Inactivation of microsomal 17β-hydroxysteroid dehydrogenase by phospholipase C: Rates of phospholipid hydrolysis and enzyme inactivation, and effects of phospholipids

1982 ◽  
Vol 16 (4) ◽  
pp. 509-514
Author(s):  
Charles H. Blomquist ◽  
Claire E. Kotts ◽  
Erick Y. Hakanson
Keyword(s):  
Phospholipase C ◽  
Hydroxysteroid Dehydrogenase ◽  
Enzyme Inactivation ◽  
Phospholipid Hydrolysis

Conservation of structure in ATP-depleted proximal tubules: role of calcium, polyphosphoinositides, and glycine

1993 ◽  
Vol 265 (5) ◽  
pp. F605-F623 ◽  
Author(s):  
R. Garza-Quintero ◽  
J. M. Weinberg ◽  
J. Ortega-Lopez ◽  
J. A. Davis ◽  
M. A. Venkatachalam
Keyword(s):  
Amino Acids ◽  
Phospholipase C ◽  
Cell Structure ◽  
Proximal Tubules ◽  
Atp Depletion ◽  
The Other ◽  
Antimycin A ◽  
Phospholipid Hydrolysis ◽  
Hydrolysis Of

Increases of intracellular free Ca2+ (Caf) may mediate phospholipid hydrolysis and disintegration in energy-compromised cells; on the other hand, glycine and related amino acids preserve structure. We have examined the effects of increased Caf on phospholipids and structure in ATP-depleted cells, as well as how these actions may be modified by glycine. Incubation of isolated proximal tubules with antimycin A led to ATP depletion, delayed increases of Caf to micromolar levels, polyphosphoinositide (PPI) hydrolysis by phospholipase C, and generalized disintegration of cell structure. Glycine inhibited PPI hydrolysis and preserved cell structure in entirety but did not apparently modify the Caf increases. When overwhelming increases of Caf were induced by the additional presence of a Ca2+ ionophore, glycine did not inhibit either the hydrolysis of PPI or disruption of mitochondria and microvilli. However, the cells remained integrated and unbroken. Incubation in low-Ca2+ medium prevented Caf increases, inhibited PPI hydrolysis, and preserved the structure of mitochondria and microvilli. Nevertheless, there was lethal damage by disintegration of all other membranes. This damage was prevented specifically and completely by glycine. Thus compartments of cells were shown to be differentially susceptible to injury from increased Caf or lack of glycine. Although damage by either factor occurs by distinct mechanisms, glycine also appears to have effects that suppress the deleterious effects of Ca2+ so long as Caf increases are not overwhelming. Our results also suggest that the PPI have a major structural role, which may be compromised by Caf increase during ATP depletion.

scholarly journals 11β-Hydroxysteroid Dehydrogenase Type II Inhibition Causes Cerebrovascular Remodeling and Increases Infarct Size after Cerebral Ischemia

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 713-719 ◽  
Author(s):  
Jessica M. Osmond ◽  
Anne M. Dorrance
Keyword(s):  
Blood Pressure ◽  
Cerebral Ischemia ◽  
Infarct Size ◽  
Focal Cerebral Ischemia ◽  
Hydroxysteroid Dehydrogenase ◽  
Enzyme Inactivation ◽  
Type Ii ◽  
Cerebral Vasculature ◽  
Triphenyltetrazolium Chloride ◽  
Cerebral Vessel

Direct mineralocorticoid receptor (MR) activation with deoxycorticosterone acetate has deleterious effects on the cerebral vasculature. Inhibition of 11β-hydroxysteroid dehydrogenase type II (11βHSD2) mimics the detrimental effects of elevated mineralocorticoids in the heart, but the effect of enzyme inactivation on the cerebral vasculature is unknown. Therefore, we hypothesized that systemic 11βHSD2 inhibition with carbenoxolone (CBX) would cause remodeling of the middle cerebral artery (MCA) and increase the damage caused by cerebral ischemia. Six-week-old male Sprague Dawley rats were divided into control and CBX (2.5 mg/d) + 0.9% NaCl treated. After 4 wk treatment, rats were used to assess either structure and reactivity of the MCA or the response to cerebral ischemia using the MCA occlusion technique. Cerebral damage was assessed by 2,3,5-triphenyltetrazolium chloride staining and expressed as a percentage of the hemisphere infarcted. CBX treatment increased systolic blood pressure (153.2 ± 7.3 vs. 122.1 ± 4.4 mm Hg; P < 0.05) compared with control rats. MCAs from CBX treated rats were smaller and stiffer than control MCAs over a range of intralumenal pressures, indicating inward remodeling of the vessel. CBX treatment significantly increased ischemic cerebral infarct size compared with control rats (27.1 ± 5.4% vs. 14.8 ± 4.2%; P < 0.05). These data indicate that inhibition of 11βHSD2, and, thus, disproportionate glucocorticoid activation of the MR, results in remodeling of the MCA and worsens the outcome of cerebral ischemia, further underscoring the importance of understanding the mechanism by which MR activation leads to cerebrovascular disease. 11β-Hydroxysteroid dehydrogenase inhibition increases blood pressure, altering cerebral vessel structure in a manner that increases the damaged caused by focal cerebral ischemia in rats.

Platelet Activation : Role of Exogenous and Endogenous Phospholipases

1979 ◽  
Author(s):  
B. Perret ◽  
G. Mauco ◽  
M.F. Simon ◽  
H. Chap ◽  
L. Douste-Blazy
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Alternative Pathway ◽  
Hydrolysis Product ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Platelet Response ◽  
Phospholipid Hydrolysis ◽  
Outer Leaflet

Phosoholipase A2 from bee venom induces aggregation of human platelets, provided that phospholipid hydrolysis is enabled by simultaneous incubation with sphingomyelinase C. Inhibition of the platelet response by indomethacin indicates that aggregation is due to arachidonic acid release. On another hand, this model allows to describe an asymmetrie distribution of arachidonic acid, whose only 6% is located in the outer leaflet of the plasma membrane.During platelet aggregation by phospholipase C, the diacylglycerol and its hydrolysis product 2-acyl-glycerol are phosphorylated into phosphatide and lysophosphatidic acids, respectively. As the same kinds of changes occur in the presence of thrombin, a unifying hypothesis for platelet activation is proposed, involving the stimulation of an endogenous phospholipase C, whose some properties will be reported (neutral optimal pH, Ca-requlrement, phosphatidylinositol specificity and cytosol-localization). This model can be related to the recent finding that phosphatide acid behaves as a calcium-ionophore (Gerrard, J.M. et al., Prostaglandins Med., 1978, 1, 387) and provides an alternative pathway for arachidonic acid mobilization.

scholarly journals Quantification of inositol phospholipid breakdown in isolated rat hepatocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 865-872 ◽  
Author(s):  
C J Allan ◽  
J H Exton
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Inositol Phosphates ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Phospholipid Hydrolysis ◽  
Inositol Phospholipids ◽  
Inositol Phospholipid ◽  
Inositol Phospholipid Breakdown ◽  
Hydrolysis Of

The hydrolysis of inositol phospholipids induced by vasopressin in hepatocytes during 60 min was quantified chemically. There was a large release of myo-inositol which was abolished by Li+, indicating that it was derived from inositol phosphates and not from phospholipase D action on PtdIns. There was also a large release of inositol phosphates which was increased approx. 2-fold by Li+ at 30 min, but then remained constant, suggesting that inositol phospholipid breakdown declined substantially beyond this time. In cells prelabelled with myo-[3H]inositol and treated with Li+, [3H]PtdIns(4,5)P2 decreased maximally (50%) at 15 s and then recovered to a level at 5 min that was maintained at 25% below control for 40 min. [3H]PtdIns4P and [3H]PtdIns showed slower decreases to approx. 30% below control at 15 min, but with no further changes. Labelled Ins(1,4,5)P3 and Ins(1,3,4)P3 showed 2-4-fold increases within 30 s and then declined to values that were maintained at a constant level above the control, except for [3H]Ins(1,3,4)P3, which showed a second increase. [3H]Ins(1,4)P2 showed a very large increase over 10 min, whereas [3H]Ins4P and [3H]Ins1P showed little change before 6 and 15 min respectively. The total [3H]inositol phosphates showed little further increase after 20 min. These data are consistent with a rapid, but not sustained, hydrolysis of PtdIns-(4,5)P2, but not of PtdIns, by phospholipase C, but do not exclude PtdIns4P as a substrate. Phosphatidate was rapidly increased by vasopressin, whereas diacylglycerol was increased after a 1-2 min lag. Both were maintained at levels 2-3-fold above control for 60 min. The vasopressin-induced increase in inositol phosphates plus myo-inositol (approx. 120 nmol/100 mg) was greater than the increase in diacylglycerol plus phosphatidate (approx. 60 nmol/100 mg) between 10 and 40 min. This indicates that there was substantial further metabolism of these lipids. Addition of 75 mM ethanol resulted in rapid production of phosphatidylethanol in response to vasopressin and a 35% reduction in phosphatidate, but no decrease in diacylglycerol. In summary, the results indicate that inositol phospholipid hydrolysis by phospholipase C can account for most of the diacylglycerol and phosphatidate that accumulate during 60 min of vasopressin action, but that these phospholipids are probably not the major source of the phosphatidate that is formed during the first 2 min by phospholipase D, or of the diacylglycerol and phosphatidate that are formed beyond 30 min.

scholarly journals Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3α-hydroxysteroid dehydrogenase

1990 ◽  
Vol 269 (3) ◽  
pp. 749-755 ◽  
Author(s):  
J W Ricigliano ◽  
T M Penning
Keyword(s):  
Binding Site ◽  
Kinetic Scheme ◽  
Hydroxysteroid Dehydrogenase ◽  
Enzyme Inactivation ◽  
Affinity Column ◽  
Compound I ◽  
Unsaturated Ketones ◽  
Competitive Inhibitors ◽  
Michael Acceptors ◽  
Steroid Binding

The non-steroidal allylic and acetylenic alcohols 1-(4′-nitrophenyl)prop-2-en-1-ol (I) and 1-(4′-nitrophenyl)prop-2-yn-1-ol (II) are oxidized by homogeneous 3 alpha-hydroxysteroid dehydrogenase to the corresponding alpha β-unsaturated ketones 1-(4′-nitrophenyl)prop-2-en-1-one (III) and 1-(4′-nitrophenyl)prop-2-yn-1-one (IV), which then inactivate the enzyme selectively with high affinity; low effective partition ratios are observed for the parent alcohols [Ricigliano & Penning (1989) Biochem. J. 262, 139-149]. Inactivation of 3 alpha-hydroxysteroid dehydrogenase by compound (I) displays an NAD+ concentration optimum. Scavenging experiments indicate that the enzyme-generated inactivators (III) and (IV) alkylate the enzyme via a release-and-return mechanism. Several lines of evidence suggest that compounds (III) and (IV) covalently modify the NAD(P)(+)-binding site. First, micromolar concentrations of NAD(P)H offer substantial protection against enzyme inactivation mediated by Michael acceptors (III) and (IV). In these protection studies Kd measurements for NAD(P)H approached those measured by fluorescence titration of free enzyme. Secondly, under initial-velocity conditions compounds (III) and (IV) act essentially as competitive inhibitors of NAD+ binding, and as mixed competitive or non-competitive inhibitors against androsterone binding. Thirdly, enzyme inactivated with either compound (III) or compound (IV) fails to bind to NAD+ affinity columns (e.g. Affi-gel Blue). Under the same conditions of chromatography native enzyme and enzyme affinity-labelled at the steroid-binding site with 17 β-bromoacetoxy-5 alpha-dihydrotestosterone is retained on the affinity column. A kinetic scheme that represents the inactivation of the homogeneous dehydrogenase by the enzyme-generated alkylators (III) and (IV) is presented.

scholarly journals Inhibition of Bacillus cereus phospholipase C by univalent anions

1982 ◽  
Vol 203 (3) ◽  
pp. 799-801 ◽  
Author(s):  
S E Aakre ◽  
C Little
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Bovine Brain ◽  
Erythrocyte Ghosts ◽  
Substrate Effects ◽  
Phospholipid Hydrolysis ◽  
Marked Inhibition ◽  
Soluble Substrate

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.

Activation and Regulation of Platelet Phospholipases

1979 ◽  
Author(s):  
S. Rittenhouse-Simmons
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Phospholipid Metabolism ◽  
Biologically Active ◽  
Phospholipase A ◽  
Oxidative Enzymes ◽  
Phospholipid Hydrolysis ◽  
Activated Platelets ◽  
Intracellular Ca ◽  
Secretory Tissue

The stimulated human platelet exhibits a number of rapid alterations in phospholipid metabolism which appear to have a significant role in promoting the thrombotic and hemostal, activity of the cell. Some of the earliest changes involve two types of phospholipid hydrolysis, resulting from the activation of at least two enzymes: 1)phospholipase(s) A, which frees arachidonic acid and 2)phosphatidylinositol (PI)-specific phospholipase C, which yields diglyceride. The action of phospholipase A and the factors which regulate it are of essential interest because this enzymatic activity governs the availahility of arachidonic acid to oxidative enzymes which, in turn, produce biologically active endoperoxides, thromboxane A2, and chemotactic hydroxy-derivatives. The activation of phospholipase A is promoted by elevated levels of intracellular Ca+2, is inhibited completely by cAMP, and is inhibited partially by indomethacin or aspirin. The nroductIon of diglyceride in response to thrombin is also blocked by cAMP. Since the enzyme is dependent upon Ca+2, it is possible that the mobilization of platelet Ca+2 stores mav be the activating event for this enzyme (phospholipase C). The rapidly (within 5 sec of stimulus)- Renerated diglvceride may then promote a membrane de-stabilization and thereby facilitate secretion. The observation of the rapid turnover of PI in activated platelets is consistent with earlier findings for stimulated secretory tissue, such as thyroid gland.

scholarly journals Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets

1984 ◽  
Vol 222 (2) ◽  
pp. 389-394 ◽  
Author(s):  
C Solberg ◽  
C Little ◽  
S Holme ◽  
S E Aakre
Keyword(s):  
Plasma Membrane ◽  
Lactate Dehydrogenase ◽  
Bacillus Cereus ◽  
Phospholipase C ◽  
Lipid Extraction ◽  
Human Platelets ◽  
Platelet Concentrate ◽  
Membrane Phospholipids ◽  
Phospholipid Hydrolysis

Phospholipase C (from Bacillus cereus) was used to study fresh and stored human platelets. Provided that the enzyme was inactivated before lipid extraction, no significant degradation of phospholipid in fresh cells was noted, even when platelets were activated or induced to change shape by ADP, collagen or thrombin. With platelets isolated from concentrates stored for transfusion for 4 days at 22 degrees C, membrane phospholipids were degraded by the enzyme to an extent depending on the pH in the platelet concentrate at day 4 of storage. The extent of phospholipid hydrolysis in platelets correlated well with the extent of release of lactate dehydrogenase during storage, with both being minimal for platelets from concentrates of final pH 6.5-6.9. Under non-lytic conditions, phosphatidylcholine was the phospholipid most degraded (40%), with no significant degradation of phosphatidylserine being detected. Storage does not seem to alter the distribution of phospholipids at the external leaflet of the plasma membrane.

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