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Biomedical subjects

F Murad

Publications and source records attributed to F Murad.

At least 163 records · Page 9Linked to original sources
PubMed1986

Regulation of cyclic GMP synthesis and the interactions with calcium.

The formation of cyclic GMP (cGMP) by guanylate cyclase, and the properties of the soluble and particulate isoenzymes, are reviewed. Regulation of guanylate cyclase and cGMP accumulation in intestinal mucosa and vascular smooth muscle with and without endothelium are summarized. The effects of E. coli heat-stable enterotoxin, nitrovasodilators, endothelium-dependent vasodilators, and atrial natriuretic factors in these systems are discussed. A potential role for free radicals, Ca, and unsaturated fatty acids in cGMP synthesis is reviewed. These tissue systems (intestinal mucosa and vascular smooth muscle with or without intact endothelium) are presented as model systems and as examples of the regulation of cGMP accumulation in a single cell type by peptides, drugs, and cell-cell interactions that are required to generate cGMP with a hormone or drug.

Animals↗
PubMed1986

Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase.

A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa. Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX. Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes. The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes. Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction. This activity was completely resistant to solubilization with a variety of detergents and chaotropes. Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively. However, ST failed to activate particulate guanylate cyclase in detergent extracts. In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures. The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored. ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose. Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography. Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST. These data suggest that guanylate cyclase and the ST receptor are independent proteins coupled by cytoskeletal components in membranes of intestinal mucosa.

Animals↗
PubMed1985

Effects of atrial natriuretic factor, sodium nitroprusside, and acetylcholine on cyclic GMP levels and relaxation in rat aorta.

The purpose of this study was to investigate the mechanisms whereby an endothelium-dependent vasodilator, acetylcholine, a nitrovasodilator, sodium nitroprusside and atrial natriuretic factor (atriopeptin II), elevate cyclic GMP levels and induce relaxation in rat thoracic aorta. Methylene blue inhibited the elevated cyclic GMP levels and relaxation due to sodium nitroprusside and acetylcholine, but not those to atriopeptin II. Cyanide inhibited relaxations to all three vasodilators, but inhibited the elevated cyclic GMP levels in response to only nitroprusside and acetylcholine. The reducing agents sodium borohydride, dithiothreitol, sucrose and isoproterenol all inhibited the elevated cyclic GMP levels due to nitroprusside and acetylcholine, while the increased cyclic GMP levels with atriopeptin II were unaffected by sodium borohydride, sucrose and isoproterenol. The effects of the reducing agents on relaxation induced by the vasodilators were difficult to interpret due to their nonspecific contractile and relaxant properties. Agents and procedures known to inhibit the Na+, K+-pump and relaxation to endothelium-dependent vasodilators and nitroprusside, including ouabain, K+-free, Mg2+-free and low Na+ Krebs-Ringer bicarbonate solution, all partially inhibited relaxations to atriopeptin II. Relaxations to atriopeptin II were also inhibited in tissues contracted with KCl. The present results suggest that the mechanism of atrial natriuretic factor-induced increased cyclic GMP levels, in contrast to that of nitroprusside and acetylcholine, does not involve the formation of free radicals, a reducible species or interaction with heme. Furthermore, the cyclic GMP formed in response to nitroprusside, acetylcholine and atrial natriuretic factor mediates relaxation through a common mechanism that may be functionally antagonized by agents and procedures which result in membrane depolarization.

Acetylcholine↗
PubMed1985

Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta.

Synthetic atriopeptin II, an atrial natriuretic factor with potent vasodilatory effects, was studied in isolated strips of rat thoracic aorta to determine its actions on contractility, cyclic nucleotide concentrations and endogenous activity of cyclic nucleotide-dependent protein kinases. Atriopeptin II was found to relax aortic strips precontracted with 0.3 microM norepinephrine whether or not the endothelial layer was present. Relaxation to atriopeptin II was closely correlated in a time- and concentration-dependent manner with increases in cyclic GMP concentrations and activation of cyclic GMP-dependent protein kinase (cyclic GMP-kinase). The threshold concentration for all three effects was 1 nM. Atriopeptin II (10 nM for 10 min) produced an 80% relaxation, an 8-fold increase in cyclic GMP concentrations and a 2-fold increase in cyclic GMP-kinase activity ratios. Atriopeptin II did not significantly alter cyclic AMP concentrations or cyclic AMP-dependent protein kinase activity. These data suggest that cyclic GMP and cyclic GMP-kinase may mediate vascular relaxation to a new class of vasoactive agents, the atrial natriuretic factors. Similar effects have been observed with the nitrovasodilator, sodium nitroprusside, and the endothelium-dependent vasodilator, acetylcholine. Therefore, a common biochemical mechanism of action that includes cyclic GMP accumulation and activation of cyclic GMP-kinase may be involved in vascular relaxation to nitrovasodilators, endothelium-dependent vasodilators and atrial natriuretic factors.

Animals↗
PubMed1985

Effects of atriopeptin on particulate guanylate cyclase from rat adrenal.

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

Adrenal Cortex↗
PubMed1985

Effects of Na+,K+-pump inhibitors and membrane depolarizing agents on acetylcholine-induced endothelium-dependent relaxation and cyclic GMP accumulation in rat aorta.

The purpose of this study was to investigate the effects of inhibitors of the Na+,K+-pump and membrane depolarizing agents on endothelium-dependent relaxation and elevated cyclic GMP levels induced by acetylcholine in rat thoracic aorta. Ouabain or exposure to K+-free or Mg2+-free Krebs-Ringer bicarbonate solution, agents and procedures known to inhibit the Na+,K+-pump, inhibited acetylcholine-induced relaxation and the associated increased levels of cyclic GMP. However, the inhibitory effect of ouabain on cyclic GMP levels was abolished in the absence of norepinephrine or in the presence of norepinephrine and the alpha-adrenergic receptor antagonist phentolamine. The membrane depolarizing agents KCl and tetraethylammonium also inhibited the acetylcholine-induced relaxation and the elevated cyclic GMP levels. Exposure to norepinephrine reduced the increased levels of cyclic GMP due to acetylcholine as compared to rested controls. This effect was inhibited by prior exposure to phentolamine, but not by the beta-adrenergic receptor antagonist, propranolol. These results suggest that increased activity of the Na+,K+-pump may mediate, in part, endothelium-dependent relaxation; inhibition of relaxation may be due to membrane depolarization; the endothelium-dependent increased levels of cyclic GMP may increase Na+,K+-pump activity; a complex interaction exists between membrane polarization, the Na+,K+-pump and alpha-adrenergic stimulation in regulation of cyclic GMP accumulation and relaxation.

Acetylcholine↗
PubMed1985

Effect of sodium-potassium pump inhibitors and membrane-depolarizing agents on sodium nitroprusside-induced relaxation and cyclic guanosine monophosphate accumulation in rat aorta.

The purpose of this study was to investigate the relationship between sodium nitroprusside-induced relaxation, inhibition of the sodium-potassium pump, and cyclic guanosine monophosphate. Exposure of rat thoracic aorta to ouabain, or potassium- or magnesium-free Krebs-Ringer bicarbonate solution, procedures which presumably inhibit the sodium-potassium pump, or to potassium chloride or tetraethylammonium, membrane-depolarizing agents, inhibited relaxation to nitroprusside. These conditions had little or no effect on the elevated cyclic guanosine monophosphate levels at a concentration of nitroprusside (0.1 microM) that relaxed norepinephrine contracted tissues by 80%. However, at a maximum relaxant concentration of nitroprusside (1.0 microM), these conditions decreased the elevation of cyclic guanosine monophosphate. The inhibition of elevated cyclic guanosine monophosphate levels was independent of the endothelium, extracellular calcium, and the cyclic guanosine monophosphate phosphodiesterase inhibitor, M&B 22,948. The inhibitory effects of ouabain and of potassium- and magnesium-free solution on the increased levels of cyclic guanosine monophosphate caused by 1.0 microM nitroprusside were abolished when tissues were incubated without norepinephrine, or with norepinephrine in the presence of the alpha-adrenergic blocker, phentolamine. In contrast, a beta-adrenergic blocker, propranolol, had no effect on the ouabain-induced inhibition of elevated cyclic guanosine monophosphate levels, with norepinephrine present. These results are consistent with the hypothesis that membrane events regulate cyclic guanosine monophosphate synthesis. At nitroprusside concentrations greater than 0.1 microM, the formation of cyclic guanosine monophosphate appears to be coupled to the status of the smooth muscle cell membrane and integrity of the sodium-potassium pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Role of cyclic-GMP in relaxations of vascular smooth muscle.

Relaxation of rat aorta segments with sodium nitroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) accumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiography. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachidonic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

A common antigenic determinant found in two functionally unrelated toxins.

The heat-stable enterotoxin ST Ib produced by enterotoxigenic E. coli strains shares a sequence homology with the sea snail neurotoxin, conotoxin GI. Rabbit antisera were raised against synthetic analogs of these toxins and to a six-residue peptide representing the region common to both toxins. Results from enzyme-linked immunosorbent assays indicate that the homologous region of both toxins represents part of their antigenic site. The lack of cross-reactivity exhibited by the six-residue common domain with serum directed against either toxin suggests that this region probably retains a similar conformation in the intact toxins but not in the isolated fragment.

Amino Acid Sequence↗
PubMed1984

Effect of cyanide on nitrovasodilator-induced relaxation, cyclic GMP accumulation and guanylate cyclase activation in rat aorta.

The effects of sodium cyanide on relaxation, increases in cyclic GMP accumulation and guanylate cyclase activation induced by sodium nitroprusside and other nitrovasodilators were examined in rat thoracic aorta. Cyanide abolished nitroprusside-induced relaxation and the associated increase in cyclic GMP levels. Basal levels of cyclic GMP and cyclic AMP were also depressed. Reversal of nitroprusside-induced relaxation by cyanide was independent of the tissue level of cyclic GMP prior to addition of cyanide. Incubation of nitroprusside with cyanide prior to addition to aortic strips did not alter the relaxant effect of nitroprusside. Sodium azide-, hydroxylamine-, N-methyl-N'-nitro-N-nitrosoguanide-, nitroglycerin- and acetylcholine-induced relaxations and increased levels of cyclic GMP were also inhibited by cyanide. Relaxations induced by nitric oxide were also inhibited by cyanide, although the relaxation with the low concentration of nitric oxide employed was not accompanied by detectable increases in cyclic GMP. Relaxation to 8-bromo-cyclic GMP was essentially unaltered by cyanide; however, isoproterenol-induced relaxation was inhibited. Guanylate cyclase in soluble and particulate fractions of aorta homogenates was activated by nitroprusside and the activation was prevented by cyanide. The present results suggest that cyanide inhibits nitrovasodilator-induced relaxation through inhibition of guanylate cyclase activation; however, cyanide may also have nonspecific effects which inhibit relaxation.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

A simple, sensitive, and specific assay for the heat-stable enterotoxin of Escherichia coli.

A simple assay for the heat-stable enterotoxin (ST) of Escherichia coli was developed on the basis of ST activation of guanylate cyclase in membranes from the intestinal mucosa of mice. ST activated guanylate cyclase in mucosal membranes in a linear fashion over a 50-fold range of toxin concentrations with Mg++-guanosine 5'-triphosphate as substrate. Activation of guanylate cyclase was detectable at concentrations of ST that were five- to 10-fold lower than those resulting in increases in the ratio of gut weight to carcass weight of mice. This assay was used to quantify ST in crude and purified samples from culture filtrates of wild-type strains and recombinant strains of E coli containing the gene for ST. Activation of guanylate cyclase was specific for ST; purified cholera toxin and E coli heat-labile enterotoxin did not activate guanylate cyclase. Thus, this assay for ST is sensitive, specific, and will facilitate rapid analysis of samples for quantification of ST.

Animals↗