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F Murad

Publications and source records attributed to F Murad.

At least 181 records · Page 10Linked to original sources
PubMed1984

Aminophylline potentiates sodium nitroprusside-induced hypotension in the dog.

The biochemical mechanisms by which nitroso-vasodilators cause smooth muscle relaxation remain controversial. One theory states that the effects of nitroso-vasodilators are mediated by increased intracellular levels of cyclic GMP due to activation of guanylate cyclase. To test this hypothesis, the authors examined the effects of sodium nitroprusside (SNP) in anesthetized dogs with an without pretreatment with the phosphodiesterase inhibitor aminophylline. Aminophylline pretreatment resulted in a 2.8-fold potentiation of the hypotensive effects of a continuous infusion of SNP. Potentiation also was seen for the effects of SNP on stroke volume, heart rate, and plasma cyclic GMP levels. These results support the hypothesis that nitroso-vasodilators exert their effects via guanylate cyclase activation. The authors advise caution when vasodilator therapy with agents such as SNP, nitroglycerin, or hydralazine is instituted in patients receiving aminophylline and when aminophylline is either instituted or discontinued in patients on vasodilator therapy.

Aminophylline↗
PubMed1984

Mechanisms of adenosine triphosphate-, thrombin-, and trypsin-induced relaxation of rat thoracic aorta.

The mechanisms by which adenosine triphosphate, thrombin, and trypsin cause relaxation of vascular smooth muscle were investigated. Relaxation of the rat thoracic aorta with adenosine triphosphate, thrombin, and/or trypsin was associated with increased levels of cyclic guanosine monophosphate in both time- and concentration-dependent manners. Thrombin and trypsin did not alter cyclic adenosine monophosphate levels, whereas adenosine triphosphate increased cyclic adenosine monophosphate levels after significant relaxation occurred. Removal of the endothelium abolished adenosine triphosphate-, thrombin-, and trypsin-induced relaxation and the associated increased levels of cyclic nucleotides. Relaxation due to these agents was also inhibited by exposure to nordihydroguaiaretic acid, a lipoxygenase inhibitor, and eicosatetraynoic acid, a lipoxygenase and cyclooxygenase inhibitor. Indomethacin, a cyclooxygenase inhibitor, potentiated relaxation to these agents, whereas the increased levels of cyclic nucleotides due to adenosine triphosphate were unaltered. Bromophenacyl bromide, a phospholipase A2 inhibitor, decreased relaxation due to adenosine triphosphate, thrombin, and trypsin and the associated increased levels of cyclic nucleotides. Removal of extracellular calcium, which also presumably inhibits phospholipase A2, prevented the elevated levels of cyclic nucleotides and the inhibitory effects of adenosine triphosphate and trypsin on contraction. In contrast, sodium nitroprusside-induced relaxation and/or increased levels of cyclic guanosine monophosphate were unaltered by nordihydroguaiaretic acid, eicosatetraynoic acid, bromophenacyl bromide, and removal of extracellular calcium. After incubation of intact tissue with 32P-orthophosphate, the patterns of protein phosphorylation caused by adenosine triphosphate, thrombin, and trypsin were indistinguishable from those of acetylcholine, sodium nitroprusside and 8-bromo cyclic guanosine monophosphate. All these agents dephosphorylated myosin light chain. Thus, the present study supports the hypothesis that relaxation induced by adenosine triphosphate, thrombin, and trypsin is mediated through the formation of an endothelial factor which elevates cyclic guanosine monophosphate levels and causes cyclic guanosine monophosphate-dependent protein phosphorylation and dephosphorylation of myosin light chain.

Acetophenones↗
PubMed1983

Highly purified particulate guanylate cyclase from rat lung: characterization and comparison with soluble guanylate cyclase.

Guanylate cyclase was purified 1000-fold from washed rat lung particulate fractions to a final specific activity of 500 nmoles cyclic GMP produced/min/mg protein by a combination of detergent extraction and chromatography on concanavalin A-Sepharose, GTP-agarose, and blue agarose. Particulate guanylate cyclase has a molecular weight of 200 000 daltons, a Stokes radius of 48 A and a sedimentation coefficient of 9.4 while the soluble form has a molecular weight of 150 000 daltons, a Stokes radius of 44 A, and a sedimentation coefficient of 7.0. Whereas the particulate enzyme is a glycoprotein with a specific affinity for concanavalin A and wheat germ agglutinin, the soluble form of guanylate cyclase did not bind to these lectins. Purified particulate guanylate cyclase did not cross-react with a number of monoclonal antibodies generated to the soluble enzyme. While both forms of the enzyme could be regulated by the formation of mixed disulfides, the particulate enzyme was relatively insensitive to inhibition by cystine. With GTP as substrate both forms of the enzyme demonstrated typical kinetics, and with GTP analogues negative cooperativity was observed with both enzyme forms. These data support the suggestion that the two forms of guanylate cyclase possess similar catalytic sites, although their remaining structure is divergent, resulting in differences in subcellular distribution, physical characteristics, and antigenicity.

Animals↗
PubMed1983

Effect of ouabain and alterations in potassium concentration on relaxation induced by sodium nitroprusside.

Relaxation of the rat thoracic aorta induced by sodium nitroprusside, 8-bromo-cyclic GMP and cyclic nucleotide phosphodiesterase inhibitor M & B 22,948 was inhibited by exposure to K+-free solution and ouabain in a concentration-dependent manner. Relaxation occurring with the change in potassium concentration in media from 1 to 2 to 10 mM was increased by sodium nitroprusside, 8-bromo-cyclic GMP and M & B 22,948. Thus, agents and procedures known to decrease and increase the activity of the Na+,K+-pump, and presumably alter the membrane potential, inhibited and enhanced relaxation, respectively. Exposure to 1 mM K+ solution increased the relaxation to low concentrations of sodium nitroprusside, but had no effect on relaxation induced by 8-bromo-cyclic GMP or M & B 22,948. Thus, another procedure which inhibits the Na+,K+-pump enhanced the effect of low concentrations of sodium nitroprusside on relaxation. Ouabain had no effect on sodium nitroprusside-induced accumulation of cyclic GMP. These results suggest that sodium nitroprusside may induce relaxation through cyclic GMP formation, effects on the Na+,K+-pump and/or hyperpolarization of the smooth muscle cell membrane.

Animals↗
PubMed1983

Agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cGMP.

The present study investigates the hypothesis that endothelium-dependent relaxation of vascular smooth muscle may be mediated through the formation of cGMP. Relaxation of the rat thoracic aorta to acetylcholine, histamine, and Ca++ ionophore A23187 was associated with increased levels of cGMP in a time- and concentration-dependent manner, whereas cAMP levels were unaltered. Removal of the endothelium prevented relaxation to these agents and prevented the increased levels of cGMP. Removal of the endothelium after exposure to acetylcholine only partially decreased the elevated levels of cGMP, suggesting that the changes in cGMP occurred within the smooth muscle cells. Eicosatetraynoic acid, an inhibitor of lipoxygenase and cyclooxygenase, and quinacrine, an inhibitor of phospholipase, prevented and reversed acetylcholine-induced relaxation, respectively, and inhibited acetylcholine-induced increased levels of cGMP. In contrast, sodium nitroprusside-induced relaxation and increased levels of cGMP were independent of the presence of the endothelium, exposure to eicosatetraynoic acid, and quinacrine. The present results support the hypothesis that vascular smooth muscle relaxation induced by some agents is dependent on the presence of the endothelium and is mediated through the formation of an endothelial factor that increases cGMP levels in smooth muscle.

Acetylcholine↗
PubMed1983

Endothelium-dependent and nitrovasodilator-induced relaxation of vascular smooth muscle: role of cyclic GMP.

The mechanisms by which endothelium-dependent relaxants and nitrovasodilators cause relaxation of vascular smooth muscle has been reviewed. A model explaining these observations is summarized in Fig. 1. The endothelium-dependent vasodilators through interaction with their appropriate receptors are thought to activate phospholipase A2 and cause the release of an unsaturated fatty acid. The released unsaturated fatty acid or a metabolite is thought to be the "endothelial relaxant factor" that interacts with the smooth muscle component to cause relaxation. While the unsaturated fatty acid may be oxidized in either the endothelial cell or smooth muscle cell, the lability of the endothelial relaxant factor suggests that at least some of this processing occurs before its release from the endothelium. the model in Figure 1 suggests that an oxidized fatty acid or a derived free radical is responsible for activation of smooth muscle guanylate cyclase and increases in cyclic GMP levels. As pointed out above, the use of various inhibitors of fatty acid release and metabolism has not allowed us or others to predict the structure of the active material. To date the best evidence suggests that the unsaturated fatty acid is a product of either the lipoxygenase or P-450 pathways. Nitrovasodilators are thought to form nitric oxide free radical and directly activate guanylate cyclase as described above. Activated guanylate cyclase, whether by endothelium dependent agents or the nitrovasodilators, then increases the formation of cyclic GMP, which activates cyclic GMP-dependent protein kinase. The phosphorylation state of various proteins is then altered and, eventually, myosin light chain is dephosphorylated and relaxation occurs. Whether this mechanism involves cyclic GMP-dependent changes in activities of myosin light chain kinase and/or myosin light chain phosphatase remains to be determined. Although the altered phosphorylation state of myosin light chain that results from cyclic GMP accumulation may explain the mechanisms of action of cyclic GMP in smooth muscle relaxation, other mechanisms can not be excluded. For example, some additional studies which we have not summarized here indicate that the integrity of the membrane and Na+-K+ pump can modify both cyclic GMP synthesis and relaxation in rat aorta (38 and unpublished observations). Apparently complex interactions may exist in smooth muscle and other tissues which regulate cyclic GMP accumulation and/or its expression on some process. While several functions for cyclic GMP have been suggested, there is considerable evidence which suggests that one of its roles is relaxation of airway and vascular smooth muscle.

Animals↗
PubMed1982

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 less than 0.05). In the suckling mouse model, subcutaneous quinacrine (0.1 mumole/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 mumole/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 mumole/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.

Animals↗
PubMed1982

Effect of GTP analogues on purified soluble guanylate cyclase.

In our studies with purified soluble guanylate cyclase from rat lung, we have tested a number of guanosine 5'-triphosphate (GTP) analogues as substrates and inhibitors, 5'-Guanylylimidodiphosphate (GMP-P(NH)P), guanylyl (beta, gamma-methylene) diphosphate (GMP-P(CH2)P), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were found to be substrates for guanylate cyclase. GTP gamma S supported cyclic GMP formation at 20 or 75% of the rate seen with Mn2+-GTP and Mg2+-GTP, respectively. GMP-P(NH)P and GMP P(CH2)P supported cyclic GMP formation at 10-20% of the GTP rate with either cation cofactor. These analogues were found to have multiple Km values; one Km value was similar to GTP (150 microM with Mg2+, 20-70 microM with Mn2+), but an additional high affinity catalytic site (3 microM) was also observed. Guanosine tetraphosphate (Ki = 10 microM), adenosine triphosphate (Ki = 9 microM) and the 2'3'-dialdehyde derivative of GTP (dial GTP) (Ki = 1 microM) were not good substrates for the enzyme; however, they were potent competitive inhibitors. These GTP analogues will be useful tools for the study of GTP binding sites on guanylate cyclase and they may also help elucidate the effects of free radicals and other agents on guanylate cyclase regulation.

Animals↗
PubMed1982

Sodium nitroprusside-induced protein phosphorylation in intact rat aorta is mimicked by 8-bromo cyclic GMP.

The effects of sodium nitroprusside, 8-bromo cyclic GMP, 8-bromoguanosine 5'-monophosphate, 8-bromo cyclic AMP, dibutyryl cyclic AMP, and isoproterenol on incorporation of (32)P into proteins in intact rat thoracic aorta were studied. Aortas were incubated in [(32)P]orthophosphate in order to label endogenous adenosine triphosphate. Agents were then added for various times and the tissues were homogenized and fractionated (100,000 x g for 60 min) into soluble and particulate fractions. Soluble and particulate fractions were subjected to isoelectric focusing followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiographs were made. Nitroprusside induced a concentration-dependent increase in incorporation of (32)P into nine proteins and a decrease in (32)P incorporation into two proteins. Some of these proteins appeared in both the soluble and particulate fractions of homogenates; others appeared only in the soluble fraction. The pattern of (32)P incorporation was identical after 2- or 15-min exposure to nitroprusside and was mimicked by exposure to 50-500 muM 8-bromo cyclic GMP. 8-Bromoguanosine 5'-monophosphate did not alter (32)P incorporation. Dibutyryl cyclic AMP at 50 muM had no effect upon (32)P incorporation whereas a higher concentration (0.5 mM) caused increased or decreased (32)P incorporation into some, but not all, of the same proteins. 8-Bromo cyclic AMP (5 mM) produced only small changes in (32)P incorporation. The pattern of (32)P incorporation induced by a relatively high concentration of isoproterenol 0.1 mM was similar but not identical to that seen with 0.5 mM dibutyryl cyclic AMP. The present study indicates that the incorporation of (32)P into endogenous proteins of intact rat aorta can be regulated by nitroprusside. These effects can be mimicked by cyclic GMP analogues and only partially by cyclic AMP analogues or isoproterenol. Presumably, these effects of nitroprusside are mediated through a cyclic GMP-dependent process (protein kinase or phosphatase) which may play a role in the relaxant properties of nitroprusside and cyclic GMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

Immunohistochemical localization of guanylate cyclase within neurons of rat brain.

The immunohistochemical localization of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been examined in rat neocortex, caudate-putamen, and cerebellum by using specific monoclonal antibodies. Immunofluorescence could be seen within somata and proximal dendrites of neurons in the these regions. A nuclear immunofluorescence reaction to guanylate cyclase was characteristically absent. The staining pattern for guanylate cyclase was coincident with previously described localizations of cyclic GMP immunofluorescence within medium spiny neurons of the caudate-putamen and pyramidal cells of the neocortex. Cerebellar guanylate cyclase immunoreactivity was primarily confined to Purkinje cells and their primary dendrites, similar to the pattern reported for cyclic GMP-dependent protein kinase localization. Guanylate cyclase immunofluorescence was abolished when the monoclonal antibodies were exposed to purified enzyme prior to incubation of the tissue slices or when control antibody was substituted for the primary antibody. Immunohistochemical localization of cyclic AMP in these same tissues was readily distinguished from that of guanylate cyclase or cyclic GMP, showing uniform fluorescence throughout the cell bodies of neurons and glial elements.

Animals↗
PubMed1982

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.

Animals↗
PubMed1982

Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside.

Highly purified rat lung soluble guanylate cyclase was activated with nitric oxide or sodium nitroprusside and the degree of activation varied with incubation conditions. With Mg2+ as the action cofactor, about 2- to 8-fold activation was observed with nitric oxide or sodium nitroprusside alone. Markedly enhanced activation (20-40 fold) was observed when 1 muM hemin added to the enzyme prior to exposure to the activating agent. The activation with hemin and sodium nitroprusside was prevented in a dose-dependent manner by sodium cyanide. The level activation was also increased by the addition of 1 mM dithiothreitol, but unlike hemin which had no effect on basal enzyme activity, dithiothreitol led to a considerable increase in basal activity. Activated guanylate cyclase decayed to basal activity within one hour at 2 degrees C and the enzyme could be reactivated upon re-exposure to nitroprusside or nitric oxide. Under basal conditions, Michaelis-Menten kinetics were observed, with a Km for GTP of 140 muM with Mg2+ cofactor. Following activation with nitroprusside or nitric oxide, curvilinear Eadie-Hofstee transformations of kinetic data were observed, with Km's of 22 MuM and 100 MuM for Mg-GTP. When optimal activation (15-40 fold) was induced by the addition of hemin and nitroprusside, multiple Km's were also seen with Mg-GTP and the high affinity form was predominant (22 MuM). Similar curvilinear Eadie-Hofstee transformations were observed with Mn2+ as the cation cofactor. These data suggest that multiple GTP catalytic sites are present in activated guanylate cyclase, or alternatively, multiple populations of enzyme exist.

Animals↗
PubMed1982

Partial purification and characterization of particulate guanylate cyclase from rat liver after solubilization with trypsin.

Guanylate cyclase from 105,000 X g particulate fractions of rat liver homogenates (20 pmoles of cyclic GMP formed/min/mg protein) was solubilized in the absence of detergents by incubating fractions 12 min at 37 degrees with 5 ug/ml trypsin. Optimal solubilization was dependent upon trypsin and particulate preparation concentrations. Virtually no activation of particulate guanylate cyclase was observed at any time point or trypsin concentration tested. Guanylate cyclase solubilized with trypsin was purified about 500-fold (9.4 nmoles/min/mg protein) using ammonium sulfate precipitation, GTP-affinity chromatography, and preparative polyacrylamide gel electrophoresis. Activity eluted as a single peak on Sepadex G-200 (Stokes radius = 40 A) and migrated as a single peak on sucrose density gradients (S20,w = 4.6). Thus, the tryptic fragment was estimated to be about 80,000 daltons (Mr) with a frictional ratio (f/fo) of 1.4. These partially purified preparations exhibited linear double reciprocal plots with Mn-GTP and Hill coefficients of 1.0. This is in contrast to the crude membrane-associated enzyme which had a Hill co-efficient of 1.5. Membrane-bound and trypsin-solubilized guanylate cyclase were activated 3- and 4-fold with nitric oxide and were inhibited with 1mM cystamine. Cystamine inhibition could be partially reversed with 7.5 mM dithiothreitol. These studies indicate that particulate guanylate cyclase solubilized by limited proteolysis is amenable to purification by "classical" chromatographic techniques. The partially purified fragment contains the catalytic site, the site for nitric oxide activation, and at least one sulfhydryl group required for activity.

Animals↗
PubMed1982

Bioequivalence of a slow-release potassium tablet and a liquid potassium supplement.

The rate and extent of potassium absorption from a slow-release potassium chloride tablet and a 10% potassium chloride solution were compared in 12 healthy male volunteers, 18-25 years old. A single 48 mEq potassium dose of the tablet released potassium 30 min slower than the same dose of potassium chloride solution. Potassium was completely absorbed from both preparations within 24 h. Complete absorption was confirmed by quantitative determination of fecal potassium excretion. Gastrointestinal tolerance was good with both preparations, although the incidence of adverse experiences was slightly higher with the liquid preparation. "Bad taste" was not associated with the slow-release tablet; 92% of the subjects reported "bad taste" with the potassium chloride solution.

Adolescent↗
PubMed1981

Reversible inactivation of guanylate cyclase by mixed disulfide formation.

Highly purified preparations of guanylate cyclase from rat lung were inactivated by several disulfide compounds in a time- and dose-dependent manner. Cystamine and cystine were the most potent disulfides tested, but other compounds which contained the cysteamine moiety (NH2CH2CH2S-), including pantethine and oxidized coenzyme A, were also able to partially inactivate the enzyme. In addition to the decrease in basal activity (measured with either Mg2+-GTP or Mn2+-GTP), disulfide-inhibited enzyme was activated to a lesser extent by nitric oxide. Treatment with dithiothreitol or other reducing agents restored basal activity and increased the level of cGMP production following nitric oxide activation. Control enzyme samples exhibited a single GTP Km of 25 microM or 150 microM with Mn2+ or Mg2+, respectively. However, cystamine-treated enzyme showed these same Km values as well as an additional GTP Km of 2 to 3 microM using either metal ion as cofactor. When [35S]cystine was incubated with purified enzyme, radioactivity was incorporated into the trichloroacetic acid-precipitable protein, and the counts were released following dithiothreitol treatment. In addition, [35S]cystine-labeled enzyme co-migrated with native guanylate cyclase on nondenaturing polyacrylamide gels. These data indicate that mixed disulfides can be formed between guanylate cyclase and certain naturally occurring compounds, and that disulfide formation leads to a reversible loss of enzyme activity.

Animals↗
PubMed1981

Effects of pyruvate and other metabolites on cyclic GMP levels in incubations of rat hepatocytes and kidney cortex.

Pyruvate increased cyclic GMP levels in rat hepatocytes. The effects were observed without or with 1-methyl-3-isobutylxanthine. Lactate, acetate, oxaloacetate, alpha-ketoglutarate, succinate, acetoacetate and beta-hydroxybutyrate also increased cyclic GMP levels. Some compounds increased cyclic GMP in kidney cortex slices. The effects were dependent upon Ca2+ in the medium. Cyclic AMP was increased 30-50% by some of these substances with 2.6 mM Ca2+. Rotenone, oligomycin, antimycin, dinitrophenol, KCN, and arsenate decreased GTP and ATP, basal cyclic GMP and the pyruvate effect, but did not alter cyclic AMP. Although fluoroacetate alone had no effect on cyclic nucleotides, GTP, or ATP, it potentiated the pyruvate effect on cyclic GMP. Adenosine and guanosine increased cyclic GMP and GTP to a similar extent of 30-50%. Aminooxyacetate, cycloserine, pentenoic acid and mepacrine decreased the pyruvate effect while cycloserine or mepacrine alone increased cyclic GMP. Citrate and mepacrine inhibited soluble and particulate guanylate cyclase from rat liver while cycloserine and acetoacetate increased guanylate cyclase activity. None of the other compounds altered guanylate cyclase activity. These results indicate that various metabolites and inhibitors can alter cyclic GMP accumulation in hepatocytes and renal cortex slices. Several mechanisms may be involved in these effects.

1-Methyl-3-isobutylxanthine↗