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PubMed1987

Alterations in Ca2+/Mg2+ ATPase activity upon treatment of heart sarcolemma with phospholipases.

In order to examine the role of phospholipids in the activation of membrane bound Ca2+/Mg2+ ATPase, the activities of Ca2+ ATPase and Mg2+ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg2+ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg2+ ATPase activity by each phospholipase treatment was associated with a decrease in the Vmax value without any changes in the Ka value. The depression of Mg2+ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg2+ ATPase, the sarcolemmal Ca2+ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg2+ ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg2+ ATPase activity to a greater extent in comparison to that for the Ca2+ ATPase activity and the phospholipids associated with Mg2+ ATPase are predominantly exposed at the outer surface of the membrane.

Animals↗

PubMed1976

Neurotoxic and myotoxic effects of crotalus phospholipase A and its complex with crotapotin.

1. The acidic peptide crotapotin potentiated the toxicity of the basic crotalus phospholipase A in all species tested. The order of sensitivity to the lethal action of the phospholipase was: chick greater than mouse larger than or equal to rabbit greater than rat. After a latency period of a least 20 min the animals died of respiratory paralysis. Rabbits survived for more than 10 h, if they were artificially respired. The animals recovered very slowly from respiratory depression. 2. In rabbits even high doses of the basic crotalus phospholipase A or its complex with crotapotin did not affect the respiratory center nor its reactivity to asphyxia. Conduction of action potentials in the phrenic nerve was not changed. 3. Phospholipase-crotapotin complexes decreased the contractile response of isolated phrenic-hemidiaphragms of rats to direct and indirect stimulation in an irreversible manner. A latency period 20-100 min preceded the paralysis. 4. A similar block of neuromuscular transmission developed in vivo. After i. v. injection of PC-complexes the contractile response of isolated rat phrenic diaphragms to nerve stimulation was considerably lower than to direct stimulation. 5. Crotalus phospholipase A alone as well as its complex with crotapotin reduced the contractions of the isolated chick biventer cervicis muscle but did not cause a contracture, thus indicating that the phospholiphase PC-complexes act not as depolarizing blockers. 6. Blood pressure, heart rate and electrocardiogram were not substantially altered, when phospholipase A or PC-complex were injected slowly i.v. into rabbits or rats. No cardiotoxic effect was observed in the Langendorff preparation of rat hearts perfused with phospholipase A alone (6 x 10(-6) M) or together with crotapotin (10(-5) M). Rapid i.v. injection of the venom produced hypotension. The degree of haemolysis correlated with the enzymatic activity, and was low for the highly toxic PC-complexes.

Action Potentials↗

PubMed1996

Detection of extracellular phospholipase activity in Candida albicans and Rhodotorula rubra.

Phospholipases are important pathogenicity determinants in Candida albicans. They play a significant role in damaging cell membranes and invading host cells. High phospholipase production is correlated with an increased ability of adherence and a higher mortality rate in animal models. By means of an egg yolk-containing agar and the Pz (= phospholipase activity zone) value according to Price, the present study investigated phospholipase production in 170 strains of C. albicans. At an incubation temperature of 37 degrees C, Pz values ranged from 0.395 to 1; no clear relationship was found between clinical origin of the isolates and severity of the disease. In addition to C. albicans, a total of 110 strains of 16 other yeast species were investigated for possible phospholipase production. Only yeasts of the species Rhodotorula rubra showed phospholipase activity, with mean values exceeding those observed in C. albicans. This result was confirmed by an assay using sterile culture filtrates and phosphatidyl-[3H-methyl]-choline-dipalmitoyl as a substrate. Since Rh. rubra has only rarely been demonstrated as a pathogen in humans, we believe that factors such as reduced growth at 37 degrees C, absence of dimorphism and low ability of adherence lessen the importance of high phospholipase activity in Rh. rubra as a pathogenicity determinant. Therefore, potential virulence factors should always be considered in the context of the whole spectrum of pathogenic determinants.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Increased phospholipase D activity in human breast cancer.

Phospholipase D is believed to play an important role in cell proliferation and tumorigenesis. One of its major functions is to cause a sustained activation of protein kinase C through the primary production of phosphatidic acid from phosphatidylcholine by the enzyme, followed by dephosphorylation forming diacylglycerol. Protein kinase C is known to be activated or translocated in some tumors including breast tumors. In order to examine phospholipase D activity in breast tumors, surgical specimens of human breast tumors were obtained by mastectomy or wide excision, and their phospholipase D activities were assayed by determining the formation of phosphatidylethanol from phosphatidylcholine and ethanol. Phospholipase D activity was predominantly localized in the microsomal fraction of the tumor tissue and markedly stimulated by oleic acid. We observed a significant increase in phospholipase D activity in 17 out of 19 spontaneous human breast tumors as compared to adjacent histologically normal breast tissue. The mean specific activity in the tumors was 52.9 +/- 41.8 (SD) pmol min-1 mg protein-1 whereas the value for the normal breast tissue was 34.0 +/- 36.2 (SD) pmol min-1 mg protein-1 (P < 0.01; paired Wilcoxon's rank-sum test). The mean tumor/normal activity ratio was 2.37. Among prognostic factors, the nuclear grade, evaluated according to Schnitt et al., was found to be correlated with the activity ratio. Our results suggest a role for phospholipase D in human breast tumors. An elevation in phospholipase D activity is useful as a potential marker for malignant disease in the breast.

Adult↗

PubMed1998

Phospholipase D hydrolyzes short-chain analogs of phosphatidylcholine in the absence of detergent.

Phospholipase D is an important enzyme in signal transduction in neuronal tissue. A variety of assays have been used to measure phospholipase D activity in vitro. The most typical measure of phospholipase D activity is the production of phosphatidylethanol in the presence of ethanol. Phosphatidylethanol is a product of transphosphatidylation activity that is considered a unique property of phospholipase D. To support transphosphatidylation activity, high concentrations of ethanol may be required. Furthermore, most assays in the literature utilize a detergent. These extreme conditions, detergent and ethanol, may alter phospholipase D and hinder the study of its regulation. In this manuscript we describe an assay that eliminates these potentially confounding conditions. It utilizes high specific activity [3H]butanol as a nucleophilic receptor. This eliminates the need for high concentrations of alcohol. The substrate is an analog of phosphatidylcholine that contains short-chain fatty acids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine. Phospholipase D readily hydrolyzes this substrate in the absence of detergent. This novel assay should be useful in the further characterization of phospholipase D.

Animals↗

PubMed1987

Ethanol does not stimulate guanine nucleotide-induced activation of phospholipase C in permeabilized hepatocytes.

Guanine nucleotides are thought to mediate the interaction of the receptors for calcium-mobilizing hormones and phosphoinositide-specific phospholipase C. In the present study the characteristics of guanine nucleotide-dependent phospholipase C activation were studied in [3H]inositol-labeled permeabilized hepatocytes. The nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanyl-5'-yl imidodiphosphate stimulated the production of inositol phosphates by phospholipase C. The effect was concentration-dependent with half-maximal and maximal stimulation occurring with 0.6 and 10 microM GTP gamma S, respectively. The guanine nucleotide-induced stimulation of phosphoinositide breakdown was selective for phosphatidylinositol (4,5)-bisphosphate over phosphatidylinositol (4)-phosphate. The individual inositol phosphates formed after maximal GTP gamma S exposure were analyzed by high-performance liquid chromatography. Inositol 1,4,5-trisphosphate was rapidly produced, followed by the formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate. Ethanol is known to activate hormone-sensitive phospholipase C in intact rat hepatocytes. Ethanol (0.3 M) was ineffective in altering the characteristics of GTP gamma S-stimulated phospholipase C activation, in both digitonin-treated and sonicated hepatocytes. The metabolism of the various inositol phosphate isomers was unaffected by ethanol. The findings demonstrate the potential for the use of permeabilized hepatocytes in the analysis of phospholipase C activation by guanine nucleotides. Ethanol does not activate phospholipase C by altering this process.

Animals↗

PubMed1975

Changes in phosoholipid susceptibility toward phospholipases induced by ATP depletion in avian and amphibian erythrocyte membranes.

About half of the sphingomyelin content of fresh and ATP-depleted chicken erythrocytes is hydrolysed by sphingomyelinase. Removal of spingomyelin exposes the rest of the membrane phospholipids to hydrolysis by phospholipase C only in ATP-depleted but not in fresh cells. Addition of both sphinogomyelinase and phospholipase C to ATP-depleted cells causes about 60-70 percent hydrolysis of the total phospholipids accompanied by extensive (90 percent) hemolysis. The phospholipids of toad erythrocytes are partially available to phospholipase C activity in fresh cells (17-25 percent hydrolysis) without prior sphingomyelinase treatment. However, in ATP-depleted toad cells phospholipase C hydrolyses 66 percent of phospholipids and causes extensive lysis. Treatment of either fresh or ATP-depleted toad erythrocytes by sphingomyelinase together with phospholipase C induces hydrolysis of most of the phospholipds with complete lysis. Restoration of ATP to ATP-depleted cells endows them with resistance to the attack of phospholipase C. The correlation between changes in ATP level and membrane organization as revealed by increased susceptibility toward phospholipases is discussed.

Adenosine Triphosphate↗

PubMed1979

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Adenosine Triphosphate↗

PubMed1977

Lysosomal phospholipase A activities of rat ovarian tissue.

1.1. Lysosome-enriched fractions were prepared by differential centrifugation of homogenates of luteinized rats ovaries. Acid phospholipase A activities were characterized with [U-14C]diacyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-[9,10-3H]- or [1-14C]oleoyl-sn-glycero-3-phosphocholine as substrates. Acid phospholipase A1 activity had properties similar to other hydrolases of lysosomal origin; subcellular distribution, latency and acidic pH optimum. Acid phospholipase A2 activity with similar characteristics was also tentatively identified. We were unable to exclude the possibility that the combined action of phospholipase A1 and lysophospholipase contributed to the release of acyl moieties from the 2-position of the synthetic substrates. 2. Lysophospholipase activity was present in the lysosome-enriched fractions. This activity had an alkaline pH optimum. 3. Phospholipase A1 and A2 activities solubilized from lysosome fractions by freeze-thawing were inhibited by Ca2+ and slightly activated by EDTA. A Ca2+- stimulated phospholipase A2 activity, with an alkaline pH optimum, remained in the particulate residue of freeze-thawed lysosome preparations. This activity is believed to represent mitochondrial contamination. 4. Activities of acid phospholipase A, as well as other acid hydrolases, increased approx. 1.5-fold between 1 and 4 days following induction of luteinizatin, suggesting a hormonal influence on lysosomal enzyme activities.

Animals↗

PubMed1986

A cytosolic phospholipase in human neutrophils that hydrolyzes arachidonoyl-containing phosphatidylcholine.

In stimulated neutrophils the production of eicosinoids and the lipid mediator, platelet-activating factor, is thought to be initiated by the activation of a phospholipase A2 which cleaves arachidonic acid from choline-containing glycerophospholipids. Accordingly, studies were undertaken in human neutrophils to characterize phospholipase enzymes that can hydrolyze 1-acyl- and 1-alkyl-linked arachidonoyl-containing phosphatidylcholine (PC). Cellular homogenates were incubated with sonicated dispersions of the arachidonoyl-labeled phospholipid substrates and the hydrolysis of radiolabeled arachidonate was measured. The phospholipase activity was cytosolic, optimal at pH 8.0, and calcium dependent. The homogenization conditions used were important in determining the amount of recoverable enzymatic activity. Vigorous sonication and the presence of calcium during homogenization were strongly inhibitory, whereas the presence of EGTA, heparin and proteinase inhibitors during homogenization increased the activity. Competitive experiments with unlabeled substrates suggested that the phospholipase hydrolyzed arachidonic acid equally well from either 1-acyl- or 1-alkyl-linked PC. However, the phospholipase did show specificity for arachidonic acid, compared to oleic or linoleic acids, at the sn-2 position of 1-acyl-linked PC. When neutrophils were first stimulated with the ionophore A23187, the phospholipase activity against 1-O-hexadecyl-2-[3H]arachidonoylglycerophosphocholine (GPC) increased in a time-dependent fashion up to 3.5-fold over the unstimulated level. The activity against 1-palmitoyl-2-[3H]arachidonoyl-GPC also increased after ionophore stimulation but to a lesser extent. The results demonstrate the presence of a cytosolic, activatable phospholipase that may be involved in PC turnover, arachidonic acid release, and platelet-activating factor production in human neutrophils.

Arachidonic Acid↗

PubMed1986

GTP and GDP will stimulate platelet cytosolic phospholipase C independently of Ca2+.

The hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by cytosolic phospholipase C from human platelets was determined. Cytosolic fractions were prepared from platelets that had or had not been preactivated with thrombin. Thrombin pretreatment did not affect cytosolic phospholipase C activity. In both cytosolic fractions, phospholipase C was activated by GTP and GTP gamma S. This action is observed in the presence of 2 mM EGTA. GDP was as effective as GTP in stimulating cytosolic phospholipase C in the presence of Ca2+ or EGTA. Partially purified phospholipase C obtained from platelet cytosol is activated by GTP, but not by GTP gamma S, in the presence of 2 mM EGTA. However, in the presence of 6 microM Ca2+, both GTP and GTP gamma S stimulated the partially purified phospholipase C. Our present information indicates that GTP and GDP have a direct effect on the cytosolic phospholipase C.

Blood Platelets↗

PubMed1987

GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein.

Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with [3H]inositol. Ca2+ has opposite effects on the formation of [3H]inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of [3H]inositol phosphates is inhibited by Ca2+, action of thrombin is stimulated by Ca2+. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca2+ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca2+ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.

Blood Platelets↗

PubMed1996

A tyrosine kinase regulates alpha-adrenoceptor-stimulated contraction and phospholipase D activation in the rat aorta.

Since previous studies had indicated a role for tyrosine kinases in alpha 2-adrenoceptor-induced contractile responses in other blood vessels, as well as in the activation of phospholipase D, we examined the sensitivity of these responses in rat aorta to the tyrosine kinase inhibitor genistein. Contractions induced by both noradrenaline and the alpha 2-adrenoceptor-selective agonist UK14304 (5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline) were fully inhibited by genistein, with the latter responses being more sensitive. Contractions induced by high K+ buffer were also inhibited, but to a lesser extent. Both agonists caused a stimulation of phospholipase D activity, which could be blocked by pretreatment with pertussis toxin, indicating involvement of either Gi or Go. Genistein completely inhibited the agonist-induced phospholipase D activity and also substantially reduced the basal level of phospholipase D activity. Pretreatment with either the alpha 1-adrenoceptor antagonist prazosin or the alpha 2-adrenoceptor antagonist rauwolscine was also effective in eliminating the agonist-induced increase of phospholipase D. These results indicate that a tyrosine kinase-regulated phospholipase D plays a critical role in alpha-adrenoceptor-induced contractions of the rat aorta and that stimulation of both alpha 1- and alpha 2-adrenoceptors is essential to allow phospholipase activation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1990

Phospholipase C and diacylglycerol lipase in human gallbladder and hepatic bile.

A phospholipase C in bile, free of bacterial infection, has recently been identified from cholesterol gallstone patients. Because of the importance of phosphatidylcholine in solubilizing cholesterol in bile, this study further investigates the metabolism of phosphatidylcholine in delipidated gallbladder and common bile duct biles. Phospholipase C activity, as measured by the release of phosphoryl[3H]choline from the substrate 1,2-dipalmitoyl-sn-glycero-3-phospho [N-methyl-3H]choline, was identified in both hepatic and gallbladder biles. Similar levels of activity (nmol.h-1.mg-1 of delipidated protein) were found in common bile duct (11.25 +/- 14.23) and gallbladder bile (19.07 +/- 22.24), although per milliliter of bile, the mean gallbaldder levels were 6.4 times greater than those found in common duct bile. With the tow substrates, 1-palmitoyl-2[9,10-3H] palmitoyl-sn-glycero-3-phosphocholine and 1,2(1-14C) dipalmitoyl-sn-glycero-3-phosphocholine, the majority of organically extracted label, after thin-layer chromatography, was recovered as radiolabeled diglyceride, confirming the presence of phospholipase C. Diglyceride levels were found to be closely correlated with [3H]choline (slope, 0.9820; r = 0.9844). In addition to diglyceride, both radiolabeled free fatty acid and monoglyceride were identified in common bile duct and gallbladder biles, although their levels were an order of magnitude less than measurable phospholipase C activity. To determine whether the free fatty acid release was due to either a diacylglycerol-lipase or a phospholipase A2, the effect of adding unlabeled diglyceride on free fatty acid formation from the substrate [14C]DPPC was examined. As the concentration of unlabeled diglyceride was increased, the amount of free fatty acid and monoglyceride released were both reduced in parallel. Direct measurement of diacylglycerol-lipase activity by incubating the diglyceride, sn-2[3H]dipalmitoyl, resulted in release of both products in a ratio similar to that found with sn-2[3H]DPPC. Finally, no radiolabeled lysolecithin was identified with [3H]choline-DPPC or [14C]DPPC as substrate indicating the free fatty acid was the product of a diacylglycerol-lipase rather than a phospholipase A2. Phospholipase C and diacyl-glycerol-lipase activities were significantly correlated (P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1995

Vasopressin Vla receptor-stimulated phospholipase D: differential regulation of transphosphatidylation and phospholipid hydrolysis by protein kinase C [corrected].

Phospholipase D belongs to a group of membrane associated phospholipases which have been shown to be activated by G-protein coupled neurotransmitter receptors. Phosphatidylcholine is the primary substrate for phospholipase D generating phosphatidic acid (PA) and choline. In the presence of 1% ethanol, phospholipase D catalyzes a transphosphatidylation reaction generating phosphatidylethanol (PEt) which is an indicator of phospholipase D activation. In the present study, we utilized Chinese hamster ovary (CHO) cells stably transfected with and expressing a rat V1a vasopressin receptor to study the regulation of phospholipase D by protein kinase C and calcium. Arginine-vasopressin (AVP) stimulated the release of 3H-PEt and 3H-PA in cells pre-labelled overnight with 3H-palmitic acid. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the release of PEt and PA that was additive with AVP over 15 min. However, long-term stimulation with PMA, which desensitizes protein kinase C, decreased PEt production while simultaneously increasing PA production. Differential regulation of PEt and PA production by PMA suggests the existence of more than one phospholipase D isoenzyme. Though differentially regulated by protein kinase C, both AVP-stimulated PEt and PA production required extracellular and not intracellular calcium.

Animals↗

PubMed1994

Activation of phospholipase D in human platelets by collagen and thrombin and its relationship to platelet aggregation.

Stimulation of phospholipase D after activation of cell surface receptors has been reported in many cell types. We have investigated the mechanism of activation of this enzyme by collagen in the human platelet by assaying the release of [3H]methylcholine from [3H]methylphosphatidylcholine. Results from these studies suggest that phospholipase D activity is regulated by reversible phosphorylation. Phospholipase D activity was stimulated when platelet-rich plasma was preincubated with collagen and was not inhibited by aspirin. Among various aggregating agents tested, collagen and thrombin but not ADP activated phospholipase D activity (2- to 3-fold). The addition of sphingosine inhibited phospholipase D activity. Preincubation of platelet-rich plasma with sphingosine inhibited collagen- and thrombin-induced platelet aggregation and the release of ATP. The inhibitory effect of sphingosine on collagen- and thrombin- induced platelet aggregation and release of ATP was dose-dependent. The functional significance of phospholipase D activation was also tested by examining the effect of the product, phosphatidic acid, on collagen-induced platelet aggregation and release of ATP. Platelet shape change and the reversibility of platelet aggregation resulted by the addition of phosphatidic acid to platelet-rich plasma. Furthermore, the simultaneous addition of phosphatidic acid and collagen shortened the latency period but had no effect on platelet aggregation. Two platelet proteins (47 kDa and 22 kDa) increased in phosphorylation after the addition of 1 microM phosphatidic acid which did not cause platelet aggregation. These results suggest that collagen stimulates phospholipase D activity which plays a secondary role in platelet aggregation and the release reaction.

Adenosine Triphosphate↗

PubMed1994

Oxidative stress modifies the activity of cardiac sarcolemmal phospholipase C.

We have examined the direct effects of oxidant metabolites on cardiac sarcolemmal phosphoinositide phospholipase C which transduces signals from various receptors for the modulation of intracellular Ca2+ levels. The enzyme activity in rat cardiac sarcolemmal membranes that had been preincubated (10 min; 37 degrees C) with xanthine-xanthine oxidase, a superoxide anion generating system, was not significantly affected. The addition to this system of superoxide dismutase, which converts superoxide anion to hydrogen peroxide (H2O2), resulted in a significant decrease of the enzyme activity in comparison with control values. Such decrease was fully prevented by catalase. Preincubation of sarcolemma with hypochlorous acid also gave a significant inhibition of phospholipase C, which was counteracted by the synthetic thiol reducer dithiothreitol. H2O2-pretreatment induced a concentration-dependent inhibition of the enzyme which was prevented by catalase but not by the iron chelator deferoxamine. Dithiothreitol was able to protect against, as well as to recover the enzyme activity from the H2O2 effects. These data suggest that superoxide anions and hydroxyl radicals did not interfere with phospholipase C activity, and that the nonradical oxidants, H2O2 and hypochlorous acid, may have acted through oxidation of thiol (SH) groups. The existence of reactive SH groups associated with the enzyme was confirmed by the inhibitory effects of SH modifiers (p-chloromercuriphenylsulfonic acid, 5'5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide and methyl methanethiosulfonate), which were prevented and in some cases also reversed by dithiothreitol. The biological reducer glutathione (GSH) was not able to recover the H2O2-induced inhibition of phospholipase C, whereas its oxidized form (GSSG) decreased the enzyme activity both in control and H2O2-pretreated membranes. The enzyme was active in a wide range of GSH/GSSG redox states, but H2O2 pretreatment narrowed this range. The results showed that oxidative stress changed the redox state of sarcolemmal phospholipase C, and this deactivated the enzyme. The oxidants' concentrations that significantly impaired phospholipase C in this study were compatible with those occurring in vivo during ischemia-reperfusion [Am. J. Med. 91(Suppl. 3C):235, 1991]. This supports the possibility that alteration of the receptor-associated phospholipase C may be a factor in the oxidant-related dysfunction of the ischemic-reperfused heart.

Animals↗

PubMed1995

Activation of phospholipase C by G-protein beta gamma subunits in DDT1MF-2 cells.

Adenosine A1 receptors directly stimulate inositol phospholipid hydrolysis and Ca2+ mobilization through a pertussis toxin sensitive mechanism in DDT1MF-2 cells. In the present study we have investigated whether G protein beta gamma subunits (G beta gamma) are capable of stimulating phospholipase C in DDT1MF-2 cell membrane preparations using lipid vesicles containing [3H]phosphatidylinositol 4,5-bisphosphate. DDT1MF-2 cell membrane and soluble fractions were found to contain phospholipase C activity which was stimulated by increases in free Ca2+ ion concentration. G beta gamma purified from bovine retinal transducin produced significant increases in phospholipase C activity in DDT1MF-2 cell membranes. G beta gamma-dependent activation of phospholipase C, while virtually absent in the presence of low Ca2+ ion concentrations, increased markedly with increasing free Ca2+ ion concentration. These data suggest that membrane bound phospholipase C in DDT1MF-2 cells is sensitive to Ca2+, and may be stimulated conditionally by G beta gamma subunits, i.e. G beta gamma subunits activate the enzyme only in the presence of Ca2+. G beta gamma subunits also stimulated soluble phospholipase C in DDT1MF-2 cells. These findings support the hypothesis that Gi beta gamma subunits are involved in adenosine A1 receptor stimulated phospholipase C/Ca2+ signaling in DDT1MF-2 cells.

Animals↗