Rat Liver Plasma Membranes Research Articles

Biosynthesis of the endogenous cyclic adenosine monophosphate (AMP) antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), from prostaglandin E and activated inositol polyphosphate in rat liver plasma membranes

Biosynthesis of the endogenous cyclic adenosine monophosphate (AMP) antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), from prostaglandin E and activated inositol polyphosphate in rat liver plasma membranes

Effect of endotoxin on bile acid transport in rat liver: a potential model for sepsis-associated cholestasis

Intrahepatic cholestasis in the setting of extrahepatic bacterial infection has been attributed to the effects of endotoxin and cytokines such as tumor necrosis factor-alpha (TNF-alpha) on bile acid transport. To define the mechanism of sepsis-associated cholestasis, taurocholate transport was examined in basolateral (bLPM) and canalicular (cLPM) rat liver plasma membrane vesicles derived from control and endotoxin [lipopolysaccharide (LPS)]-treated animals and in plasma membrane vesicles prepared after TNF-alpha treatment. Na(+)-dependent [3H]taurocholate uptake and both membrane-potential-dependent and ATP-dependent [3H]taurocholate transport were reduced in bLPM and cLPM vesicles, respectively, after LPS treatment. In membrane vesicles from TNF-alpha-treated animals, Na(+)-dependent [3H]taurocholate uptake was also reduced. Northern blot hybridization, using cDNA probes for the putative sinusoidal bile acid transporter (Ntcp) and canalicular ecto-adenosinetriphosphatase, demonstrated decreased mRNA levels after LPS and TNF-alpha treatment. Immunoblot analysis of membrane extracts from LPS-treated animals revealed decreased levels of these putative bile acid transporters. Impaired bile acid transport at the sinusoidal and canalicular membrane domains by these and other mediators of the inflammatory response may account for sepsis-associated cholestasis.

Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

Characterization of a nucleotide stimulated aspartic proteinase in rat liver plasma membranes.

Inositol phosphoglycan molecules are believed to mediate multiple intracellular actions of insulin. They are released from plasma membranes in response to insulin binding and are transported into the cell. Release of insulin mediators is stimulated by MnATP and MgATP and is inhibited by p-aminobenzamidine. Inositol phosphoglycan mediators may be released by a poorly characterized mechanism requiring proteolytic cleavage of an attached protein from the mediator and phospholipase cleavage of the mediator from its membrane anchor. We examined rat liver plasma membranes for proteinase activity stimulated by insulin and MnATP. Although we could not demonstrate insulin stimulation, we have found and characterized a nucleotide-stimulated aspartic proteinase bound to rat liver plasma membranes. We also detected and separated a soluble activating factor for the proteinase. The activating factor appears to be a protein with M(r) approximately 70 kDa.

Biosynthesis of the endogenous cyclic adenosine monophosphate (AMP) antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), from prostaglandin E and activated inositol polyphosphate in rat liver plasma membranes.

The endogenous cyclic adenosine monophosphate (AMP) antagonist, cyclic PIP, has been identified as a prostaglandylinositol cyclic phosphate. It inhibits protein kinase A 100% and activates protein serine phosphatase about sevenfold. It is biosynthesized by an enzyme of the plasma membrane when the assay mixture contains adenosine triphosphate (ATP), Mg2+, prostaglandin E and a novel inositol polyphosphate, which cannot be substituted by commercially available inositol phosphates. This novel inositol polyphosphate is a very labile compound. On anion exchange chromatography it elutes in the range of ATP, which may indicate the presence of three phosphate groups. It adsorbs on charcoal, which suggests the presence of a hydrophobic component, possibly a guanosine. Pyrophosphates obtained from inositol 1,4- and inositol 2,4-bisphosphate are accepted by cyclic PIP synthetase for the synthesis of cyclic PIP. The biosynthesis is characterized by enzyme kinetic parameters like dependence on time, enzyme and substrate concentration. The pH optimum of the enzyme is in the range 7.5-8. The enzyme functions optimally with prostaglandin E and poorly with prostaglandin A as the substrate. The presence of fluoride in the assay causes a three- to fourfold increase in cyclic PIP synthesis, which may be correlated with activation via G proteins. These data support previous reports on the chemical structure and action of cyclic PIP. With respect to the possible isomers of cyclic PIP, these indicate that it is most likely the C4-hydroxyl group of the inositol which binds the C15-hydroxyl group of prostaglandin E. A model of hormone-stimulated synthesis of cyclic PIP is proposed: phospholipase A2 and phospholipase C, activated by G proteins upon alpha-adrenergic stimulation, liberate either unsaturated fatty acids or inositol phosphates, which are transformed to prostaglandins and to novel inositol polyphosphate with an energy-rich bond. The cyclic PIP synthetase combines these two substrates to cyclic PIP.

Effect of endurance physical training on rat liver adenylyl cyclase system

The adaptive response to endurance exercise of the catecholamine- and glucagon-sensitive adenylyl cyclase system was studied in rat liver plasma membranes. Endurance exercise enhanced adenylyl cyclase system activation by cellular agonists (glucagon, isoproterenol), by stimulators of the enzyme catalytic subunit (forskolin, Mn 2+), and by G s-protein activators (GppNHp, fluoride). In addition, endurance exercise increased the levels of G α, G iα, and G β subunts. These results show that the adenylyl cyclase system becomes sensitized in response to physical training.

Characterization of the binding of diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) to rat liver cell membranes

Diadenosine polyphosphates present in the extracellular environment can, through interaction with appropriate purinoceptors, influence a range of cellular activities. Here we have investigated the nature of the ligand:receptor interactions involved in diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A)-mediated stimulation of glycogen breakdown in isolated rat liver cells. [2-3H]Ap4A showed specific binding to both intact isolated liver cells and plasma membrane fractions prepared from isolated liver cells. HPLC analysis confirmed that binding was mediated by intact Ap4A and not by potential breakdown products (e.g. ATP, adenosine, etc.). Binding of [2-3H]Ap4A, to isolated liver cell plasma membrane preparations, was successfully displaced by a range of both naturally occurring and synthetic diadenosine polyphospates with the rank order potency Ap4A > or = Ap5A > Ap6A > Ap3A > Ap2A. [2-3H]Ap4A binding was not displaced by P1 effectors but was successfully displaced by a range of P2 effectors with the rank order potency 2-methylthio-ATP > ATP > ATP > or = adenosine 5'-[alpha beta-methylene]triphosphate > adenosine 5'-[beta gamma-methylene]triphospate. These findings are consistent with the interaction of Ap4A with a P2y-like subclass of purinoceptor and are discussed in relation to (1) the known purinoceptor populations in liver cell plasma membranes and (2) observations concerning the binding of diadenosine polyphosphates to purinoceptors in other tissues.

Effects of long-term alcohol intake on ADP ribosylation in rat liver plasma membranes.

Mono-ADP-ribosylation, In which the ADP-ribosyl moiety is transferred from NAD to an acceptor protein, is one of the important posttranslational modifications of cellular proteins. Because mounting evidence suggests significant biological roles of this reaction in transmembrane signal transduction and other cell metabolic reactions, we assessed how long-term alcohol intake alters toxin catalyzed- and endogenous mono-ADP-ribosylation in the liver of a rat model. We first found that thiol-preactivated cholera toxin-catalyzed ADP-ribosylation of the alpha-subunit of the stimulatory GTP-binding protein was enhanced after long-term alcohol intake. Unexpectedly, but interestingly, this enhancement was not accompanied by a concomitant increase of cholera toxin-catalyzed stimulation of the adenylate cyclase activity. We also found that long-term alcohol intake remarkably enhanced endogenous mono-ADP-ribosylation of a 58 kDa protein in plasma membranes. Thus, long-term alcohol intake stimulated endogenous, as well as, toxin-catalyzed mono-ADP-ribosylations. Characterization of the 58 kDa protein may uncover pathophysiological roles of this interesting phenomenon in alcohol-induced liver damage.

The role of thiols in ATP-dependent transport of S-(2,4-dinitrophenyl) glutathione by rat liver plasma membrane vesicles

The effect of thiol/disulfide exchange on ATP-dependent S-(2,4-dinitrophenyl)glutathione (GS-DNP) transport was studied in sodium nitrate treated rat liver plasma membrane vesicles. Transport followed Michaelis-Menten kinetics with an apparent K m of 9.6 μM for GS-DNP and 124 μM for ATP. 5,5′-Dithiobis(2-nitrobenzoate) (DTNB) and N-ethylmaleimide (NEM) efficiently inactivated GS-DNP transport activity in a dose- and time-dependent manner. Half-maximal inactivation occurred in 10 min at 40 μM for DTNB and 550 μM for NEM. Inactivation by DTNB was reversed by dithiothreitol. S-(N-Ethyl)maleimyl glutathione and/or ATP-Mg 2+, but neither S-(N-ethyl)maleimyl cysteinylglycine nor oxidized glutathione could protect transport activity from inactivation by NEM or cystamine. These results suggest that reactive thiols are located near the active site of the transporter and that S-alkyl and the γ-glutamyl residues of glutathione are important for protection. Biological disulfides which were tested included cystine, oxidized glutathione, oxidized Coenzyme-A, oxidized lipoic acid, and oxidized lipoamide; cystamine was the most potent reversible inactivator. Molecular oxygen also inactivated transport activity, which was recovered on addition of dithiothreitol, suggesting intramolecular disulfide formation by vicinal thiols. We interpret these results to indicate that the ATP-dependent GS-DNP transporter contains two or more thiols which are necessary for the maintenance of transport activity. The reversible inactivation of the activity by biological disulfides suggests that the transporter may be regulated by thiol/disulfide exchange in vivo.

Fatty acid uptake by Caco-2 human intestinal cells

The Caco-2 human enterocytic cell line was used to study the kinetics and mechanism of intestinal long chain fatty acid uptake. Initial rates of palmitate (16:0), oleate (18:1), and octanoate (8:0) uptake were determined for adherent cells at greater than 7 days confluence. Uptake of long chain 18:1 and 16:0 by cells grown on coverslips was saturable with an apparent Km of 0.3 microM, but also included a notable diffusive component. Uptake of short chain 8:0, on the other hand, was linear up to 10 microM. Cells grown on permeable Transwell filters were used to study uptake at the apical versus the basolateral membrane. Uptake of long chain (18:1 and 16:0), but not short chain (8:0), fatty acid was saturable at both surfaces with a similar Km of 0.3 microM. In addition, long chain but not short chain fatty acid uptake was competitively inhibitable. Western blot analysis demonstrated that Caco-2 cells express a protein immunoreactive with antibodies to the rat liver plasma membrane fatty acid binding protein (FABPpm), which is thought to be involved in long chain fatty acid transport. Nevertheless, long chain fatty acid uptake was not inhibited by pretreatment of the cells with an FABPpm antibody, nor by pretreatment with two proteases. These data support a saturable component in the transport of long chain but not short chain fatty acids by human intestinal epithelial cells, which may involve an as yet unknown plasma membrane protein.

Interactions of malondialdehyde and 4-hydroxynonenal with rat liver plasma membranes and their effect on binding of prostaglandin E2 by specific receptors.

We investigated effect of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE) on prostaglandin (PG) E2 receptors of liver plasma membranes. The modification of the membranes by MDA diminished PGE2 binding, decreasing receptor affinity for PGE2 and receptor density whereas HNE increased PGE2 binding, enhanced receptor density but did not changed receptor affinity. ESR study showed the decrease of the whole membrane fluidity after modification by MDA whereas HNE lowered membrane fluidity only in the internal zone of lipid bilayer and increased it in the surface area. The possible effects of membrane changes caused by MDA and HNE on PGE2 receptor parameters are discussed.

Activatory effect of regucalcin on hepatic plasma membrane (Ca(2+)-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats.

The alteration of the plasma membrane (Ca(2+)-Mg2+)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl4) solution was investigated. Rats received a single oral administration of CCl4 (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl4 administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca(2+)-Mg2+)-ATPase activity, indicating that the increased Ca2+ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca(2+)-Mg2+)-ATPase activity increased by CCl4 administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25-1.0 uM) to activate (Ca(2+)-Mg2+)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl4-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca(2+)-Mg2+)-ATPase activity in the liver obtained from CCl4-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca2+ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl4 administration.

Covalent Modification of Rat Liver Dipeptidyl Peptidase IV (CD26) by the Nonsteroidal Anti-Inflammatory Drug Diclofenac

Diclofenac is a nonsteroidal anti-inflammatory drug that has been implicated in several cases of severe hepatotoxicity. Our previous study showed that diclofenac metabolites bound covalently and selectively to rat liver plasma membrane proteins with estimated monomeric masses of 110, 140, and 200 kDa. We report here that we have identified the 110 kDa diclofenac-labeled protein in rat liver as dipeptidyl peptidase IV, also known as CD26. In addition, we found that the activity of dipeptidyl peptidase IV in liver plasma membrane fractions was lowered after diclofenac treatment of rats. These results suggest that the hepatotoxicity associated with diclofenac might be due, in part, to the covalent modification of dipeptidyl peptidase IV.

Calmodulin-Dependent Cyclic AMP Phosphodiesterase in Liver Plasma Membranes: Stimulated by Insulin

In vivoinsulin consistently stimulates the plasma membrane, high-affinity (lowKm) cyclic AMP phosphodiesterase (PDE) from diabetic rat liver or adipose tissue.In vitrostimulation of membrane PDE by insulin has been reported to be inconsistent. Also, the involvement of calmodulin (CaM) in insulin stimulation of PDE has been controversial. In this report, conditions for the isolation of rat liver plasma membranes containing PDE has been controversial. In this report, conditions for the isolation of rat liver plasma membranes containing PDE that is sensitive toin vitroinsulin stimulation and the involvement of CaM in insulin stimulation of PDE were investigated.In vitroinsulin raised theVmaxof the enzyme without altering its apparentKmand was dose dependent. Insulin stimulation was lost after freezing, sonication, solubilization with detergents, or storage of the membranes at 4° for 4 h after isolation, Insulin stimulation was completely blocked by the CaM antagonist compound 48/80, EGTA, or trifluoperazine. Two isoforms of membrane-bound PDE were separated by ion-exchange chromatography following solubilization of the plasma membranes. The activities of both isoforms were stimulated by exogenous CaM. Plasma membrane PDE eluted after the application of exogenous CaM plus Ca2+. The data support the concept of a critical involvement of CaM in insulin activation of liver membrane PDE.

Dissociation of protein kinase C activities and diacylglycerol levels in liver plasma membranes of rats on coconut oil and safflower oil diets

The activation of protein kinase C (PKC) is affected differently in vitro by different fatty acids. Whether this event occurs in response to fatty acid has not heretofore been determined in animal tissues. We addressed this question using the liver of rats on diets containing saturated or polyunsaturated fats. Rats on coconut oil, which is rich in saturated fatty acids, had a markedly lower PKC activity in liver plasma membranes with a slight but significant reduction of the activity in the cytosol than did rats fed safflower oil rich in linoleic acid. Ingestion of coconut oil resulted in a higher content of diacylglycerols (DG) in these membranes than did ingestion of safflower oil, whereas the proportions of saturated fatty acids and phospholipids and membrane fluidity were similar between rats ingesting different fats. These results are the first evidence that ingestion of coconut oil disproportionately affects PKC activation and the DG level in mammalian membranes. It seems likely that saturated fats exert various physiological effects on lipid and lipoprotein metabolism, in part through PKC pathways.

Characterization of the human Na +-dependent bile acid transporter (NTCP) using a recombinant vaccinia system : [formula omitted] Dept. of Pharmacology, Vanderbilt University, Nashville, TN

The transport of bile acids across sinusoidal and canalicular membranes of hepatocytes is characterized as carrier mediated. Such a carrier should specifically bind bile acids at physiological concentrations. We examined the binding of [3H]cholate to rat liver plasma membranes using a microcentrifugation technique and detected high (KD = 1.23 ± 0.44 μM, Bmax = 21.8 ± 3.3 pmol/mg protein) and low (KD = 1.97 ± 1.33 mM, Bmax = 41.5± 25.3 nmol/mg protein) affinity binding sites. Maximal binding was achieved within 15–45 sec and was stable for 2 min at 37°. Binding to the high affinity site was reversible, was not Na+ dependent or attributable to vesicular uptake, and exhibited a broad pH optimum. Binding to this site was negligible or not detected in liver mitochondrial and microsomal fractions, was saturable, and was inhibited by other bile acids. The IC50 values for bile acids as inhibitors of [3H]cholate binding at the high affinity site were: taurocholate, 1.9 nM; glycodeoxycholate, 3.1nM; chenodeoxycholate, 5.6nM; taurochenodeoxycholate, 7.3nM; glycochenodeoxycholate, 11 nM; lithocholate, 13 nM; taurodeoxycholate, 20 nM; glycocholate, 3.6 μM; and deoxycholate, 5.6 μM. [3H]Cholate specific binding was inhibited by 10−5 M bromosulfophthalein, bilirubin and indocyanin green. These data support the hypothesis that the high affinity binding site represents a carrier which is shared by bile acids and nonbile acid organic anions.

Insulin-Induced Proteolysis of the Insulin Receptor alpha-Subunit from Rat Liver does not Occur in vivo but is Prevented in vitro by Blood Serum Proteinase Inhibitors

Insulin binding to rat liver plasma membranes promotes the action of a plasma membrane proteinase that degrades the 135-kDa alpha-subunit of the insulin receptor to a 120kDa product. It has been proposed that this proteolysis may be the initial step leading to down-regulation of the hepatic insulin receptor [Lipson, K. E., Kolhatkar, A. A. & Donner, D. B. (1988) J. Biol. Chem. 263, 10495-10501]. Our results confirm that liver plasma membrane fractions from intact or perfused rat liver and from isolated rat hepatocytes do have a proteinase that degrades the alpha-subunit of the insulin receptor, whose activity increases significantly in the presence of insulin. In contrast to these in vitro results, plasma membranes and Golgi fractions isolated from liver at different times after a single intravenous injection of insulin to rats only contained intact alpha-subunits of the insulin receptor. Insulin administration was associated with a rapid and reversible translocation of insulin receptors from the plasma membranes to Golgi fractions but did not affect their total numbers or the receptor half-life. Incubation of rat liver plasma membranes with human, bovine or rat blood sera totally blocked alpha-subunit proteolysis both in the absence or presence of insulin. The three major serum proteinase inhibitors, alpha 1-antitrypsin, alpha 2-macroglobulin and antithrombin III, blocked alpha-subunit proteolysis of the insulin receptor to a varying extent. alpha 1-antitrypsin exhibited the highest potency, decreasing the amount of 120-kDa product by 70%. When both alpha 1-antitrypsin and antithrombin III were present, inhibition increased to 85%. Thus, the absence of proteolysis of the hepatic insulin receptor in vivo could be mostly accounted for by the antiproteolytic activity of blood serum. These findings suggest that insulin receptor alpha-subunit proteolysis does not represent a mechanism involved in the down-regulation of the insulin receptor in liver.

Regulatory interaction between calmodulin and the epidermal growth factor receptor.

Growth factor-stimulated cell proliferation is preceded by a transient increase in the cytoplasmic Ca concentration,' and calmodulin, an intracellular calcium receptor protein,' appears to intervene in the modulation of this process.3 Calmodulin binds to nuclear proteins and could therefore affect their functions! However, calmodulin does not control cell proliferation by acting exclusively at the nuclear level. Several lines of evidence obtained in our laboratory indicate that the epidermal growth factor receptor (EGFR) is a calmodulin-binding protein and that calmodulin acts as a modulator of its intrinsic tyrosine kinase activity.'.' We have demonstrated that the EGFR can be isolated from solubilized rat liver plasma membranes by calmodulin-affinity chromatography.' Binding of the EGFR to calmodulin-agarose occurs in the presence of Ca', and its elution is achieved upon addition of EGTA. The preparation obtained contains a set of calmodulinbinding proteins associated with the plasma membrane. However, the major autophosphorylated protein in this preparation corresponds to the EGFR. This autophosphorylation is stimulated by epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) with essentially the same effi~iency.~ Furthermore, the isolated receptor presents both EGFand TGFa-stimulated tyrosine kinase activity towards the exogenous substrate PoIY-L-(GIu : Tyr).'.' The identity of the isolated EGFR

Purinergic agonist and G protein stimulation of phospholipase D in rat liver plasma membranes. Independence from phospholipase C activation

Hormonal regulation of phospholipase D (PLD) was studied in isolated rat liver plasma membranes. Purinergic agents and a submaximal concentration of guanosine 5′-0-(3-thiotriphosphate) (GTPγS), a non-hydrolyzable analog of GTP, synergistically stimulate phosphatidylethanol formation, a measure of PLD activity. The rank order of efficacy for stimulation of PLD activity in the presence of 0.2 μM GTPγS was β, γ-methylene-ATP > adenosine 5′-0-(3-thiotriphosphate) = ATP = ADP = 2-methylthio-ATP > α, β-methylene-ATP = UTP. This pattern of activation does not conform to the series at known P 2 receptors. GTPγS stimulated PLD activity in a dose-dependent manner, and the GTPyS dose-response curve for phosphatidylethanol formation was shifted to the left by an analog of ATP. Activation of PLD by purinergic agents in the presence of GTPγS supports the involvement of a purinergic receptor of the P 2 class and a GTP-binding protein. Purinergic agents competitively inhibited [ 35S]adenosine 5′-0-(3-thiotriphosphate) binding to plasma membranes in the rank order adenosine 5′-0′(3-thiotriphosphate) > ATP > α, β-methylene-ATP = UTP ⪢ β γ-methylene-ATP = ADP. Stimulation of phosphoinositide phospholipase C (PI-PLC) by purinergic agents, as measured by release of radioactivity from endogenously myo[ 3H] inositol-labeled plasma membranes, occurred in the order α, β- methylene- ATP ⪢ 2- methylthio- ATP. β, γ-meth-ylene-ATP had little effect on PI-PLC activity. Different dose-response relationships for agonist-stimulation of PI-PLC and PLD indicate that activation of PI-PLC is not involved in stimulation of PLD in rat liver plasma membranes, and suggest that purinergic activation of PLD occurs via a pathway involving a G protein and a heretofore uncharacterized P 2 receptor.

Purification and Characterization of a Novel Plasma Membrane Phosphatidate Phosphohydrolase from Rat Liver

An N-ethylmaleimide-insensitive phosphatidate phosphohydrolase, which also hydrolyzes lysophosphatidate, was isolated from the plasma membranes of rat liver. The specific activity of an anionic form of the enzyme (53 kDa, pI < 4) was increased 2700-fold. A cationic form of enzyme (51 kDa, pI = 9) was purified to homogeneity, but the -fold purification was low because the activity of the highly purified enzyme was unstable. Immunoprecipitating antibodies raised against the homogeneous protein confirmed the identity of the cationic protein as the phosphohydrolase and were used to identify the anionic enzyme. Both forms are integral membrane glycoproteins that were converted to 28-kDa proteins upon treatment with N-glycanase F. Treatment of the anionic form with neuraminidase allowed it to be purified in the same manner as the cationic enzyme and yielded an immunoreactive protein with a molecular mass identical to the cationic protein. Thus, the two ionic forms most likely represent different sialated states of protein. An immunoreactive 51-53-kDa protein was detected in rat liver, heart, kidney, skeletal muscle, testis, and brain. Little immunoreactive 51-53-kDa protein was detected in rat thymus, spleen, adipose, or lung tissue. This work provides the tools for determining the regulation and function of the phosphatidate phosphohydrolase in signal transduction and cell activation.