-receptor complex was stable to the action of Triton X-100. At concentrations of Triton X-100. At concentrations of Triton X-100 (0.1--0.2%). in which only the presynaptic membrane disintegrated, the 3H-HDE specific radioactivity was reduced. With a more drastic treatment that disintegrated the postsynaptic membrane, 3H-DHE binding was further reduced. These results suggest that alpha-adrenergic receptors may be localized at both the pre- and postsynaptic membranes of central synapses.     

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides in guinea pig tracheal smooth muscle

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides were investigated in epithelium-denuded guinea pig tracheal rings, treated with indomethacin (5 μM) and phosphoramidon (1 μM) and contracted with histamine (3 μM). Atrial natriuretic peptide (ANP) was a more potent relaxant than C-type natriuretic peptide whereas ANP-(4–23) was inactive suggesting the involvement of ANP_A receptors in the relaxant effect of ANP. ODQ (1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one, 10 μM), a selective inhibitor of soluble guanylyl cyclase, markedly inhibited the relaxant response to sodium nitroprusside. The relaxant response to ANP was not altered by ODQ demonstrating the involvement of particulate guanylyl cyclase. ANP-induced relaxations, as well as sodium nitroprusside-induced relaxations, were similarly potentiated by rolipram (4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one, 3 μM), a type IV phosphodiesterase inhibitor, and by zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one, 10 μM), a type V phosphodiesterase inhibitor. ANP-mediated response was unaffected by glibenclamide (10 μM), a selective blocker of ATP-sensitive K⁺ channels, and by apamin (1 μM), a selective blocker of small-conductance Ca²⁺-activated K⁺ channels. Iberiotoxin (100 nM) extensively prevented the relaxant effect of ANP suggesting the activation of large-conductance Ca²⁺-activated K⁺ channels. In addition, ANP (10 nM) and ANP-(4–23) (100 nM) significantly reduced forskolin (1 μM)-stimulated cAMP accumulation suggesting, for the first time, the presence of functional ANP_C receptors in guinea pig airway smooth muscle. However, relaxations to forskolin and to isoproterenol were not altered in the presence of ANP-(4–23) or ANP demonstrating that the inhibitory effect of ANP-(4–23) and ANP on adenylyl cyclase was not sufficient to alter the functional response induced by these two activators of adenylyl cyclase.     

Inhibition by HS-142-1, a novel nonpeptide atrial natriuretic peptide antagonist of microbial origin, of atrial natriuretic peptide-induced relaxation of isolated rabbit aorta through the blockade of guanylyl cyclase-linked receptors.

HS-142-1, a specific nonpeptide antagonist for the atrial natriuretic peptide (ANP) receptor, equally blocked rat ANP (rANP)-, porcine brain natriuretic peptide-, or porcine C-type natriuretic peptide-stimulated GMP production in cultured bovine aortic smooth muscle (BASM) and bovine aortic endothelial (BAE) cells in a concentration-dependent fashion, at concentrations of 1-300 micrograms/ml. But, even at 300 micrograms/ml, HS-142-1 only weakly inhibited the specific binding of 125I-rANP to the BASM and BAE cells, where only a small portion of the binding sites are linked to guanylyl cyclase. Further, with BAE cell membranes, HS-142-1 recognized only the 135-kDa ANP receptor, which is thought from 125I-rANP affinity cross-linking studies to be the guanylyl cyclase-linked receptor. HS-142-1 also, if anything, inhibited the labeling of 135-kDa ANP receptors in the affinity cross-linking studies with BASM membranes, suggesting that a major portion of the 135-kDa ANP receptors are HS-142-1 insensitive and only a small portion of the 135-kDa ANP receptors are responsible for the blockade by HS-142-1 of GMP production in BASM cells. At a concentration of 100 micrograms/ml, HS-142-1 reversibly prevented ANP-induced relaxation of the isolated rabbit thoracic aorta induced to contract with 3 x 10(-7) M phenylephrine, but not the relaxation induced by sodium nitroprusside, isoproterenol, or papaverine. These results suggest that HS-142-1 specifically inhibits natriuretic peptide-induced vasorelaxation through the blockade of guanylyl cyclase-linked natriuretic peptide receptors. HS-142-1 thus will be a powerful tool for understanding the physiological roles, in vasculature, of natriuretic peptides, which contribute to the homeostasis of blood pressure and intravascular volume.     

Activation of adenylate cyclase by dopamine, GTP, NaF and forskolin in striatal membranes of neonatal, adult and senescent rats

Dopamine (DA) caused a significant activation of striatal adenylate cyclase in neonatal and adult but not in senescent rats. GTP activated cyclase at the adult stage but not at both neonatal and senescent stages. NaF and forskolin activated cyclase at every stage. The coupling mechanism between DA1 receptors and catalytic units of cyclase seems to become functional at the neonatal stage but GTP recognition and/or binding sites lack in stimulatory GTP binding protein in neonatal and senescent membranes.     

Epygid: a new Soviet antioxidant promoted the selective inhibition of membrane-dependent protein synthesis in the brain

Intraperitoneal injection of different amounts of Epygid (2-ethyl-6-methyl-3-oxypyridine) to 3- and 18-month-old rats led to significant reduction of translating activity in vitro of membrane-bound polysomes of brain cells, but not of free polysomes, more so for bound polysomes from 18-month-old animals than those from 3 months old. Separation of polysomes from membranes by Triton X-100 resulted in restoration of template activity to the level of free polysomes. The phenomenon may be related to incorporation of Epygid into the membranes of endoplasmic reticulum which contain a part of cell polysomes on their surface.     

Improvement of 5-HT3 receptor binding assay: enhancement of specific [3H]quipazine binding with Triton X-100-treated membranes from rat cortex.

The 5-hydroxytryptamine (5-HT)3 receptor binding assay using [3H]quipazine was examined. It was impossible to obtain specific [3H]quipazine binding with the membrane fractions from rat cortex prepared by the usual procedure. When the membranes were pretreated with detergent Triton X-100, the ratio of specific [3H]quipazine binding markedly increased, depending upon the concentration of Triton X-100 in the range of 0.01-0.1% (w/v). At a concentration of more than 0.05%, the specific binding reached a maximum of 55 to 60% of the total binding. The specific [3H]quipazine binding to the Triton X-100-treated membranes was reversible and was potently inhibited by several 5-HT3 antagonists, while 5-HT1, 5-HT2 receptor antagonists and other receptor-specific ligands had no effect on the binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.62 nM and Bmax of 97 fmol/mg protein. Thus, the Triton X-100-treated membranes retained the characteristics of 5-HT3 binding sites, making it possible to use [3H]quipazine for a 5-HT3 receptor binding assay with a high ratio of specific binding.     

Discrimination of monoamine uptake by membranes of adrenal chromaffin granules.

1 The accumulation of various radioactive monoamines by isolated membranes of bovine adrenal chromaffin granules was measured by equilibrium dialysis. 2 Adenosine-5'-triphosphate (ATP) in the presence of Mg++ stimulated the uptake of all the amines tested, but the accumulation of dopamine, (-)-noradrenaline (NA), 5-hydroxytryptamine (5-HT), (plus or minus)-adrenaline and (plus or minus)-octopamine was greater than that of tyramine, (plus or minus)-metaraminol, tryptamine, beta-phenylethylamine and histamine. 3 At the higher concentration levels of the amines in the medium the ATP-dependent accumulation of dopamine, NA, adrenaline and 5-HT in the membranes reached a saturation level, whereas in the absence of the nucleotide no saturation level was attained. 4 Octopamine and 5-HT competitively inhibited the ATP-dependent uptake of NA, 5 Decrease in the incubation temperature or the presence of N-ethylameimide greatly reduced the ATP-stimulated amine accumulation. Ouabain had no effect on uptake. 6 Reserpine virtually abolished the ATP-dependent uptake of dopamine, NA and 5-HT, caused a partial inhibition of the metaraminol, octopamine and tyramine accumulation, but did not interfere with the uptake of tryptamine. 7 The content of endogenous catecholamines of the membranes was changed very little by incubation of NA and 5-HT in the presence of ATP. However, the membranes lost over 80% of their endogenous amines if incubated for 30 min without ATP. 8 The ATP content of the medium progressively decreased during the incubation of granular membranes. 9 It is concluded that the membrane of adrenal chromaffin granules discriminates between the various monoamines with regard to the magnitude of their uptake and that two mechanisms of ATP-stimulated uptake, one responsive and the other resistant to reserpine, exist at the level of this membrane. The ATP-stimulated transport at the granular membrane level may be an important factor in determining the intraneuronal storage of a physiological or false neurotransmitter.     

Efficacy of isatin analogues as antagonists of rat brain and heart atrial natriuretic peptide receptors coupled to particulate guanylyl cyclase

Isatin is an endogenous indole and an inhibitor of atrial natriuretic peptide (ANP) receptors coupled with particulate guanylyl cyclase (GC). In this study, several isatin analogues were tested as inhibitors of ANP-stimulated GC in rat brain and heart membranes. None of these analogues affected activity in the absence of ANP, or stimulated ANP-induced activity. In both tissues, some 5-substituted isatins (5-hydroxyisatin, 5-methylisatin, and 5-aminoisatin) exhibited more effective inhibitory activity than isatin itself, with IC50 values in the range 1.3-20 microM. The efficacy of other analogues varied and was not consistent between the two tissues, raising the possibility of receptor heterogeneity and relative selectivity of inhibition. Some substituted isatins may have a role as pharmacological tools for investigating the physiological roles of natriuretic peptides and their receptors.     

Developmental changes in the effect of atrial natriuretic peptide on tissue cyclic GMP content and particulate guanylate cyclase activity of aorta, kidney and lung of rats.

To investigate possible developmental changes in the physiological effect of atrial natriuretic peptide (ANP) after birth, we studied the effect of ANP on the slice cGMP content and the particulate guanylate cyclase activity of aorta, kidney and lung in neonate, 2-week-old and adult rats of both sexes. Incubation with human ANP(99-126) (hANP) increased significantly the slice cGMP content of aorta, kidney and lung in three ages of rats. The hANP-stimulated fraction of cGMP contents of kidney decreased, that of lung increased with development, whereas that of aorta showed no significant change. Consistently, the hANP-responsive particulate guanylate cyclase activity decreased in kidney, increased in lung during development, without significant developmental change in aorta. These results indicate a differential change in the effect of hANP on the slice cGMP content among tissues during development. The developmental change in the effect of hANP on slice cGMP content is probably caused by the ontogenetic change in activation of ANP receptor-linked guanylate cyclase.     

Kinetic characterization of atrial natriuretic factor-sensitive particulate guanylate cyclase

The present investigation describes kinetic characteristics of membrane-bound and Triton X-100-solubilized atrial natriuretic factor (ANF)-sensitive guanylate cyclase from bovine adrenal cortex. The kinetic analysis of both enzyme forms suggests that in the presence of manganese, ANF induces or stabilizes at least two apparent GTP*Mn2(+)- and in addition two Mn2(+)-binding sites. Addition of the natriuretic drug amiloride favors this state. ATP increases the vmax in the presence of ANF for GTP*Mg2+, but not for GTP*Mn2+ as a substrate. With GTP*Mg2+, amiloride has no effect on basal or ANF-stimulated activity, but slightly reduces the effect of ATP. Under all conditions tested, the enzyme follows regular Michaelis-Menten kinetics in the presence of Mg2+ and exhibits positive cooperativity with Mn2+. Positive cooperativity is also retained after Triton extraction. The results indicate that Triton extraction has no major influence on the kinetic properties of particulate guanylate cyclase when the extraction procedure is done carefully. The data also support the suggestion that multiple interactions of subunits might occur upon activation of the enzyme by ANF in the presence of Mn2+.