Particulate Guanylate Cyclase Activity Research Articles

Epidermal growth factor enhances guanylate cyclase activity in vivo and in vitro.

Epidermal growth factor (EGF) increases DNA synthesis and cell division both in vivo and in vitro. The mechanism by which EGF increases growth and DNA synthesis is unknown. Since the intracellular messenger cGMP stimulates DNA synthesis, the present investigation was designed to determine if EGF might have part of its mechanism of action through activating guanylate cyclase [EC 4.6.1.2], the enzyme that catalyzes the formation of cGMP. EGF enhanced soluble and particulate guanylate cyclase activities as well as cGMP levels 2- to 3-fold in hypophysectomized and nonhypophysectomized tissues both in vivo and in vitro. EGF increased guanylate cyclase activity 0.5 h after ip injection in mice, and this increased activity was still present 12 h later. Guanylate cyclase activity was increased to a greater extent secondary to EGF in hypophysectomized cecum compared to nonhypophysectomized cecum. Dose-response curves revealed that maximal stimulation of guanylate cyclase by EGF occurred at 1 nM. There was no augmented guanylate cyclase activity when the concentration of EGF was decreased to 0.01 nM. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of EGF.

Pharmacology and receptor binding of atrial natriuretic factor in vascular smooth muscle.

Characterization of the vascular pharmacology and receptor binding of atrial natriuretic factor (ANF) has been achieved utilizing a synthetic peptide which contains the sequence and biological activity of ANF. The synthetic ANF (sANF) relaxes aortic segments contracted by agonists or by low (15 to 20 mM) but not high (greater than or equal to 40 mM) concentrations of K+. The relaxation to sANF is well maintained, reversible, independent of the vascular endothelium and correlated with increases in cyclic GMP (with no change in cyclic AMP). Plasma membranes prepared from rabbit aorta and kidney possess high affinity (Kd = 100 pM) specific binding sites for sANF. An excellent correlation exists between the receptor binding and pharmacology for several synthetic analogs of ANF. The presence of these receptors appears consistent with the activation of particulate (but not soluble) guanylate cyclase by sANF. sANF does exhibit a profound regional vasodilator selectivity which can be explained, in part, by changes in receptor density.

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.

Atrial natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle.

A 26 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) relaxed isolated rabbit aortic segments in which the endothelium was either intact or functionally destroyed. The relaxations were temporally associated with increases in levels of cGMP with no change in the levels of cAMP. The ANF-induced increases in cGMP were also observed in aortic segments pretreated with calcium-free buffer or the cGMP phosphodiesterase inhibitor M&B 22,948. Qualitatively similar results were obtained for sodium nitroprusside. ANF selectively activated particulate guanylate cyclase, having no effect on the soluble form of the enzyme. Thus, the direct (endothelium-independent) vasodilator effect of ANF may be mediated via increased tissue levels of cGMP. ANF appears to increase vascular cGMP levels by activation of particulate guanylate cyclase.

Solubilization and partial characterization of the intestinal receptor for Escherichia coli heat-stable enterotoxin.

Binding of Escherichia coli strain 431 heat-stable enterotoxin (STa) and activation of intestinal particulate guanylate cyclase by E. coli STa were studied with rat intestinal epithelial cells and brush border membranes (BBMs). The rates of guanylate cyclase stimulation by 431 STa in cells and BBMs were rapid, with maximal levels of cyclic GMP observed within 5 min. Specific binding of 125I-labeled STa from E. coli 431 (431 125I-STa) and activation of guanylate cyclase by unlabeled 431 STa were observed with intestinal BBMs; however, neither was detected with membranes from nonintestinal tissues. The STa receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a nondenaturing dipolar ionic detergent, in yields of approximately 50%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the detergent-solubilized receptor-431 125I-STa complex, followed by autoradiography, showed that 431 125I-STa bound to a single BBM component with a molecular weight of about 100,000. Binding of 431 STa to its solubilized receptor was saturable, specific, and essentially irreversible. Pretreatment of the soluble receptor with trypsin and pronase but not chymotrypsin decreased binding of 431 125I-STa. The 431 STa-receptor complex was dissociated by boiling in the presence of 1% sodium dodecyl sulfate, incubation with 0.5 M acetic acid, or reduction with dithiothreitol. In contrast to the residual particulate guanylate cyclase activity of detergent-treated membranes, solubilized guanylate cyclase was not stimulated by STa. Membrane structure appears to play an important role in the coordination of STa binding and stimulation of guanylate cyclase activity.

Selective activation of particulate guanylate cyclase by a specific class of porphyrins.

Guanylate cyclase was activated 3- to 10-fold by hemin in a dose-dependent manner in membranes prepared from homogenates of rat lung, C6 rat glioma cells, or B103 rat neuroblastoma cells. Maximum activation was observed with 50 to 100 microM hemin with higher concentrations being inhibitory. Activation was observed when Mg2+-GTP but not when Mn2+-GTP was used as the substrate. Increased enzyme activity reflected selective activation of the particulate form of guanylate cyclase; hemin inhibited the soluble form of guanylate cyclase 70 to 90% over a wide range of concentrations. Activation was not secondary to proteolysis since a variety of protease inhibitors failed to alter stimulation by hemin. Protophorphyrin IX had little effect on particulate guanylate cyclase activity and sodium borohydride almost completely abolished hemin-dependent activation. These data suggest a requirement for the ferric form of the porphyrin-metal chelate for activation. However, agents which interact with the iron nucleus of porphyrins, such as cyanide, had little effect on the ability of hemin to activate guanylate cyclase. The stimulatory effects of hemin were observed in the presence of detergents such as Lubrol-PX, and highly purified particulate enzyme could be activated to the same extent as enzyme in native membranes. These data suggest that the interaction of porphyrins with particulate guanylate cyclase is complex in nature and different from that with the soluble enzyme.

Cation-dependent vitamin D activation of human renal cortical guanylate cyclase.

The objective of this investigation was to determine whether physiological levels of vitamin D and its metabolites have part of their mechanisms of action through stimulation of guanylate cyclase (EC 4.6.1.2). These sterols enhanced both soluble and particulate guanylate cyclase activities as well as cGMP levels two- to threefold in human and rat tissues. At a concentration of 1 nM, 1,25(OH)2D3 greater than 25(OH)D3 greater than vitamin D3 greater than 24,25(OH)2D3 = 25,26(OH)2D3 = vitamin D2. Dose-response curves revealed that maximal stimulation of guanylate cyclase by these sterols was at 1 nM and that there was no augmented guanylate cyclase activity at 0.01 nM. The precursors of vitamin D, cholesterol and 7-dehydrocholesterol, had no effect on guanylate cyclase activity. The activation of guanylate cyclase activity by the vitamin D sterols required the presence of manganese ion. Calcium was not as efficient as manganese in optimizing basal or hormone-stimulated guanylate cyclase activity. Vitamin D and its metabolites failed to stimulate adenylate cyclase (EC 4.6.1.1) activity. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of vitamin D at the cellular level.

Inhibition of Escherichia coli heat-stable enterotoxin effects on intestinal guanylate cyclase and fluid secretion by quinacrine

Enterotoxigenic Escherichia coli may produce a heat-stable enterotoxin (ST) that causes diarrheal disease in humans and in animals ST activates particulate guanylate cyclase in intestinal mucosal cells and causes intestinal fluid secretion. In this study, we examined the effects of quinacrine on ST activation of guanylate cyclase and ST-mediated intestinal fluid secretion. Quinacrine significantly reduced ST activation of particulate guanylate cyclase in rat intestinal tissue. Additionally, quinacrine reduced ST-mediated fluid secretion in a rat intestinal loop assay (P < 0.05). In the suckling mouse model, subcutaneous quinacrine (0.1 μmole/mouse) reduced ST-induced fluid secretion at a submaximally effective dose of the toxin, but it did not reduce ST-mediated fluid secretion at a near maximally effective dose. Quinacrine (0.1 μmole/mouse) did not significantly reduce intestinal fluid secretion induced by the analog of cyclic GMP, 8-bromo cyclic GMP. However, at a higher concentration of quinacrine (1 μmole/mouse), significant inhibition of 8-bromo cyclic GMP-induced secretion was observed. Inhibition by the antimalarial agent quinacrine of ST-induced fluid secretion, by a block prior to guanylate cyclase activation, suggests a possible role for a phospholipase early in the sequence of events of ST activation of guanylate cyclase. The results suggest that ST may activate membrane phospholipases prior to ST activation of guanylate cyclase.

Involvement of sulfhydryl groups in the oxidative modulation of particulate lung guanylate cyclase by nitric oxide and N-methyl- N′-nitro- N-nitrosoguanidine

Particulate guanylate cyclase from rat lung was activated by nitric oxide or if N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) in a dose-dependent manner that was enhanced by dithiothreitol. Nitric oxide-stimulated guanylate cyclase activity decayed during a 60-min preincubation at 37°, but did not decay at 24° or 4°. Dithiothreitol enhanced the decay of nitric oxide-stimulated enzyme at all temperatures by potentiating the reversal of nitric oxide activation. Following the reversal of nitric oxide activation at 24° by dithiothreitol, the particulate enzyme could be reactivated by a second exposure to nitric oxide. Preincubation of basal particulate guanylate cyclase activity at 37° resulted in the loss of enzyme responsiveness to activation by nitric oxide or MNNG that was potentiated by diamide or oxidized glutathione. The inhibitory effects of the thiol oxidants on enzyme responsiveness to activation by MNNG were prevented by dithiothreitol. The results suggest that activation of particulate guanylate cyclase by nitric oxide or MNNG involves the oxidation of key enzyme sulfhydryl groups.

Lanthanum chloride inhibition of the secretory response to Escherichia coli heat-stable enterotoxin

Escherichia coli heat-stable enterotoxin (ST) appears to cause intestinal fluid secretion by activating intestinal particulate guanylate cyclase. Recent studies suggest that chlorpromazine and quinacrine reduce the intestinal secretory response to ST and activation of guanylate cyclase by ST. We have examined the effects of lanthanum chloride, another agent that has been shown to inhibit calcium-dependent cellular processes, on the intestinal secretory response to ST and on the inhibition of ST by chlorpromazine and quinacrine. Lanthanum (2.5 to 10 mumol per mouse) reduced ST-mediated intestinal fluid secretion in the suckling mouse assay by 40 to 56%, respectively, but did not reduce basal fluid accumulation or ST activation of particulate guanylate cyclase. Intestinal fluid secretion in suckling mice induced by 8-bromocyclic GMP was also reduced by lanthanum. When subeffective doses of lanthanum and chlorpromazine were combined, they blocked both ST- and 8-bromocyclic GMP-mediated gut secretion in suckling mice. Likewise, the combination of subeffective doses of lanthanum and quinacrine reduced ST-mediated gut secretion in suckling mice. However, 8-bromocyclic GMP-induced secretion was not synergistically inhibited by lanthanum and quinacrine. These results suggest that lanthanum blocks ST-induced secretion after ST activation of guanylate cyclase. Additionally, lanthanum potentiates the inhibitory effects of quinacrine and chlorpromazine on ST and suggests that combination antisecretory therapy deserves further exploration.

Relationship between calcium stimulation of cyclic GMP and lipid peroxidation in the rat kidney: Evidence for involvement of calmodulin and separate pathways of peroxidation in cortex versus inner medulla

The relationship between Ca 2+ stimulation of renal cGMP accumulation, release of endogenous arachidonic acid (AA) from lipid stores, lipid peroxidation and prostaglandin (PG) synthesis were examined in rat renal cortex and inner medulla. In slice incubates of each tissue, increases in slice cGMP induced by Ca 2+ plus ionophore A23187 were preceded by or occurred concurrently with Ca 2+ induced increases in (1) release of [ 14C] AA from prelabeled lipid stores, (2) lipid peroxidation, as monitored by accumulation of malondialdehyde (MDA) in the media, and (3) inner medullary slice PGE content. Ca 2+ induced increases in cGMP, MDA and PGE required O 2. Exogenous AA also increased MDA, PGE and cGMP in the presence but not in the absence of O 2. In inner medulla, the cyclooxygenase inhibitors indomethacin or meclofenamate suppressed or abolished the actions of Ca 2+, Ca 2+ plus A23187 or exogenous AA to increase MDA, PGE and cGMP, thus implicating products of the prostaglandin synthetic pathway as potential mediators of Ca 2+ effects on cGMP in this tissue. By contrast, in renal cortex, the cyclooxygenase inhibitors did not alter Ca 2+, A23187 or AA induced increases in MDA or cGMP. However, preformed AA hydroperoxide significantly stimulated soluble and particulate guanylate cyclase activities from both regions of the kidney, suggesting that oxygenation of AA by the lipoxygenase pathway could result in generation of products capable of enhancing cGMP accumulation in cortex. Trifluoperazine (TFP), a phenothiazine that binds to and inhibits many of the biologic actions of the Ca 2+-calmodulin complex, suppressed increases in [ 14C] AA release, MDA and cGMP induced by Ca 2+ or Ca 2+ plus A23187 in both cortex and medulla. By contrast, TFP did not alter increases in MDA or cGMP in response to exogenous AA or the increase in cGMP induced by nitroprusside. Promethazine, a phenothiazine which binds poorly to Ca 2+-calmodulin, had no effect on Ca 2+ induced increases in MDA or cGMP in cortex or medulla, TFP, but not promethazine, also suppressed Ca 2+ induced increases in acyl hydrolase activities in the 100,000 xg particulate fractions from cortex and medulla. Reduction of the endogenous calmodulin-like activity of particulate fractions from inner medulla by extraction with EGTA was associated with loss of Ca 2+ responsive acyl hydrolase activity. Ca 2+-responsiveness was restored by addition of purified exogenous calmodulin. The data are consistent with the proposal that Ca 2+ induced increases in cGMP involve (1) Ca 2+ stimulation of Ca 2+-calmodulin responsive acyl hydrolase activity with liberation of AA from lipid stores, and (2) oxygenation of AA by cyclooxygenase (medulla) or lipoxygenase (cortex) pathways to products which activate guanylate cyclase.

Adrenocorticotropic hormone-responsive guanylate cyclase in the particulate fraction of rat adrenal glands.

Low concentrations of ACTH, 7 x 10(-12) M, caused a marked stimulation of the 100,000 x g particulate guanylate cyclase without any detectable change in the adenylate cyclase activity. The lowest concentration of the hormone that elicited adenylate cyclase stimulation was 7 x 10(-10) M, a concentration 100--fold higher than that required to stimulate the guanylate cyclase. Although calcium was found to be obligatory in the hormonally--dependent guanylate cyclase activity, calcium alone could not duplicate the ACTH effect. Sodium nitroprusside and ascorbic acid inhibited the particulate guanylate cyclase activity. While ACTH was unable to stimulate the soluble guanylate cyclase, sodium nitroprusside markedly stimulated this enzyme. From these data, we conclude that the adrenal guanylate cyclase exists in two forms, particulate and soluble. The particulate form is specifically responsive to ACTH, and calcium is one of the essential coupling factors of this hormonally--responsive guanylate cyclase.

797 CYCLASES IN NORMAL AND LEUKEMIC LYMPHOCYTES

Persistent low level of c-AMP and persistent high level of cGMP in leukemic lymphocytes suggest a modified activity of the cyclases which synthetize them. Soluble and particulate guanylate cyclase (GC) and adenylate cyclase (AC) activities were studied in normal B-enriched and T-enriched lymphocytes, in lymphocytes of children with acute lymphocytic leukemia (ALL) and adults with chronic lymphocytic leukemia (CLL). GC activity was greater in normal T than B cells (10.2 vs 5.3 pmol/min/mg protein), increased little with isoproterenol and prostaglandins stimulation, and much more with sodium azide (NaN3) and dehydroascorbic acid (DHA) stimulation (19 and 23 pmol for T cells and 9.1 and 17.2 pmol for B cells). GC activity was greater in both types of leukemic lymphocytes (23 pmol for ALL cells and 18 pmol for CLL cells) and was insensitive to stimulation with DHA and NaN3. Particulate AC activity was greater in normal B than T cells (215 vs 80 pmol) and increased in both after stimulation with isoproterenol (300 pmol) and prostaglandins (400 pmol). In leukemic lymphocytes AC showed depressed activity (20 pmol in ALL cells and 55 pmol in CLL cells) and was insensitive to stimulation. These findings suggest that circulating T cells are more actively cycling or functioning than circulating B cells. They also suggest a derangement of the activity of both cyclases and/or their receptors in leukemic cells: this derangement may be a cause of leukemic cell escape from their microenvironment control.

Physiological calcium ion concentration and a cholinergic drug on activity of particulate guanylate cyclase in guinea pig taenia caecum.

The microsomal fraction derived from the taenia caecum of guinea pigs was used as particulate guanylate cyclase preparation. The guanylate cyclase activity was not influenced by butyltrimethylammonium bromide and Ca2+ (4.0 to 24 X 10-7 M). The results suggest that the particulate guanylate cyclase is not activated by the drug-receptor interaction and also by the increase of intracellular Ca2+ concentration. Therefore, the increase of tissue level of cyclic GMP brought about in the guinea pig taenia caecum might be due to activation of soluble guanylate cyclase following the increase in the intracellular Ca2+ concentration.

Activation of rat lung particulate guanylate cyclase by cholesterol-sequestering agents

Naturally occurring cholesterol-sequestering agents, digitonin, cereolysin and streptolysin O, activated rat lung particulate guanylate cyclase. Particulate enzyme treated with digitonin and cereolysin was further activated by sodium nitroprusside. Digitonin and cereolysin lowered sodium nitroprusside activation of the rat lung soluable guanylate cyclase. Activation of the particulate guanylate cyclase by digitonin and cereolysin was not due to the solubilization of the enzyme.

The role of insulin, glucagon, and cAMP in the regulation of hepatocyte guanylate cyclase activity.

Rat liver regeneration is regulated by a humoral signal that includes insulin and a sustained elevation in glucagon. The intracellular response is characterized by a rise in cAMP as well as altered cGMP metabolism, i.e. increased particulate guanylate cyclase activity. To evaluate the role of hormones in the regulation of guanylate cyclase during liver regeneration, the enzyme activity of primary cultures of rat hepatocytes was examined. Hepatocytes were maintained for 22 h in medium containing various combinations of insulin, glucagon, and cAMP. The cells were then harvested and homogenized and the guanylate cyclase activity was assessed in vitro. Hepatocytes maintained in 100 nM insulin exhibited a 42% (p < 0.001) increase in guanylate cyclase activity when compared to cells cultured in medium alone. Incubation with glucagon (100 nM) produced a 52% (p < 0.01) rise. In the presence of insulin (100 nM), culturing with as little as 5 nM glucagon resulted in increased activity, and 100 nM glucagon produced a 161% (p < 0.001) rise above cultures maintained in insulin alone. Thus, the combination of the two hormones produced an effect that was significantly (p < 0.01) greater than additive. Dibutyryl cAMP and 8-bromoadenosine 3':5'-monophosphoric acid were at least as effective as glucagon; the enzyme activity of cells maintained in 5 microM N6,02'-dibutyryl adenosine 3':5'-monophosphoric acid and 100 nM insulin was 243% (p < 0.001) above those in insulin alone. The findings suggest that the activity of hepatocyte guanylate cyclase is regulated by a synergistic action of insulin and glucagon and that positive interactions between the two cyclic nucleotide second messenger systems exist.

Activation of rat lung particulate guanylate cyclase due to filipin induced fluidity change

Involvement of cholesterol in the regulation of rat lung particulate guanylate cyclase was studied with filipin. The enzyme was not activated to a great extent by sodium nitroprusside alone; however, in presence of filipin nitroprusside activated the enzyme about 12–16 fold over the basal. Filipin did not affect the soluble enzyme significantly. The changes induced by filipin did not cause solubilization of proteins or enzyme. The Arrhenius plot of filipin-treated particulate enzyme did not have a “break” which was evident with untreated enzyme. The results suggest that the sequestering of cholesterol by filipin can modulate the activity of membrane-associated guanylate cyclase probably by changing the membrane fluidity.

Effects of heat-stable enterotoxin of Yersinia enterocolitica on ion transport and cyclic guanosine 3',5'-monophosphate metabolism in rabbit ileum.

Strains of Yersinia enterocolitica produce a heat-stable enterotoxin which is positive in the suckling mouse bioassay. Partial purification by a procedure previously worked out for heat-stable Escherichia coli enterotoxin yielded a substance which increases particulate guanylate cyclase activity and short-circuit current and inhibits active Cl-absorption in rabbit ileal mucosa. These effects of Y. enterocolitica enterotoxin are similar to those of heat-stable E. coli enterotoxin, suggesting a common mechanism of action.

Fatty acid activation of guanylate cyclase from fibroblasts and liver

Sodium arachidonate and sodium oleate increased particulate guanylate cyclase activity from homogenates of Balb 3T3 cells or rat liver. The fatty acids were about equipotent and were maximally effective at about 100 μ m concentrations. Higher concentrations were less effective or inhibitory. Activation was similar in an air or nitrogen atmosphere and was unaltered by KCN, aspirin, or indomethacin. The dose-response curve was shifted to the right when arachidonate was preincubated prior to its addition to guanylate cyclase assays. Agents that facilitate fatty acid oxidation and the formation of malonyldialdehyde during preincubation such as glutathione, hemoglobin, Mn 2+, Fe 3+, or lipoxygenase shifted the dose-response curve further to the right. In contrast, agents that decreased or prevented arachidonate oxidation and malonyldialdehyde formation during preincubation such as butylated hydroxyanisole, propyl gallate, hydroquinone, and diphenylfuran prevented the shift in the dose-response curve or in some instances shifted the dose-response curve to the left. Activation of guanylate cyclase by arachidonate was reversed by the addition of lipoxygenase to incubations. These studies indicate that unsaturated fatty acids and not their oxidation products activate particulate enzyme from Balb 3T3 cells. The mechanism of fatty acid activation appears to be different from activation by nitro compounds. Fatty acids but not nitro compounds activated fibroblast preparations, and the effect of fatty acids in contrast to the activation by nitroprusside in liver preparations was not prevented with Lubrol PX.

Activation of guanylate cyclase during the oxidation of arylamine carcinogens

When added alone, the arylamine procarcinogens N-acetyl-aminofluorene, 4-acetyl-aminobiphenyl or their N-hydroxy derivatives failed to alter partially purified soluble guanylate cyclase from rat liver or particulate guanylate cyclase activity from colonic mucosa. However, addition of linoleic acid hydroperoxide to the enzyme preparation in the presence N-OH-acetyl-aminofluorene or N-OH-acetyl-aminobiphenyl significantly increased guanylate cyclase activity. With linoleic acid hydroperoxide plus N-OH-acetyl-aminofluorene, both the activation of hepatic guanylate cyclase and the formation of the carcinogen oxidation product 2-nitrosofluorene required hematin but not molecular O 2. Both processes were inhibited by ascorbic acid. These data strongly imply that guanylate cyclase activation was dependent upon hematin catalyzed oxidation of N-OH-acetyl-aminofluorene by the lipid peroxide. The results provide the first evidence that guanylate cyclase activation can occur during the conversion of a procarcinogen to a more reactive chemical species, and thereby emphasize the importance of examining carcinogen interaction with the GC system under conditions which permit such chemical conversion.