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Biomedical subjects

F Murad

Publications and source records attributed to F Murad.

At least 127 records · Page 7Linked to original sources
PubMed1989

ANP relaxes bovine tracheal smooth muscle and increases cGMP.

Effects of atrial natriuretic peptides (ANP) on the tension, content of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) and activity of particulate and soluble forms of guanylate cyclase were examined in bovine tracheal smooth muscle. Atrial natriuretic factor (ANF), atriopeptin II, and atriopeptin III were found to induce relaxation of tracheal smooth muscle precontracted with 5 x 10(-8) M carbachol (an approximate median effective concentration) in a concentration-dependent manner. However, atriopeptin I failed to induce significant relaxation of the muscle. Similar results were obtained when 3 x 10(-6) M histamine or 5 x 10(-7) M serotonin was used as the contracting agent. However, the relaxant effects of ANF, atriopeptin II, and atriopeptin III were much less when a higher concentration of carbachol or 30 mM K+ was used as the contractile agent. Maximally inhibitory concentration (IC50) values of ANF, atriopeptin II, and atriopeptin III for inhibition of muscle contraction induced by 5 x 10(-8) M carbachol ranged from 3.8 to 8.3 x 10(-9) M, indicating that these peptides have intermediate potency between isoproterenol (IC50, 2.0 x 10(-9) M) and sodium nitroprusside (IC50, 2.0 x 10(-8) M). Treatment of the muscle with 3 x 10(-7) M ANF slowed the rate of tension development of the muscle by 10(-7) M carbachol. Tissue levels of cAMP were not influenced by any of the atrial peptides at concentrations of 10(-9)-10(-6) M; however, cGMP levels were increased about five- to ninefold.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1989

Effects of melittin on endothelium-dependent relaxation and cyclic GMP levels in rat aorta.

The present study investigates the mechanism of endothelium-dependent relaxation of vascular smooth muscle. Melittin, a polypeptide found in honeybee venom and a known activator of phospholipase A2, induced transient, endothelium-dependent relaxations of rat thoracic aortae contracted with norepinephrine. Higher concentrations of melittin induced relaxations followed by contractions. Prior incubation of melittin with trypsin abolished the changes in relaxation and contraction due to melittin. Melittin (10 micrograms/ml)-induced relaxations were associated with transiently elevated levels of cyclic GMP with a peak increase of 30-fold, which occurred 30 seconds after melittin exposure. Melittin (10 micrograms/ml) elevated cyclic AMP levels less than twofold and this effect was variable. A lower concentration of melittin (1 microgram/ml) elevated cyclic GMP levels approximately twofold, while exposure to 1 microgram/ml melittin in the presence of the cyclic GMP phosphodiesterase inhibitor, M&B 22948 (1 mM), increased cyclic GMP levels fivefold. Removal of the endothelium prevented the increased levels of cyclic GMP and cyclic AMP due to melittin. Exposure to the guanylate cyclase inhibitor, methylene blue, prevented the increased levels of cyclic GMP. Methylene blue, nordihydroguaiaretic acid, and the phospholipase A2 inhibitor, parabromophenacyl bromide, inhibited melittin-induced relaxations, while the cyclo-oxygenase inhibitor, indomethacin, was without effect. Arachidonic acid increased cyclic AMP levels but had no effect on cyclic GMP levels in the presence or absence of indomethacin. Relaxations to melittin, and to the endothelium-dependent vasodilators acetylcholine, trypsin, histamine, and the Ca2+ ionophore A23187, and/or the associated increased cyclic GMP levels, were reduced following exposure to melittin. Prior exposure to polyarginine (10 micrograms/ml), which induced endothelium-dependent relaxations that were prevented by methylene blue, also inhibited relaxations to the endothelium-dependent vasodilators. In contrast, relaxations to sodium nitroprusside were potentiated in tissues previously exposed to melittin. Removal of the endothelium by rubbing the intimal surface also potentiated relaxations to sodium nitroprusside. Scanning electron micrographs of the intimal surface demonstrated that melittin and polyarginine greatly damaged the endothelial cells. The present results suggest that polycation containing peptides induce endothelium-dependent relaxation through elevation of cyclic GMP levels within the smooth muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Relationship between biotransformation of glyceryl trinitrate and cyclic GMP accumulation in various cultured cell lines.

We assessed glyceryl trinitrate (GTN) biotransformation and cyclic GMP accumulation in cultured rat lung fibroblasts (RLF), porcine kidney epithelial (PK1), bovine aortic endothelial (BAE) and bovine aortic smooth muscle (BASM) cells. Biotransformation of 0.1 microM GTN was linear over 30 min and the percentage of glyceryl dinitrate (GDN)/10(6) cells for BAE, BASM, RLF and PK1 at 30 min was 3.1, 2.3, 5.8 and 21.7%, respectively. At low GTN concentration (0.01-0.1 microM) there was a highly selective formation of 1,2-GDN, whereas at higher GTN concentration (greater than 1 microM) this selectivity was lost. Cyclic GMP accumulation did not occur in BAE or BASM at any GTN concentration, whereas for RLF and PK1 it was highly correlated to the rate of GDN formation. Upon re-exposure to GTN after treatment of RLF or PK1 cells for 3 hr with 0.1 mM GTN, there was an almost complete loss of the cyclic GMP response, GTN biotransformation was attenuated markedly and the selective formation of 1,2-GDN at low GTN concentration was absent. However, when GTN-treated cells were incubated for 18 hr in GTN-free media, there was a recovery of the cyclic GMP response, GTN biotransformation and selective 1,2-GDN formation toward control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung.

A complementary DNA clone corresponding to the 70 kDa subunit of soluble guanylate cyclase (EC 4.6.1.2) of rat lung has been isolated. The primary structure of the cDNA consisted of 3063 nucleotides including a 1857-nucleotide coding region for 619 amino acids, and the calculated molecular weight was 70476. Blot hybridization of total poly(A)+RNAs from rat tissues detected a mRNA of about 3.4 kilobases. The amount of mRNA was abundant in lung, cerebrum and cerebellum, moderate in heart and kidney, and low in liver and muscle. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated the presence of one gene in the rat haploid genome. The amino acid sequence of the 70 kDa subunit has partial homology with particulate guanylate cyclase from sea-urchin sperm, and protein phosphatase inhibitor I.

Amino Acid Sequence↗
PubMed1988

Brain natriuretic factor. Augmentation of cellular cyclic GMP, activation of particulate guanylate cyclase and receptor binding.

The newly discovered peptide, brain natriuretic factor (BNF), caused a concentration-dependent increase (up to 400-fold) in intracellular cyclic GMP levels in cultured endothelial, smooth muscle and fibroblast cells. The extent of cGMP augmentation was comparable to that produced by atrial natriuretic factor (ANF). The activity of the membrane-bound guanylate cyclase of different rat tissues and cultured cells was markedly stimulated by the peptide and the addition of ATP potentiated the stimulation. As opposed to tissue particulate guanylate cyclase, the enzyme in cell membranes was slightly more sensitive to activation by BNF than to stimulation by ANF. On bovine aortic smooth muscle (BASM) cells, specific high-affinity binding sites (Bmax = 398 fmol/10(6) cells, Kd = 0.52 nM) for BNF were observed for which ANF could compete with apparently equal affinity. These results suggest that activation of the cGMP pathway constitutes a common mechanism of action for both BNF and ANF.

Adenosine Triphosphate↗
PubMed1988

Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells.

The stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity by atrial natriuretic peptide (ANP) was compared to the affinity and number of ANP receptors in eight cultured cell types. At 100 nM, ANP increased cyclic GMP by 13-fold in bovine adrenal cortical, 35-fold in human lung fibroblast, 58-fold in canine kidney epithelial, 60-fold in bovine aortic smooth muscle, 120-fold in rat mammary epithelial, 260-fold in rat Leydig, 300-fold in bovine kidney epithelial, and 475-fold in bovine aortic endothelial cells. ANP (1 microM) increased particulate guanylate cyclase activity by 1.5-, 2.5-, 3.1-, 3.2-, 5.0-, 7.0-, 7.8-, and 8.0-fold in bovine adrenal cortical, bovine aortic smooth muscle, human lung fibroblast, canine kidney epithelial, rat mammary epithelial, rat Leydig, bovine kidney epithelial, and bovine aortic endothelial cells, respectively. Specific 125I-ANP binding to intact rat Leydig (3,000 sites/cell; Kd = 0.11 nM), bovine aortic endothelial (14,000 sites/cell; Kd = 0.09 nM), bovine adrenal cortical (50,000 sites/cell; Kd = 0.12 nM), human lung fibroblast (80,000 sites/cell; Kd = 0.32 nM), and bovine aortic smooth muscle (310,000 sites/cell; Kd = 0.82 nM) cells was saturable and high affinity. No specific and saturable ANP binding was detected in bovine and canine kidney epithelial and rat mammary epithelial cells. Two ANP-binding sites of 66,000 and 130,000 daltons were specifically labeled by 125I-ANP after cross-linking with disuccinimidyl suberate. The 130,000-dalton ANP-binding sites bound to a GTP-agarose affinity column, and the specific activity of guanylate cyclase was increased by 90-fold in this fraction. Our results demonstrate that the increase in cyclic GMP accumulation and particulate guanylate cyclase activity by ANP does not correlate with the affinity and number of ANP-binding sites. These results suggest that multiple populations of ANP receptors exist in these cells and that only one receptor subtype (130,000 daltons) is associated with particulate guanylate cyclase activity.

Animals↗
PubMed1988

Effects of ethacrynic acid and cystamine on sodium nitroprusside-induced relaxation, cyclic GMP levels and guanylate cyclase activity in rat aorta.

1. Ethacrynic acid, an agent that alkylates sulfhydryl residues, inhibited sodium nitroprusside- and 8-bromo cyclic GMP-induced relaxations. 2. Sodium nitroprusside-induced increased levels of cyclic GMP were unaltered by ethacrynic acid. 3. Concentrations of ethacrynic acid that inhibited sodium nitroprusside-induced relaxation did not affect sodium nitroprusside-activation of crude soluble and particulate fractions of guanylate cyclase, while a higher concentration of ethacrynic acid did inhibit the activation. 4. Cystamine, an agent that oxidizes sulfhydryl residues, inhibited sodium nitroprusside-activation of crude soluble and particulate fractions of guanylate cyclase. Exposure of intact rat aorta to cystamine inhibited basal guanylate cyclase activity in the particulate fraction but, in general, not in the soluble fraction. 5. These results are consistent with the hypothesis that vascular smooth muscle relaxation requires sulfhydryl groups. The sulfhydryl groups that presumably are alkylated by ethacrynic acid are not contained within guanylate cyclase and are involved at a regulatory step after the formation of cyclic GMP. The sulfhydryl groups altered by cystamine may be located on particulate guanylate cyclase and a role for particulate guanylate cyclase in nitrovasodilator-induced relaxation needs to be examined further.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Hemodynamic, renal and endocrine effects of atrial natriuretic peptide infusion in severe heart failure.

The cardiac release and total body and renal clearances and the hemodynamic, renal and endocrine effects of increasing doses of atrial natriuretic peptide were investigated in 12 patients with severe chronic congestive heart failure. Immunoreactive arterial plasma levels of atrial natriuretic peptide were 10-fold higher than normal and there was no correlation between aortic atrial natriuretic peptide and cardiac filling pressures. The heart released atrial natriuretic peptide into the coronary sinus. The kidney, though a major clearance site, accounted for only 33% of the total body clearance. Administration of 0.3 micrograms/kg per min atrial natriuretic peptide produced significant changes in heart rate (95 +/- 4 to 85 +/- 4 beats/min) and mean arterial (92 +/- 8 to 77 +/- 9 mm Hg), right atrial (13 +/- 3 to 8 +/- 2 mm Hg) and mean pulmonary artery occluded (27 +/- 3 to 14 +/- 3 mm Hg) pressures. Atrial natriuretic peptide increased cardiac index (2.25 +/- 0.18 to 2.83 +/- 0.3 liters/min per m2) and stroke work index (21 +/- 1.5 to 29 +/- 3.4 g/m2), whereas systemic vascular resistance (1,424 +/- 139 to 1,033 +/- 97 dynes.s.cm(-5)) decreased. Infusion of 0.1 microgram/kg per min atrial natriuretic peptide increased urinary flow 128%, fractional excretion of sodium 133% and fractional excretion of potassium 35%. The filtration fraction increased from 29 +/- 2 to 31 +/- 4%. This represented a disproportionate rise in glomerular filtration rate over renal plasma flow. Plasma aldosterone and norepinephrine decreased whereas plasma renin activity remained unchanged. In association with these hemodynamic, excretory and endocrine changes, the urinary excretion of cyclic guanosine monophosphate doubled. Placebo had no effect. These results showed that, despite high circulating levels of atrial natriuretic peptide, administration of this hormone in heart failure is associated with potentially beneficial hemodynamic, renal and endocrine effects.

Adult↗
PubMed1988

Effects of nitrovasodilators, endothelium-dependent vasodilators, and atrial peptides on cGMP.

The interactions of the nitrovasodilators, endothelium-dependent vasodilators, and atrial peptides with cGMP synthesis have proved to be useful and important leads to the functions of cGMP and signal transduction mechanisms. Although considerable progress has been made, many important questions remain to be answered. Nevertheless, the guanylate cyclase-cGMP system clearly represents an important second-messenger system that mediates the effects of numerous agents.

Atrial Natriuretic Factor↗
PubMed1988

Role of cardiac atria in the human renal response to changing plasma volume.

We examined the role of cardiac atria in the renal response to sequential volume expansion and contraction, during and directly following water immersion. In immersed healthy volunteers (group 1, n = 9) atrial diameter, plasma levels of atrial natriuretic peptide (ANP), and natriuresis increased, whereas renal vascular resistance (RVR) and filtration fraction declined. Each parameter changed in an opposite direction postimmersion. An analysis of transglomerular dextran transport suggests that transglomerular hydraulic pressure difference (delta P) changed in parallel with filtration fraction. Baseline atrial diameter, plasma ANP, RVR, and filtration fraction were significantly elevated in nine recipients of denervated cardiac allografts (group 2). Atrial diameter and plasma ANP changed in parallel with group 1 during and after immersion. However, corresponding reciprocal changes in RVR were smaller and filtration fraction remained constant throughout. From transglomerular dextran transport, we compute that delta P increased progressively during and after immersion, suggesting predominant efferent arteriolar tone. The postimmersed state was associated also with enhanced sodium retention despite sixfold higher plasma ANP than in group 1. These findings are consistent with an effect of cardiac denervation to leave unopposed efferent sympathetic nervous traffic to the kidney. They suggest that the latter is an important modulator of the renal response to changing plasma volume in humans.

Atrial Function↗
PubMed1988

Effect of in vitro organic nitrate tolerance on relaxation, cyclic GMP accumulation, and guanylate cyclase activation by glyceryl trinitrate and the enantiomers of isoidide dinitrate.

Previously, it was shown that the D enantiomer of isoidide dinitrate was 10-fold more potent than the L enantiomer and 10-fold less potent than glyceryl trinitrate for stimulating cyclic GMP accumulation and relaxation of isolated rat aorta. In the present study, these organic nitrates were tested for their ability to induce tolerance to organic nitrate-induced relaxation, cyclic GMP accumulation, and guanylate cyclase activation in rat aorta in vitro. To compensate for the differences in vasodilator potency, tolerance was induced by incubating isolated rat aorta with concentrations of organic nitrates 1,000-fold greater than the EC50 for relaxation. Under these conditions, the EC50 for relaxation was increased significantly for each organic nitrate and to a similar degree on subsequent reexposure. These data suggest that the potential for inducing in vitro tolerance to relaxation was the same for the three organic nitrates tested. When activation of soluble guanylate cyclase by these compounds was assessed, the enantiomers of isoidide dinitrate were equipotent, but less potent than glyceryl trinitrate, suggesting that the site of enantioselectivity is not guanylate cyclase itself. In blood vessels made tolerant to organic nitrates by pretreatment with glyceryl trinitrate, vasodilator activity, cyclic GMP accumulation, and guanylate cyclase activation were attenuated on reexposure to each organic nitrate. In addition, differences in the potency of the three organic nitrates and the enantioselectivity of isoidide dinitrate for relaxation were abolished in tolerant tissue, whereas the potency difference between glyceryl trinitrate and isoidide dinitrate for activation of guanylate cyclase was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Atrial natriuretic peptide, oxytocin, and vasopressin increase guanosine 3',5'-monophosphate in LLC-PK1 kidney epithelial cells.

The effect of atrial natriuretic peptide (ANP), arginine vasopressin (AVP), and oxytocin (OT) on cAMP and cGMP accumulation was investigated in LLC-PK1 kidney epithelial cells. The addition of ANP, AVP, and OT to intact cells produced a time- and concentration-dependent increase in cGMP accumulation. ANP produced a 1.7-fold increase in cGMP at 10 pM and a maximal 28-fold increase in cGMP at 1 microM. ANP had no effect on basal or AVP-induced stimulation of cAMP accumulation. OT was 10-fold more potent than AVP at increasing cGMP levels, producing a 2.1-fold increase in cGMP at 0.1 nM, whereas AVP was 100-fold more potent at increasing cAMP levels. At a concentration of 1 microM, AVP and OT produced a maximal 12 to 14-fold increase in cGMP, while OT and AVP produced 50- and 90-fold increase in cAMP, respectively. The selective OT agonist [Thr4, Gly7]oxytocin was very effective at increasing cGMP, but not at increasing cAMP levels. The V2-vasopressin agonist [deamino-Pen1,Val4, D-Arg8]vasopressin did not increase cGMP levels, but produced a 20-fold increase in cAMP levels. The addition of ANP together with either AVP or OT produced an additive increase in cGMP content. Simultaneous addition of AVP and OT did not lead to a greater increase in cAMP or cGMP levels. These results suggest that the AVP- and OT-induced increase in cGMP is mediated by OT receptors, whereas the increase in cAMP is probably mediated by vasopressin receptors. ANP increased the activity of particulate guanylate cyclase by 6-fold, while AVP and OT has no effect on particulate guanylate cyclase activity. The relatively selective inhibitor of soluble guanylate cyclase, methylene blue, had no effect on the ANP-induced increase in cGMP content in intact cells, but produced a 50% inhibition of the increase in cGMP by AVP and OT. Methylene blue did not alter the stimulation of cAMP by AVP or OT. These results demonstrate that ANP, AVP, and OT increase cGMP in LLC-PK1 kidney epithelial cells. The increase in cGMP by ANP is mediated by particulate guanylate cyclase, whereas AVP and OT probably increase cGMP by interacting with OT receptors coupled to soluble guanylate cyclase.

Animals↗
PubMed1988

Glyceryl trinitrate-induced desensitization of guanylate cyclase in cultured rat lung fibroblasts.

Cultured rat lung fibroblasts were used to explore desensitization of guanylate cyclase to nitrovasodilators. The effect of pretreatment with glyceryl trinitrate (GTN) on the concentration-response curves of GTN and sodium nitroprusside (SNP) for cyclic GMP accumulation in intact cells and activation of guanylate cyclase in broken cell preparations was measured. Pretreatment of cells with 1 microM GTN for 3 h decreased cyclic GMP accumulation induced by GTN but had no effect on SNP-induced cyclic GMP accumulation. Pretreatment of cells with 100 microM GTN decreased the efficacy of GTN and SNP for cyclic GMP elevation by 89% and 40%, respectively. In contrast to results obtained with GTN, SNP slightly desensitized cyclic GMP accumulation induced by GTN and SNP. Pretreatment of cells with 100 nM atrial natriuretic peptide resulted in a 44% decrease in cyclic GMP accumulation induced by subsequent exposure to 10 nM atrial natriuretic peptide but had no effect on cyclic GMP elevation induced by nitrovasodilators. In experiments with crude preparations of soluble guanylate cyclase from cells pretreated with 1 mM GTN, activation of the enzyme by GTN and SNP was inhibited almost completely. Tolerance to GTN in intact cells could not be reversed by subsequent incubation with thiols such as cysteine, N-acetylcysteine or glutathione. However, overnight incubation of GTN-tolerant cells in media without added thiols resulted in complete recovery of responsiveness to GTN. Recovery of GTN-induced cyclic GMP accumulation was inhibited in a concentration-dependent manner by cycloheximide, suggesting that reversal of organic nitrate tolerance requires de novo synthesis of gyanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1988

Biochemical mechanisms underlying vascular smooth muscle relaxation: the guanylate cyclase-cyclic GMP system.

Several classes of vasodilators have been demonstrated to elicit their affects by activating guanylate cyclase and elevating intracellular concentrations of cyclic GMP. The nitrovasodilators, such as nitroglycerin, generate nitric oxide, which directly activates the soluble isoenzyme of guanylate cyclase resulting in increased intracellular concentrations of cyclic GMP. A second class of agents, the endothelium-dependent vasodilators, such as acetylcholine, requires an intact endothelium to elicit vascular smooth muscle relaxation, in contrast to the nitrovasodilators. These agents stimulate the release of an endothelium-derived relaxing factor (EDRF), which also activates the soluble form of guanylate cyclase, triggering the production of cyclic GMP. The third class of agents includes atrial natriuretic peptides (ANPs). These low-molecular-weight, heat-stable peptides bind to specific receptors on vascular smooth muscle. These receptors appear unique in that they have a dual function possessing both ANP binding and particulate guanylate cyclase activities. Binding to and activation of particulate guanylate cyclase, in the absence of endothelium, results in the elevation of intracellular concentrations of cyclic GMP and relaxation.

Animals↗
PubMed1987

Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase.

We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37 degrees C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 +/- 0.077 nM and the Bmax. was 216 +/- 33 fmol bound/10(6) cells, which corresponds to 130,000 +/- 20,000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 microM-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increased the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demonstrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.

Animals↗