PLC Enzyme Research Articles

Synergistic Activation of PLC‐dependent Diacylglycerol (DAG) Production by Mu‐Opioid and Gq‐Coupled Receptor Signaling

The µ-opioid receptor (MOR) is a Gi-protein coupled receptor (GPCR) responsible for opioid-induced analgesia and undesired effects such as constipation, respiratory depression, and addiction. Upon binding of an agonist, the Gαi and the Gβγ subunits dissociate to signal downstream effectors. One such effector is phospholipase-Cβ3 (PLCβ3) that can be activated by both Gβγ subunits and Gαq. PLC enzymes hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG) and inositol triphosphate (IP3). Knockout of PLCβ3 in mice, and small molecule inhibition of Gβγ-PLC interactions potentiate opioid-induced antinociception. Based on these data we hypothesized that MOR-dependent activation of PLCβ3 opposes opioid-induced analgesia. However, activation of PLC by MORs is difficult to observe in cellular models. Since PLCβ is synergistically activated by Gαq and Gβγ subunits, we hypothesized that MOR-dependent PLC activation may require a coincident signal from a Gq-signaling pathway. To test this, we established an assay for PLC activation using a fluorescent DAG sensor in HEK-293 cells stably expressing MOR with or without co-expression of exogenous M1 muscarinic receptors. In cells, coexpressing M1 receptors, saturating concentrations of carbachol strongly increased DAG production, but saturating concentrations of DAMGO or morphine gave no response. However, synergistic activation of PLC was observed upon simultaneous addition of subsaturating concentrations of the muscarinic agonist carbachol, and MOR agonists morphine and DAMGO. Strong synergy was also observed when activating endogenous muscarinic receptors (likely M3). When cells were treated with pertussis toxin (PTX), the synergy driven by the co-activation of both types of GPCRs was lost demonstrating that Gβγ activation from MOR is necessary for PLC synergy. We used newly designed small molecule inhibitors of Gβγ-PLC interaction to demonstrate that this synergistic activation is due to Gβγ and PLCβ activation. Physiologically, MOR containing neurons respond to multiple neurotransmitters including those coupled to Gq coupled receptors, suggesting that this mechanism may play a role in modulation of opioid-induced analgesia. In the future, we will explore this mechanism using electrophysiological and in vivo models.

Verapamil prevents the effect of calcium-sensing receptor activation on the blood glucose and insulin levels in rats.

The Ca2+ triggered insulin exocytosis in β cells of the pancreatic islets may be the result of Ca2+ influx through L-type voltage dependent calcium channels (VDCC) localized in the plasma membrane, as well as of liberation of Ca2+ from intracellular storages, induced by activation of the calcium receptor (CaR) coupled with the PLC enzyme present in the pancreatic islets. The present study was designated to determine, in in vivo experiments, the effects of CaR activation by R-568 and inhibition of the receptor by NPS 2143 on the plasma glucose and insulin levels in the presence of verapamil, a calcium channel blocker. Wistar rats, after fasting for 14 h before the experiment, were anesthetized with inactin and loaded ip with 1 g/kg glucose. In comparison to the control group, the verapamil-induced blockade of the calcium channels in glucose loaded animals increased the blood glucose level and decreased the insulin level, whereas CaR activation with R-568 induced opposite effects. However, in the presence of verapamil, R-568 did not change the concentration of glucose or insulin versus the control animals. Verapamil infusion did not alter elevated glucose concentration in the NPS 2143 animals. At the same time, verapamil reduced the plasma insulin level and potentiated the drop of insulin concentration induced by NPS 2143. The observations suggest that under the in vivo conditions, calcium channel blockade may prevent changes in the blood glucose and insulin concentrations induced by the CaR activation.

Conformational Dynamics Contribute to Phospholipase Cβ Activity

Phospholipase Cβ (PLCβ) enzymes hydrolyze the lipid phosphatidylinositol‐4,5‐bisphosphate (PIP2) to produce the second messengers inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). Production of these second messengers leads to many diverse physiological responses, including vascular smooth muscle contraction and inflammation. Previous structural and functional studies of PLCβ have revealed a highly conserved catalytic core that adopts a compact, globular structure and forms the minimal fragment that retains lipase activity. However, a growing body of evidence suggests that the PLCβ PH domain, which interacts with EF hands and TIM barrel of the catalytic core in crystal structures, may be flexible in solution. Using a combination of small angle x‐ray scattering (SAXS), cross‐linking studies, and biochemical assays, we provide the first structural data demonstrating that the PH is in fact conformationally flexible in solution. The PH domain adopts two major states: an open state wherein the PH domain is extended away from the core, and a closed state consistent with the crystal structures. We also provide evidence that these conformational states are functionally significant. These findings provide new evidence of conformational heterogeneity of PLC enzymes in solution and reveal new insights into their roles in cell signaling and cardiovascular disease.Support or Funding InformationAmerican Heart Association Scientist Development Grant 16SDG29920017; Purdue Center for Cancer Research; Showalter Foundation; all to A.M.LThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Zeta potential and surface charge of DPPC and DOPC liposomes in the presence of PLC enzyme

Zeta potential of liposomes is often measured in studies of their properties and/or applications. However, there are not many papers published in which zeta potential was determined during enzymatic reaction of a lipid. Therefore in this paper size, polydispersity index, and zeta potentials of 1,2-dipalmitoylsn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes were investigated in 1 mM NaCl (pH 6.2) and phosphate buffer (pH 8.1) during 60 min of phospholipase C action at 20 and 37 °C. The hydrocarbon chains saturation differ these two phospholipids what appears in some differences in the zeta potential changes during the hydrolysis reaction run. The polydispersity index of the liposomes indicates that they are relatively stable and monodisperse, except for DPPC in phosphate buffer where up to 30 % of the initial amount forms larger size moieties, possibly aggregates of the liposomes, enzyme and the hydrolysis products. However, in this buffer zeta potential of the liposomes practically does not change during PLC action. Also the changes of zeta potential of DOPC liposomes are minor, although their negative values are much smaller than those of DPPC at both temperatures. These small changes of the potential may be due to compression of the diffuse double layer by present phosphate buffer. However, distinct changes of the zeta potentials in the presence of PLC take place in NaCl solution. The observed changes can be explained by reorientation of the phospholipid polar heads and their different density on DPPC and DOPC surface of the liposomes. Although it appeared that the zeta potential is not a very sensitive parameter for tracking the hydrolysis reaction in phosphate buffer, generally zeta potential enables the characterization of such reactions through determination of electrokinetic properties of liposomes as well as the polydispersity and size distribution of the liposomes do.

Mapping the Phosphoinositide Landscape in Mammalian Cells

Polyphosphoinositides (PPIs) are universal lipid regulators that control a great variety of cellular function serving as docking sites and conformational switches for a large number of signaling proteins associated with cellular membranes. PPIs are produced from phosphatidylinositol (PtdIns) that also is a structural lipid of biological membranes. PtdIns synthesis is believed to occur primarily in the ER, although it is debated whether it also happens at the plasma membrane (PM). Considerable progress has been made to determine the localization and dynamic changes in PPIs both in live and fixed cells, yet little has been done to map the distribution and possible functional diversity of the PtdIns pools in mammalian cells. Here we devised a strategy to address this question by using a PtdIns specific PLC enzyme isolated from Listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of PtdIns in living cells. Our studies reveal that a significant metabolically highly active PtdIns pool exists associated with tiny mobile structures within the cytoplasm in addition to the known ER and PM PtdIns pools. We will show our most recent data on the consequences of PtdIns depletion within the various PtdIns pools and on PPI production and will also discuss principles of PPI modulation of multiple effectors.

Regulatory links between PLC enzymes and Ras superfamily GTPases: Signalling via PLCɛ

Regulatory links between PLC enzymes and Ras superfamily GTPases: Signalling via PLCɛ

Structural Determinants for Phosphatidic Acid Regulation of Phospholipase C-β1

Signaling from G protein-coupled receptors to phospholipase C-beta (PLC-beta) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-beta(1) through a mechanism that requires the PLC-beta(1) C-terminal domain. PA also modulates Galpha(q) stimulation of PLC-beta(1). These data suggest that PA may have a key role in the regulation of PLC-beta(1) signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-beta(1). We used a combination of enzymatic assays, PA-binding assays, and circular dichroism spectroscopy to evaluate the interaction of PA with wild-type and mutant PLC-beta(1) proteins and with fragments of the Galpha(q) binding domain. The results identify a region that includes the alphaA helix and flexible loop of the Galpha(q)-binding domain as necessary for PA regulation. A mutant PLC-beta(1) with multiple alanine/glycine replacements for residues (944)LIKEHTTKYNEIQN(957) was markedly impaired in PA regulation. The high affinity and low affinity component of PA stimulation was reduced 70% and PA binding was reduced 45% in this mutant. Relative PLC stimulation by PA increased with PLC-beta(1) concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-beta(1) mutant. These data are consistent with a model for PA regulation of PLC-beta(1) that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Galpha(q)-binding domain. PA regulation of PLC-beta(1) requires unique residues that are not required for Galpha(q) stimulation or GTPase-activating protein activity.

PLC-gamma1 enzyme activity is required for insulin-induced DNA synthesis.

Previously, we had shown that inhibition of PLC activity impaired the ability of insulin to activate ERK in 3T3-L1 adipocytes. In this study, we confirmed that the insulin receptor and PLC-gamma1 are physically associated in hIRcB fibroblasts, insulin stimulates PLC-gamma1 enzyme activity, and inhibition of PLC activity impairs activation of ERK. We subsequently investigated whether PLC-gamma1 is required for insulin-stimulated mitogenesis. First, inhibition of PLC activity using U73122 impairs the ability of insulin to stimulate DNA synthesis. Second, disruption of the interaction of the insulin receptor with PLC-gamma1 by microinjection of SH2 domains derived from PLC-gamma1 or Grb2 but not Shc similarly blocks insulin-induced DNA synthesis. Third, microinjection of neutralizing antibodies to PLC-gamma1 blocks DNA synthesis, but nonneutralizing antibodies do not. The blockade in all three cases is rescued by synthetic diacylglycerols but not by inositol-1,4,5-trisphosphate, indicating a requirement for PLC enzyme activity. These experimental data point to a requirement for PLC-gamma1 in insulin-stimulated mitogenesis in hIRcB cells.

Mechanical forces and signaling in connective tissue cells: cellular mechanisms of detection, transduction, and responses to mechanical deformation

The environment provides mechanical input to tissues through ground reaction forces, gravity, barometric pressure, vibration, and contact with bodies. When tissues are deformed, resident cells are deformed in complex modes. Cells detect “outside-in” mechanical signals by diverse means, including stretch-activated ion channels and other ion channels as the plasma membrane is deformed, through integrin linkages from the matrix to the cytoskeleton, and through cadherins or desmosomes in cell-cell connections. Ion channels, particularly sodium, chloride, potassium, and calcium channels, play an important role in the signal transduced from the mechanical stimulus to a chemical signal. This signal often results in an increase in the concentration of intracellular calcium. This part of the signaling pathway is often blocked by removal of extracellular calcium. Once an initial step in signal transduction has occurred, activation of intracellular pathways initiates phosphorylation events and protein-protein associations including association of integrin β subunits with the cytoskeleton and associated proteins, such as FAK, paxillin, filamin, integrin-linked kinase (ILK), and talin. PLA, PLC, adenyl cyclase, guanyl cyclase, iNOS, and other enzymes are activated producing the second messengers: cAMP, cGMP, ATP, GTP, NO, PGE 2 , IP 3 , and DAG that act, not only in the target cell but also may have paracrine effects. Furthermore, specific pathways may be activated that drive mitogenesis (MEK/MAPK), a stress response (JAK/STAT and JNK), or other responses that result in specific gene expression through activation of transcription factors, cell division, and contraction. The chemical signal can be passed from cell to cell by mediator secretion or through gap junctions. A cell must be able to respond, then desensitize to a mechanical stimulus, just as a receptor may desensitize to a ligand. Therefore, “stop,” or inhibitory signals, are perhaps as important as “go,” or stimulatory signals with respect to a mechanical load response. A cell must be able to attenuate its response to load. This review discusses the recent concepts in mechanical signal detection, transduction, and response pathway in mammalian connective tissue cells.

Phosphorylation and Regulation of G-protein-activated Phospholipase C-β3 by cGMP-dependent Protein Kinases

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, beta-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca(2+)] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca(2+) by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-beta) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca(2+). In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-beta2 and PLC-beta3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-activated PLC-beta3 activity by 50-70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-beta3 by PKG (Ser(26) and Ser(1105)). Mutation at these two sites (S26A and S1105A) of PLC-beta3 completely blocked the phosphorylation of PLC-beta3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-beta3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca(2+)] by the NO-cGMP-dependent signaling pathway.

Inhibition of phosphoinositide hydrolysis and cell growth of Swiss 3T3 cells by myristoylated phospholipase C inhibitor peptides.

It has been demonstrated that the phospholipase C-gamma (PLC-gamma) molecule contains within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of this region (PCI peptides) suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem. 267, 21884-21889]. In order to improve the permeability of the plasma membrane to PCI peptides, we synthesized myristoylated PCI peptides, myr-GLYRKAMRLRYPV [myr-PCI(Y)] and myr-GLFRKAMRLRFPV [myr-PCI(F)], which are identical except for the replacement of the two tyrosine residues in myr-PCI(Y) by phenylalanines in myr-PCI(F), and examined their inhibitory activity on PLC enzymes in vitro and in vivo. This fatty acid modification potentiated the inhibitory activity of the original PCI peptides and both myr-PCI(Y) and myr-PCI(F) suppressed the PIP2-hydrolyzing activity of purified PLC isoforms in vitro. The Ki values of myr-PCI(Y) and myr-PCI(F) for purified PLC-gamma1 were 3.5 and 55 microM, respectively. Myr-PCI(Y) at concentrations in the sub-micromolar range significantly suppressed IP3 formation induced by EGF, PDGF, bombesin, or serum in Swiss 3T3 cells. Furthermore, myr-PCI(Y) also strongly inhibited cell proliferation induced by these stimuli. The inhibitory effect on IP3 formation and proliferation of myr-PCI(F) was much less potent than that of myr-PCI(Y). These results suggest that myristoylated PCI peptides could be applied to living cells as specific inhibitors of PLC signaling pathways and that PLC pathways are at least in part required for growth in Swiss 3T3 cells.

Application of capillary electrophoresis to determination of enzyme activity and other properties of phosphatidylinositol-specific phospholipase C

This paper describes a method for monitoring enzymatic activity using micellar electrokinetic chromatography (MEKC). MEKC is used to analyze the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) with convenience and precision. PI specific PLC enzyme converts phosphatidylinositol (PI) to diacylglycerol (DAG) and inositol 1,2-cyclic phosphate (IP). The assay system developed is based on monitoring both the breakdown of substrate and the formation of products simultaneously. To obtain the best separation for the substrate PI and product DAG, we investigated changes in concentration of electrolyte buffer and SDS as well as in pH of the eletrolyte buffer. Since the structure of the substrate and products are different, the reaction could be monitored easily by MEKC mostly based on hydrophobicity. Under the separation conditions developed, we investigated the enzymatic activity of PI-PLC depending on the concentrations of phosphatidylinositol, various divalent cations and the reaction temperature. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to confirm the molecular masses of the substrate PI and products DAG and IP. The results of this study show that capillary electrophoresis can be widely applied to analyze and characterize many other enzymes since capillary electrophoresis has several advantages as follows, simplicity, speed, no need for radiolabelled substrate, small sample volumes and sufficient accuracy.

Mechanism and structure based inhibitors of phospholipase C enzymes

PI-specific PLC enzymes are a key component of phosphatidylinositol-mediated signaling pathways since the hydrophobic product, diacylglycerol, activates protein kinase C and the water-soluble product, inositol trisphosphate, is involved in Ca 2+ mobilization. Nonspecific, or PC-PLC, enzymes can generate diacylglycerol without Ca 2+ mobilization. A series of inhibitors, both lipophilic and water-soluble, have been synthesized to target each of these two classes of PLC enzymes. Design of the inhibitors was based on proposed enzyme mechanisms and available crystal structures. The solution conformations of the lipophilic phospholipid analogs, (diheptanoylphosphatidyl(2-O-methyl)inositol for PI-PLC and a dihexanoyl-sn-(3-N-benzylaminoglycero)phosphoramidocholine for PC-PLC, have been determined using NMR methodology and the interaction of these compounds with bacterial enzymes has been examined. Water-soluble inhibitors include strained cyclic phosphonates for PI-PLC and vanadate for PC-PLC. An eventual goal of this work is to generate compounds that specifically target each type of intracellular PLC activity.

Ras‐p21 Activates phospholipase D and A2, but not phospholipase C or PKC, in Xenopus laevis Oocytes

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.

Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.

Effect of ethanol administration and withdrawal on serotonin receptor subtypes and receptor-mediated phosphoinositide hydrolysis in rat brain.

The effect of short-term (15 days) and long-term (60 days) ethanol treatment and withdrawal on agonist-stimulated phosphoinositide (Pl) hydrolysis, serotonin receptor subtypes (5HT1A and 5HT2), and alpha 1-adrenergic receptors were studied in rat cerebral cortex. Short-term ethanol treatment had no significant effect on serotonin (5HT), norepinephrine (NE), and calcium ionophore (A23187)-stimulated [3H]-inositol-1-phosphate ([3H]-IP1) formation and 5-HT2 receptors as measured by 125I-lysergic acid diethylamide (125I-LSD) binding, in rat cerebral cortex. However, 15 days of ethanol treatment, followed by 24 hr of withdrawal resulted in a decrease in Bmax of 125I-LSD binding without significant change in KD, as well as a decrease in 5HT-stimulated [3H]-IP1 formation in rat cerebral cortex. 5HT1A and alpha 1-adrenergic receptors were determined by using [3H]-8-hydroxy-2-(di-N-propylamino)tetralin and [3H]-prazosin as radioligand, respectively. We also observed that long-term ethanol treatment had no significant effect on Bmax and KD of 5HT2, 5HT1A, and alpha 1-adrenergic receptors, as well as NE and A23187-stimulated [3H]-IP1 formation, but significantly decreased the 5HT-stimulated [3H]-IP1 formation in rat cerebral cortex. It is possible that a decrease in 5HT-induced PI turnover after long-term ethanol exposure may be due to a decrease in coupling of 5HT2 receptors to G protein or PLC enzyme, whereas the decrease in 5HT-induced PI turnover after withdrawal may be due to a decrease in functional 5HT2 receptor number.

Characterization of polyphosphoinositide-specific phospholipase C in rat parotid gland membranes

Hydrolysis of exogenously added, [ 3H]inositol-labeled, phosphatidylinositol 4,5-bisphosphate (PIP 2) by rat parotid membranes was increased, dose-dependently, by the muscarinic cholinergic agonist carbamylcholine (carbachol) in the presence of guanosine 5′- O-thiotriphosphate (GTPγS). The stimulation was inhibited by atropine and guanosine 5′- O-thiodiphosphate (GDPβS). GTPγS alone stimulated PIP 2 hydrolysis, with halfmaximal activation at 0.1 μ m. This was inhibited by GDPβS but not by atropine. Agonist stimulation of PIP 2 hydrolysis was dependent on the presence of lipids (phosphatidylserine:phosphatidylethanolamine:PIP 2 = 1:1:1). When PIP 2 was added as micelles with detergent (sodium deoxycholate) only, basal hydrolysis was elevated, thus decreasing the relative stimulation by GTPγS and carbachol. The water-soluble hydrolysis products formed under either condition were 1,4,5-inositol trisphosphate, 1,4-inositol bisphosphate, and cyclic inositol trisphosphate. Hydrolysis of exogenous phosphatidylinositol (PI) was also stimulated by carbachol in the presence of GTPγS but the extent of PI hydrolysis was 44-fold lower than PIP 2 hydrolysis. When [Ca 2+] in the medium was increased from 100 n m to 1 μ m, basal hydrolysis of both PI and PIP 2 increased (9.3- and 19.2-fold, respectively). However, levels of basal and stimulated PIP 2 hydrolysis were higher (37.9- and 29.6-fold, respectively) than those of PI hydrolysis. Antibodies (both polyclonal and monoclonal) raised against phospholipase C (PLC β 1) from bovine brain did not react with any component in either rat parotid membranes or cytosol, although a reactivity was detected in rat brain membranes. A monoclonal antibody against bovine brain PLC γ 1 detected a ~ 150-kDa protein only in the parotid cytosol, while antisera against bovine brain PLC δ 1 enzyme showed no reactivity with parotid membranes or cytosol. Together, these observations suggest that while there appears to be a protein similar to bovine brain PLC γ 1 in parotid gland cytosol, the PLC which mediates PIP 2 hydrolysis in rat parotid membranes and can be regulated by the muscarinic receptor via a G-protein is distinct from the well-characterized PLC enzymes γ 1, δ 1, and β 1.

Cloning and sequence of multiple forms of phospholipase C

Three phospholipase C isozymes (PLC-I, II, and III) have been purified from bovine brain. Here, phospholipase C-related cDNA clones corresponding to PLC-I and PLC-III were isolated from a rat brain λgt11 expression cDNA library using specific monoclonal antibodies and sequenced. Each of them encodes a distinct polypeptide with a calculated molecular mass of 138,225 (PLC-I) and 85,840 (PLC-III). Comparison of these two with the sequence of another isozyme PLC-II (M r = 148,431) that we have previously characterized revealed a low overall sequence homology. Nevertheless, a significant amino acid sequence similarity between the three enzymes was found in two regions, one of about 150 amino acids and the other of about 120 amino acids. The two conserved domains were separated by a variable region. The variable region sequence of PLC-II is relatively long and has recently been shown to contain regions homologous to the noncatalytic domain of the nonreceptor tyrosine kinases. Those of PLC-I and III were short and appeared to be unrelated to these tyrosine kinases. The physiological implications of the multiple species of PLC enzymes are discussed.

Phospholipase C I and II brain isozymes: immunohistochemical localization in neuronal systems in rat brain.

Two distinct inositol phospholipid-specific phospholipase C (PLC; phosphatidylcholine phosphatidohydrolase, EC 3.1.4.3) isozymes, PLC-I and PLC-II, have been purified and characterized from bovine brain. Monoclonal antibodies that distinguish between these isozymes are used in the present study to map isozyme distribution in the rat brain with immunohistochemical techniques. Both isozymes are localized in neurons, and, whereas PLC-II is rather ubiquitous--being expressed in most neurons, PLC-I is restricted in its distribution. The strongest immunoreactive labeling for PLC-I is in the neurons of the striatum, which provide inputs to the globus pallidus and substantia nigra, where terminals are also densely labeled. The neuronal targets of these terminals in the globus pallidus and substantia nigra do not express PLC-I immunoreactivity, but they do display PLC-II immunoreactivity. PLC-I immunoreactivity is also particularly well pronounced in the pyramidal cells of the hippocampus and, to a lesser extent, in the granule cells of the dentate gyrus. In the thalamus, PLC-I is localized to neurons in the reticular thalamic nucleus, in the medial subdivision of the mediodorsal thalamic nucleus, and in the anteromedial thalamic nucleus. Other areas displaying PLC-I immunoreactive neurons include the dorsal lateral septal nucleus and the basolateral amygdala. The expression of at least one or more forms of PLC in most neurons of the brain suggests that this enzyme may be part of a common system of signal transduction used universally by all neurons. However, the differential expression of PLC isozymes suggests further that certain neurotransmitter and receptor interactions may differ in the forms of the PLC enzyme used for signal transduction.