Phosphoinositide-specific Phospholipase C Activity Research Articles

Involvement of phospholipase C in the norepinephrine-induced hypertrophic response in Cardiomyocytes

Norepinephrine (NE) is known to mediate cardiomyocyte hypertrophy through the G protein coupled a1 -adrenoceptor (a1 -AR) and the activation of the phosphoinositide-specific phospholipase C (PLC). Since the by-products of PLC activity are important downstream signal transducers for cardiac hypertrophy, the role of and the regulatory mechanisms involved in the activation of PLC isozymes in cardiac hypertrophy are highlighted in this review. The discussion is focused to underscore PLC in different experimental models of cardiac hypertrophy, as well as in isolated adult and neonatal cardiomyocytes treated with NE. Particular emphasis is laid concerning the a1 -AR-PLC-mediated hypertrophic signalling pathway. From the information provided, it is evident that the specific activation of PLC isozymes is a primary signalling event in the a1 -AR mediated response to NE as well as initiation and progression of cardiac hypertrophy. Furthermore, the possibility of PLC involvement in the perpetuation of cardiac hypertrophy is also described. It is suggested that specific PLC isozymes may serve as viable targets for the prevention of cardiac hypertrophy in patient population at-risk for the development of heart failure.

IP3 mediates NO-enhanced chilling tolerance in postharvest kiwifruit

The function of inositol 1,4,5-trisphosphate (IP3) in nitric oxide (NO)-induced chilling tolerance in postharvest kiwifruit was revealed. The fruit were treated using sodium nitroprusside (SNP; exogenous NO donor) and neomycin (IP3 inhibitor). Data demonstrated that compared with the control, chilling injury (CI) index and firmness decreased and increased upon SNP treatment in kiwifruit. SNP treatment enhanced phosphoinositide-specific phospholipase C (PI-PLC) activity, and consequently induced IP3 production. Moreover, SNP treatment down regulated malondialdehyde (MDA) content and electrolyte leakage as well as the activity and gene expression of lipoxygenase (LOX) in kiwifruit. In addition, the gene expression of transcription factors, including C-repeat binding factor1 (CBF1), WRKY1 and NAC5 was induced by SNP treatment. The above effects induced upon SNP treatment were inhibited by neomycin treatment. Neomycin treatment alone also led to the increase in CI index, MDA content and electrolyte leakage as well as the activity and gene expression of LOX, and the decrease in firmness, PI-PLC activity and IP3 production and gene expression of CBF1, WRKY1 and NAC5. Thus, IP3 mediated the alleviation of membrane damage, and the induction of CBF1, WRKY1 and NAC5 by SNP, thereby delaying CI in kiwifruit.

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

Multiple roles for Plasmodium berghei phosphoinositide-specific phospholipase C in regulating gametocyte activation and differentiation.

Critical events in the life cycle of malaria parasites are controlled by calcium-dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10 s of gametocytes being exposed to mosquito-derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide-specific phospholipase C (PI-PLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP2 and the production of the secondary messenger IP3 in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca2+ signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5 min after activation, suggesting a requirement for PI-PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI-PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP2/IP3 probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca2+ and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process.

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

The phospholipase C inhibitor U‐73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

The sarcoplasmic reticulum (SR) releases Ca(2+) via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca(2+) release subsequently propagates as Ca(2+) waves. To clarify the role of IP(3) production in wave generation, the contribution of a key enzyme in the production of IP(3) was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122. Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) measured using fluo-3. SR Ca(2+) release was evoked either by activation of IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine). U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) transients. The drug also inhibited [Ca(2+)](cyto) increases, evoked by direct IP(3)R activation (by photolysis of caged IP(3)) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca(2+)](cyto) and slowed the rate of Ca(2+) removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP(3)R- or RyR-mediated Ca(2+) release. U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) increases. However, the drug also reduced Ca(2+) release when evoked by direct activation of IP(3)R or RyR, slowed Ca(2+) removal and increased steady-state [Ca(2+)](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca(2+) transients by inhibiting the SR Ca(2+) pump to deplete the SR of Ca(2+) rather than by inhibiting PI-PLC.

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.

Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells ofTriticum turgidum

The role of phosphoinositide-specific phospholipase C (PI-PLC) signaling in the macrotubule-dependent protoplast volume regulation in plasmolyzed root cells of Triticum turgidum was investigated. At the onset of hyperosmotic stress, PI-PLC activation was documented. Inhibition of PI-PLC activity by U73122 blocked tubulin macrotubule formation in plasmolyzed cells and their protoplast volume regulatory mechanism. In neomycin-treated plasmolyzed cells, macrotubule formation and protoplast volume regulation were not affected. In these cells the PI-PLC pathway is down-regulated as neomycin sequesters the PI-PLC substrate, 4,5-diphosphate-phosphatidyl inositol (PtdInsP(2)). These phenomena were unaffected by R59022, an inhibitor of phosphatidic acic (PA) production via the PLC pathway. Taxol, a microtubule (MT) stabilizer, inhibited the hyperosmotic activation of PI-PLC, but oryzalin, which disorganized MTs, triggered PI-PLC activity. Taxol prevented macrotubule formation and inhibited the mechanism regulating the volume of the plasmolyzed protoplast. Neomycin partly relieved some of the taxol effects. These data suggest that PtdInspP(2) turnover via PI-PLC assists macrotubule formation and activation of the mechanism regulating the plasmolyzed protoplast volume; and the massive disorganization of MTs that is carried out at the onset of hyperosmotic treatment triggers the activation of this mechanism.

The SOCS Box Protein STOPS Is Required for Phototransduction through Its Effects on Phospholipase C

Phosphoinositide-specific phospholipase C (PLC) isozymes play roles in a diversity of processes including Drosophila phototransduction. In fly photoreceptor cells, the PLCbeta encoded by norpA is critical for activation of TRP channels. Here, we describe a PLCbeta regulator, STOPS, which encodes a SOCS box protein. Mutation of stops resulted in a reduced concentration of NORPA and a defect in stopping signaling following cessation of the light stimulus. NORPA has been proposed to have dual roles as a PLC- and GTPase-activating protein (GAP). We found that the slow termination resulting from expressing low levels of wild-type NORPA was suppressed by addition of normal amounts of an altered NORPA, which had wild-type GAP activity, but no PLC activity. STOPS is the first protein identified that specifically regulates PLCbeta protein concentration. Moreover, this work demonstrates that a PLCbeta derivative that does not promote TRP channel activation, still contributes to signaling in vivo.

M2 and M3 Muscarinic Receptor Activation of Urinary Bladder Contractile Signal Transduction. I. Normal Rat Bladder

The muscarinic receptor subtype-activated signal transduction mechanisms mediating rat urinary bladder contraction are incompletely understood. M(3) mediates normal rat bladder contractions; however, the M(2) receptor subtype has a more dominant role in contractions of the hypertrophied bladder. Normal bladder muscle strips were exposed to inhibitors of enzymes thought to be involved in signal transduction in vitro followed by a single cumulative concentration-response curve to the muscarinic receptor agonist carbachol. The outcome measures were the maximal contraction, the potency of carbachol, and the affinity of the M(3) -selective antimuscarinic agent darifenacin for inhibition of contraction. Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) with 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH(3)) reduces carbachol potency and reduces darifenacin affinity, whereas inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) with O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609) attenuates the carbachol maximal contraction. Inhibition of rho kinase with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) reduces carbachol potency and increases darifenacin affinity. Inhibition of rho kinase, protein kinase A (PKA), and protein kinase G (PKG) with 1-(5-isoquinolinesulfonyl)-homopiperazine.HCl (HA-1077) reduces the carbachol maximal contraction, carbachol potency, and darifenacin affinity. Inhibition of protein kinase C (PKC) with chelerythrine increases darifenacin affinity, whereas inhibition of rho kinase, PKA, PKG, and PKC with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine.2HCl (H7) reduces the carbachol maximum and carbachol potency while increasing darifenacin affinity. Inhibition of rho kinase, PKA, and PKG with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H89) reduces carbachol maximum and carbachol potency. Both the M(2) and the M(3) receptor subtype are involved in normal rat bladder contractions. The M(3)subtype seems to mediate contraction by activation of PI-PLC, PC-PLC, and PKA, whereas the M(2) signal transduction cascade may include activation of rho kinase, PKC, and an additional contractile signal transduction mechanism independent of rho kinase or PKC.

Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

Phospholipase C in Living Cells

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin–sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor–mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.

The exocytosis induced in HL-60 cells by 4-hydroxynonenal, a lipid peroxidation product, is not prevented by reduced glutathione

The effect of reduced glutathione (GSH) was studied on exocytosis triggered by 4-hydroxynonenal in HL-60 cells induced to differentiate towards the granulocytic cell line by dimethylsulfoxide; we measured beta-glucuronidase secretion from cells incubated at 37 degrees C in the presence of 5 mM GSH. GSH addition to the cell suspensions failed to induce any significant change of the exocytosis stimulated by HNE concentrations between 10(-8) and 10(-6) M. In contrast however, 5 mM GSH was able to fully prevent the release of lactate dehydrogenase observed in the presence of 50 microM HNE, a concentration much higher than that able to stimulate the exocytotic secretion. As the activation of phosphoinositide-specific phospholipase C (PLC) has been shown to play a major role in HNE-induced exocytosis, we studied the GSH effect on the breakdown of phosphatidylinositol-4,5-bisphosphate added to plasma membranes isolated from rat neutrophils and incubated in the presence of increasing concentrations of the aldehyde. In neutrophil membranes HNE induced a significant increase of PLC activity when used in the same concentrations as those able to stimulate beta-glucuronidase secretion in DMSO-differentiated HL-60 cells; the presence of 5 mM GSH failed to prevent its action. Our results suggest that these low aldehyde concentrations, which have actually been found in exudates, may increase tissue damage in inflammation through the release of lytic enzymes by neutrophils; it seems unlikely that their effects could be influenced by the levels of -SH groups present in the exudate and by its protein concentration.

Diacylglycerol and Its Formation by Phospholipase C Regulate Rab- and SNARE-dependent Yeast Vacuole Fusion

Although diacylglycerol (DAG) can trigger liposome fusion, biological membrane fusion requires Rab and SNARE proteins. We have investigated whether DAG and phosphoinositide-specific phospholipase C (PLC) have a role in the Rab- and SNARE-dependent homo-typic vacuole fusion in Saccharomyces cerevisiae. Vacuole fusion was blocked when DAG was sequestered by a recombinant C1b domain. DAG underwent ATP-dependent turnover during vacuole fusion, but was replenished by the hydrolysis of phosphatidylinositol 4,5-bisphosphate to DAG by PLC. The PLC inhibitors 3-nitrocoumarin and U73122 blocked vacuole fusion in vitro, whereas their inactive homologues did not. Plc1p is the only known PLC in yeast. Yeast cells lacking the PLC1 gene have many small vacuoles, indicating defects in protein trafficking to the vacuole or vacuole fusion, and purified Plc1p stimulates vacuole fusion. Docking-dependent Ca(2+) efflux is absent in plc1Delta vacuoles and was restored only upon the addition of both Plc1p and the Vam7p SNARE. However, vacuoles purified from plc1Delta strains still retain PLC activity and significant 3-nitrocoumarin- and U73122-sensitive fusion, suggesting that there is another PLC in S. cerevisiae with an important role in vacuole fusion.

Molecular cloning and expression analysis of phospholipase Cδ from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach ( Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni 2+-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP 2) and its activity was Ca 2+-dependent, which was similar to mammalian PLCδ isozymes.

Phospholipase C may be involved in norepinephrine-induced cardiac hypertrophy

Cardiac hypertrophy is characterized by increased cardiomyocyte size, mRNA levels for atrial natriuretic factor (ANF), and protein synthesis. Although activation of the phosphoinositide-specific phospholipase C (PLC) leads to the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, the involvement of PLC in hypertrophic response remains to be fully understood. The present study was therefore undertaken to examine if the inhibition of PLC activity is associated with a decrease in ANF expression and protein synthesis in cardiomyocytes, due to norepinephrine (NE), a known hypertrophic agent. NE resulted in an increase in ANF gene expression and protein synthesis in adult rat cardiomyocytes, these effects of NE were attenuated by a PLC inhibitor, U73122. The NE-induced increase in ANF gene expression and protein synthesis was also inhibited by an α-adrenoceptor blocker, prazosin. Both U73122 and prazosin depressed the NE-induced increase in DAG production in cardiomyocytes. These results indicate that the α-adrenoceptor mediated PLC activation may be involved in the process of NE-induced cardiac hypertrophy.

Identification of a compound that directly stimulates phospholipase C activity.

Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in the signal transduction of various cellular responses. However, although it is undeniably important that modulators of PLC activity be identified, no direct PLC activity modulator has been identified until now. In this study, by screening more than 10,000 different compounds in human neutrophils, we identified a compound that strongly enhances superoxide-generating activity, which is well known to be PLC-dependent. The active compound 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS) stimulated a transient intracellular calcium concentration ([Ca(2+)](i)) increase in neutrophils. Moreover, m-3M3FBS stimulated the formation of inositol phosphates in U937 cells, indicating that it stimulates PLC activity. The compound showed no cell-type specificity in terms of [Ca(2+)](i) increase in the various cell lines including leukocytes, fibroblasts, and neuronal cells. We also ruled out the possible involvement of heterotrimeric G proteins in m-3M3FBS-stimulated signaling by confirming the following: 1) pertussis toxin does not inhibit m-3M3FBS-induced [Ca(2+)](i) increase; 2) m-3M3FBS does not stimulate cyclic AMP generation; and 3) the inhibition of G(q) by the regulator of G protein-signaling 2 does not affect the m-3M3FBS-induced [Ca(2+)](i) increase. We also observed that m-3M3FBS stimulated PLC activity in vitro. The purified isoforms of PLC that were tested (i.e., beta2, beta3, gamma1, gamma2, and delta1) were activated by m-3M3FBS and showed no isoform specificity. Taken together, these results demonstrate that m-3M3FBS modulates neutrophil functions by directly activating PLC. Because m-3M3FBS is the first compound known to directly activate PLC, it should prove useful in the study of the basic molecular mechanisms of PLC activation and PLC-mediated cell signaling.

Aluminium differentially modifies lipid metabolism from the phosphoinositide pathway in Coffea arabica cells

The effect of aluminium (Al) on phosphoinositide-specific phospholipase C (PLC) and lipid kinase activities was examined in a cellular suspension of coffee. Two main effects were seen when cells were treated with AlCl3. In periods as short as 1 minute, Al-exposed cells increased the activity of PLC and IP3 formation up to two fold. Over longer periods PLC activity was inhibited by more than 50%. The activity of phosphatidylinositol 4-kinase (Pl 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5-K) and diacylglycerol kinase increased when cells were incubated in the presence of different concentrations of AlCl3. The present study reports for the first time that Al may have different effects on the Pl-signaling pathway depending on the time of exposure. Our results strongly support the hypothesis that Al disrupts the metabolism of membrane phospholipids regulating not only PLC but also other enzymes that have key roles in signal-transduction pathways.

Antidepressants reduce phosphoinositide-specific phospholipase C (PI-PLC) activity and the mRNA and protein expression of selective PLC β 1 isozyme in rat brain

In order to examine whether antidepressants mediate their action by interacting with one of the key components of the phosphoinositide (PI) signaling pathway, i.e. PI-specific phospholipase C (PLC), and whether this represents a common mechanism of action of antidepressants, we determined the effects of antidepressants of various classes on PI-PLC activity and on the expression of PLC isozymes in rat brain. It was observed that chronic (21-day) but not acute (1-day) administration with desipramine (DMI), fluoxetine (FLX) and phenelzine (PHLZ), decreased PI-PLC activity in membrane and cytosol fractions of cortex and hippocampus. Similar changes were observed with alprazolam (ALP) and buspirone (BUS), who possess anxiolytic and antidepressant properties. On the other hand, an anxiogenic drug, metachlorophenylpiperazine (MCPP), increased PI-PLC activity in both membrane and cytosol fractions of cortex and hippocampus. The immunolabeling studies showed that all the antidepressants and anxiolytics that caused a decrease in PI-PLC activity also selectively decreased the protein levels of a specific isozyme of PLC, i.e. PLCβ 1, in membrane and cytosol fractions of cortex and hippocampus, whereas MCPP increased the levels of this particular isozyme. These changes were accompained with changes in the mRNA levels of PLCβ 1, as determined by quantitative RT-PCR. These antidepressants and anxiolytics did not cause significant changes in the expression of PLC δ 1 or γ 1 isozyme. Our results thus suggest that modulation of PI-PLC may be common to all classes of antidepressants, which in turn, may be associated with their mechanisms of action.