scholarly journals Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver

1976 ◽  
Vol 157 (3) ◽  
pp. 713-719 ◽  
Author(s):  
S J Sulakhe ◽  
N L Leung ◽  
P V Sulakhe
Keyword(s):  
Guanylate Cyclase ◽  
Metal Binding ◽  
Plasma Membranes ◽  
Soluble Guanylate Cyclase ◽  
Inhibitory Effect ◽  
Ion Concentration ◽  
Metal Binding Sites ◽  
Microsomal Membranes ◽  
Solubilized Enzyme ◽  
Triton X

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.

α2-Adrenergic-mediated tubular NO production inhibits thick ascending limb chloride absorption

2001 ◽  
Vol 281 (4) ◽  
pp. F679-F686 ◽  
Author(s):  
Craig F. Plato ◽  
Jeffrey L. Garvin
Keyword(s):  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Chloride Transport ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
No Production ◽  
Thick Ascending Limb ◽  
Chloride Absorption ◽  
Ly 83583

Stimulation of α2-adrenergic receptors inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (THAL) expresses α2-receptors. We hypothesized that selective α2-receptor activation decreases NaCl absorption by cortical THALs through activation of NOS and increased production of NO. We found that the α2-receptor agonist clonidine (10 nM) decreased chloride flux ( J Cl) from 119.5 ± 15.9 to 67.4 ± 13.8 pmol · mm−1 · min−1 (43% reduction; P < 0.02), whereas removal of clonidine from the bath increased J Cl by 20%. When NOS activity was inhibited by pretreatment with 5 mM N G-nitro-l-arginine methyl ester, the inhibitory effects of clonidine on THAL J Clwere prevented (81.7 ± 10.8 vs. 71.6 ± 6.9 pmol · mm−1 · min−1). Similarly, when the NOS substrate l-arginine was deleted from the bath, addition of clonidine did not decrease THAL J Cl from control (106.9 ± 11.6 vs. 132.2 ± 21.3 pmol · mm−1 · min−1). When we blocked the α2-receptors with rauwolscine (1 μM), we found that the inhibitory effect of 10 nM clonidine on THAL J Cl was abolished, verifying that α2, rather than I1, receptors mediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did not increase in response to clonidine, whereas pretreatment with 150 nM wortmannin abolished the clonidine-mediated inhibition of THAL J Cl, indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 μM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL J Cl. In conclusion, α2-receptor stimulation decreases THAL J Cl by increasing NO release and stimulating guanylate cyclase. These data suggest that α2-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diuretic effects of clonidine in vivo.

Nitric oxide stimulates guanylate cyclase and regulates sodium transport in rabbit proximal tubule

1996 ◽  
Vol 270 (1) ◽  
pp. F106-F115 ◽  
Author(s):  
A. Roczniak ◽  
K. D. Burns
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Inhibitory Effect ◽  
Na Transport ◽  
Uptake Inhibition ◽  
Proximal Tubule Cells ◽  
Cyclic Monophosphate ◽  
Tubule Cells

The proximal tubule contains the target for nitric oxide (NO), soluble guanylate cyclase, and has the capacity for NO production. Inhibition of renal NO synthesis reduces fractional excretion of lithium, suggesting an inhibitory effect of NO on proximal tubule Na+ transport. The present studies determined direct effects of donors of NO in rabbit proximal tubule. In both freshly isolated proximal tubule segments and in primary cultures of proximal tubule cells, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) caused dose-dependent increases in guanosine 3',5'-cyclic monophosphate (cGMP). SNAP was more potent than SNP in stimulating cGMP; this was associated with an enhanced production of nitrite, the stable end-product of NO. In rabbit proximal tubule cells, SNP or SNAP (10(-3) M) significantly inhibited the activity of the apical Na+/H+ exchanger, determined by assay of amiloride-sensitive 22Na+ uptake (% inhibition: SNP, 34.90 +/- 5.52%, P < 0.001; SNAP, 30.77 +/- 8.20%, P < 0.002). To determine the role of cGMP in mediating these effects, proximal tubule cells were incubated with the membrane-permeable analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). Na+/H+ exchange was significantly inhibited by 8-BrcGMP (10(3)M) (% inhibition: 32.40 +/- 9.06%: P < 0.05). The inhibitor of soluble guanylate cyclase, LY-83583, caused partial inhibition of SNP-stimulated cGMP generation and partly blocked the inhibitory effect of SNP on Na+/H+ exchange. Protein kinase A (PKA) activity was not stimulated by SNP, indicating that potential cross-activation of PKA by cGMP did not mediate the effects of NO donors. These data indicate that NO stimulates soluble guanylate cyclase in rabbit proximal tubule and causes inhibition of Na-/H+ exchange. This is at least partly mediated by generation of cGMP. We conclude that NO is an important autocrine or paracrine factor directly regulating Na+ transport in the proximal tubule.

scholarly journals Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes

1988 ◽  
Vol 250 (1) ◽  
pp. 47-52 ◽  
Author(s):  
S Corvera ◽  
K A Yagaloff ◽  
R E Whitehead ◽  
M P Czech
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Plasma Membranes ◽  
Inhibitory Effect ◽  
Insulin Like Growth Factor ◽  
Receptor Phosphorylation ◽  
Adipocyte Plasma Membranes ◽  
Triton X ◽  
Soluble Components

Insulin action in intact adipocytes leads to a rapid increase in the concentration of receptors for insulin-like growth factor (IGF) II on the adipocyte cell surface, and to a decrease in the [32P]phosphate content of these receptors on the plasma membrane [Corvera & Czech (1985) Proc. Natl. Acad. Sci. U.S.A. 82. 7314-7318]. It has been previously shown that the receptor for IGF-II can be phosphorylated on tyrosine residues by a kinase activity which is expressed in isolated adipocyte plasma membranes. It is now shown that IGF-II-receptor phosphorylation in vitro, in plasma membranes derived from insulin-treated cells, is markedly decreased compared with the phosphorylation of the receptor in membranes from control cells. This effect of insulin cannot be attributed to an increase in the activity of phosphotyrosyl phosphatase in the membranes. The tyrosine kinase that catalyses the phosphorylation of IGF-II receptors is associated with a fraction of the plasma membrane which is insoluble in Triton X-100. Removal of the Triton X-100-soluble components of the membrane markedly enhances receptor phosphorylation. Moreover, the expression of the inhibitory effect of insulin requires the presence of one or several Triton X-100-soluble components of the plasma membrane.

scholarly journals Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes.

1983 ◽  
Vol 96 (3) ◽  
pp. 802-806 ◽  
Author(s):  
S Miyata ◽  
T Akazawa
Keyword(s):  
Signal Sequence ◽  
Inhibitory Effect ◽  
Secretory Protein ◽  
Proteolytic Processing ◽  
Alpha Amylase ◽  
Rice Seed ◽  
Microsomal Membranes ◽  
Enzyme Molecules ◽  
Triton X ◽  
Polypeptide Chains

Microsomes prepared from the rice seed scutellum were incubated in wheat germ extracts (S-100 fraction) to direct the synthesis of alpha-amylase, a secretory protein subject to proteolytic processing (cleavage of the N-terminal signal sequence) as well as glycosylation during its biosynthesis. The characterization and identification of the immunoprecipitable products synthesized were performed by SDS gel electrophoresis and subsequent fluorography. The molecular weight of the alpha-amylase synthesized by the microsomes was found to be identical with that of the mature secretory form of the enzyme on the basis of electrophoretic mobilities. A significant portion of the enzyme molecules synthesized was shown to be segregated into the microsomal vesicles and protected against digestion by endo-beta-N-acetylglucosaminidase, indicating that both proteolytic processing and glycosylation of the precursor polypeptide chains take place in the microsomes. The modification of the polypeptide chains was further examined by disrupting the microsomal membranes with Triton X-100. Detergent treatment of the microsomes prior to protein synthesis caused an inhibition of both proteolytic processing and glycosylation of the polypeptide chains, leading to the synthesis of the unprocessed nascent (precursor I), processed but nonglycosylated nascent (precursor II) forms, in addition to the mature form of alpha-amylase. Furthermore, the results of time-sequence analysis of the inhibitory effect of Triton X-100 on the modification of the polypeptide chains have led us to conclude that both proteolytic processing and subsequent glycosylation occur in the microsomes during the biosynthesis of alpha-amylase.

scholarly journals Distinct Pharmacological Properties of Gaseous CO and CO-Releasing Molecule in Human Platelets

2021 ◽  
Vol 22 (7) ◽  
pp. 3584
Author(s):  
Patrycja Kaczara ◽  
Kamil Przyborowski ◽  
Tasnim Mohaissen ◽  
Stefan Chlopicki
Keyword(s):  
Carbon Monoxide ◽  
Platelet Aggregation ◽  
Energy Metabolism ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Light Transmission ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Pharmacological Properties ◽  
Light Transmission Aggregometry

Carbon monoxide (CO)—gaseous or released by CO-RMs—both possess antiplatelet properties; however, it remains uncertain whether the mechanisms involved are the same. Here, we characterise the involvement of soluble guanylate cyclase (sGC) in the effects of CO—delivered by gaseous CO–saturated buffer (COG) and generated by CORM-A1—on platelet aggregation and energy metabolism, as well as on vasodilatation in aorta, using light transmission aggregometry, Seahorse XFe technique, and wire myography, respectively. ODQ completely prevented the inhibitory effect of COG on platelet aggregation, but did not modify antiplatelet effect of CORM-A1. In turn, COG did not affect, whereas CORM-A1 substantially inhibited energy metabolism in platelets. Even though activation of sGC by BAY 41-2272 or BAY 58-2667 inhibited significantly platelet aggregation, their effects on energy metabolism in platelets were absent or weak and could not contribute to antiplatelet effects of sGC activation. In contrast, vasodilatation of murine aortic rings, induced either by COG or CORM-A1, was dependent on sGC. We conclude that the source (COG vs. CORM-A1) and kinetics (rapid vs. slow) of CO delivery represent key determinants of the mechanism of antiplatelet action of CO, involving either impairment of energy metabolism or activation of sGG.

scholarly journals A study of human erythrocyte acetylcholinesterase inhibition by chlorpromazine

1991 ◽  
Vol 278 (2) ◽  
pp. 461-463 ◽  
Author(s):  
A Spinedi ◽  
L Pacini ◽  
C Limatola ◽  
P Luly ◽  
R N Farias
Keyword(s):  
Human Erythrocyte ◽  
Inhibitory Effect ◽  
Membrane Lipid ◽  
Acetylcholinesterase Inhibition ◽  
Erythrocyte Membranes ◽  
Micellar Aggregates ◽  
Solubilized Enzyme ◽  
Erythrocyte Ache ◽  
Lipid Environment ◽  
Triton X

Membrane-bound acetylcholinesterase (AChE) from the human erythrocyte is inhibited by chlorpromazine (CPZ) in a concentration range within this amphiphilic drug has been demonstrated to interact with erythrocyte membranes, causing a large spectrum of physical and structural effects; membrane solubilization with 0.5% Triton X-100 results in a complete loss of CPZ inhibitory potency. Although these observations might suggest a role of membrane lipid environment in mediating human erythrocyte AChE inhibition, we observed that CPZ retains its full inhibitory effect on the fraction of enzyme (5-6% of total) that is solubilized from erythrocytes upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis; furthermore, Triton X-100 is able to reverse the CPZ effect also in the case of PI-PLC-solubilized enzyme. These results demonstrate unequivocally that CPZ inhibits human erythrocyte AChE through direct molecular interaction. The inhibition kinetics displayed by CPZ on human erythrocyte AChE are dependent on drug concentration: evidence is provided that this phenomenon may be related to formation of CPZ micellar aggregates.

Effect of nucleotides on UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities in microsomal membranes

1979 ◽  
Vol 57 (6) ◽  
pp. 557-565 ◽  
Author(s):  
Sailen Mookerjea
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Rat Liver Microsomes ◽  
Glycoprotein Synthesis ◽  
Microsomal Membranes ◽  
Pyrophosphatase Activity ◽  
Triton X ◽  
Hydrolysis Of

Rat liver microsomes solubilized by incubating with lysolecithin or Triton X-100 showed very active UDP-N-acetylglucosamine pyrophosphatase activity leading to the hydrolysis of the substrate into N-acetylglucosamine-1-P and N-acetylglucosamine. ATP, GTP, CDPcholine, and CDPglucose exerted a considerable inhibitory effect on the solubilized membrane pyrophosphatase activity. CDPcholine and CDPglucose, in addition, appeared to stimulate the transfer of N-acetylglucosamine into endogenous and exogenous acceptor proteins. Evidence is also presented of an inhibitory effect of ATP (and to some extent GTP) on N-acetylglucosaminyltransferase activity. This inhibitory effect of ATP and GTP became clearly evident when the pyrophosphatase activity in the membranes was virtually eliminated in the presence of CDPcholine and CDPglucose. The effect of ATP and GTP on the solubilized membrane enzymes indicated that the inhibition of pyrophosphatase activity alone did not determine the rate of transfer of sugar to protein. The results also suggested that the UDP-N-acetylglucosamine pyrophosphatase and N-acetyiglucosaminyltransferase activities were controlled independently and the effect of each nucleotide on these enzymes should, therefore, be carefully evaluated to understand its role in glycopolymer biosynthesis. Also, a possible role of choline and its derivatives in glycoprotein synthesis is discussed.

Effect of lysolecithin in solubilizing membrane-bound glycosyltransferases

1979 ◽  
Vol 57 (1) ◽  
pp. 66-71 ◽  
Author(s):  
Sailen Mookerjea
Keyword(s):  
Lysophosphatidic Acid ◽  
High Speed ◽  
Critical Concentration ◽  
Egg Yolk ◽  
Inhibitory Effect ◽  
Fatty Acyl ◽  
Supernatant Fraction ◽  
Sediment Fraction ◽  
Microsomal Membranes ◽  
Triton X

Microsomal membranes were solubilized by incubation with lysolecithin which caused considerable release of galactosyl- and N-acetylglucosaminyl-transferase into a high-speed supernatant fraction. With a critical concentration of lysolecithin (2.5 mg/10 mg protein in 1 mL microsome suspension), there was a maximal binding of radioactive lysolecithin to the sediment fraction obtained after high-speed centrifugation. Increase of lysolecithin concentration (above 2.5 mg/mL) in the incubation mixture caused a progressive release of the enzymes into the supernatant fraction.Lysolecithin binding to the membrane was greatly inhibited by 1 M NaCl, and high salt concentration also inactivated galactosyltransferase in the sediment, suggesting an electrostatic interaction between lysolecithin and membrane enzyme. In contrast, high NaCl concentration had no inhibitory effect on the enzyme activity in the sediment when the fraction was prepared by treatment with Triton X-100.Lysolecithin-treated microsomal sediment and supernatant galactosyltransferase was inactivated by oleoyllysophosphatidic acid but not by palmitoyllysophosphatidic acid or egg yolk lysophosphatidic acid. Triton X-100 treated microsomal fractions were also similarly affected by different species of lysophosphatidic acid. The results suggested a similarity of interactions of lysophosphatidic fatty acyl species with lysolecithin and Triton-treated galactosyltransferase.

scholarly journals Liberators of NO exert a dual effect on renin secretion from isolated mouse renal juxtaglomerular cells

1993 ◽  
Vol 265 (2) ◽  
pp. F180-F186 ◽  
Author(s):  
K. Schricker ◽  
A. Kurtz
Keyword(s):  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Primary Cultures ◽  
Inhibitory Effect ◽  
Extracellular Calcium ◽  
Renin Secretion ◽  
Cyclase Activity ◽  
Guanylate Cyclase Activity ◽  
Cyclic Monophosphate ◽  
Stimulation Of

This study aimed to examine the role of nitric oxide (NO) in the regulation of renin secretion from renal juxtaglomerular (JG) cells. Using primary cultures of mouse renal JG cells, we found that sodium nitroprusside (SNP) and 3-morpholino-sydnonimin-hydrochloride (SIN-1), two structurally different liberators of NO, led to a transient inhibition during the first hour followed by a marked dose-dependent stimulation of renin secretion lasting for an additional 20 h. This stimulatory effect was blunted by methylene blue (50 microM) and was reversible within minutes after removal of the NO liberators. SNP and SIN-1 also stimulated guanylate cyclase activity in the cultures with a maximum within the first hour of incubation. Increasing intracellular guanosine 3',5'-cyclic monophosphate levels by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (100 microM) or by atrial natriuretic peptide (10 nM) decreased basal renin secretion but did not inhibit the effect of SNP. The stimulatory effect of SNP was not related to adenosine 3',5'-cyclic monophosphate levels in the JG cells and was blunted after chelation of extracellular calcium by 2 mM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N'N'-tetraacetic acid. Taken together, our findings suggest that liberators of NO have two effects on renin secretion from isolated JG cells: an inhibitory effect mediated by stimulation of soluble guanylate cyclase activity and a stimulatory effect mediated by an as yet unknown pathway that requires extracellular calcium.

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