Phosphatidylinositol-specific Phospholipase C Inhibitor Research Articles

Phosphatidylinositol-specific phospholipase C-associated phospholipid metabolism mediates DcGLRs channel to promote calcium influx under CaCl2 treatment in shredded carrots during storage

The rapid activation of phosphatidylinositol-specific phospholipase C (PI-PLC) occurs early after the stimulation of biotic and abiotic stress in plants, which directly associated with the calcium channel-induced calcium ion (Ca2+) influx. Exogenous calcium chloride (CaCl2) mediates the calcium signaling transduction to promote the γ-aminobutyric acid accumulation and nutritional quality in shredded carrots whereas the generation mechanism remains uncertain. Therefore, the involvement of PI-PLC-associated phospholipid metabolism was investigated in present study. Our result revealed that CaCl2 treatment promoted the expression and activity of PI-PLC and increased the inositol 1,4,5-trisphosphate and hexakisphosphate content in shredded carrots. The transcripts of multi-glutamate receptor-like channels (DcGLRs), the glutamate and γ-aminobutyric acid (GABA) content, and Ca2+ influx were induced by CaCl2 treatment in shredded carrots during storage. However, PI-PLC inhibitor (U73122) treatment inhibited the activation of PI-PLC, the increase of many DcGLRs family genes expression levels, and Ca2+ influx. Moreover, the identification of DcPI-PLC4/6 and DcGLRs proteins, along with the analysis of characteristic domains such as PLCXc, PLCYc, C2 domain, transmembranous regions, and ligand binding domain, suggests their involvement in phospholipid catalysis and calcium transport in carrots. Furthermore, DcPI-PLC4/6 overexpression in tobacco leaves induced the Ca2+ influx by activating the expressions of NtGLRs and the accumulation of glutamate and GABA. These findings collectively indicate that CaCl2 treatment-induced PI-PLC activation influences DcGLRs expression levels to mediate cytosolic Ca2+ influx, thus, highlighting the “PI-PLC-GLRs-Ca2+” pathway in calcium signaling generation and GABA biosynthesis in shredded carrots.

Association of Phosphatidylinositol-Specific Phospholipase C with Calcium-Induced Biomineralization in the Coccolithophore Emiliania huxleyi.

Biomineralization by calcifying microalgae is a precisely controlled intracellular calcification process that produces delicate calcite scales (or coccoliths) in the coccolithophore Emiliania huxleyi (Haptophycea). Despite its importance in biogeochemical cycles and the marine environment globally, the underlying molecular mechanism of intracellular coccolith formation, which requires calcium, bicarbonate, and coccolith-polysaccharides, remains unclear. In E. huxleyi CCMP 371, we demonstrated that reducing the calcium concentration from 10 (ambient seawater) to 0.1 mM strongly restricted coccolith production, which was then recovered by adding 10 mM calcium, irrespective of inorganic phosphate conditions, indicating that coccolith production could be finely controlled by the calcium supply. Using this strain, we investigated the expression of differentially expressed genes (DEGs) to observe the cellular events induced by changes in calcium concentrations. Intriguingly, DEG analysis revealed that the phosphatidylinositol-specific phospholipase C (PI-PLC) gene was upregulated and coccolith production by cells was blocked by the PI-PLC inhibitor U73122 under conditions closely associated with calcium-induced calcification. These findings imply that PI-PLC plays an important role in the biomineralization process of the coccolithophore E. huxleyi.

Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.

Synthesis and determination of the relative structure of akaterpin, a potent inhibitor of PI-PLC

We describe the first total synthesis and structural determination of akaterpin, an inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC). The key features of the synthetic strategy include a regio- and stereoselective C-alkylation at the angular C1′ position and an exo-selective intermolecular Diels–Alder reaction. The relative stereochemistry was determined by a comparison of the NMR spectra of synthetic akaterpin with those of natural akaterpin.

Bile Acid Reflux Contributes to Development of Esophageal Adenocarcinoma via Activation of Phosphatidylinositol-Specific Phospholipase Cγ2 and NADPH Oxidase NOX5-S

Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA.

Presynaptic BK type Ca 2+-activated K + channels are involved in prostanoid TP receptor-mediated inhibition of noradrenaline release from the rat gastric sympathetic nerves

Previously, we reported that prostanoid TP receptor mediates the inhibition of electrically evoked noradrenaline release from gastric sympathetic nerves in rats. Prostanoid TP receptor has been shown to activate phospholipase C (PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate (IP 3) and diacylglycerol; IP 3 triggers the release of Ca 2+ from intracellular stores and diacylglycerol activates protein kinase C. In the present study, therefore, we examined whether these PLC-mediated mechanisms are involved in the TP receptor-mediated inhibition of gastric noradrenaline release using an isolated, vascularly perfused rat stomach. U-46619 (9,11-dideoxy-9α,11α-methanoepoxy PGF 2α) (a prostanoid TP receptor agonist)-induced inhibition of noradrenaline release from the stomach was reduced by U-73122 [1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]hexyl]-1 H-pyrrole-2,5-dine] (a PLC inhibitor) and ET-18-OCH 3 (1- O-octadecyl-2- O-methyl- sn-glycero-3-phosphorylcholine) (a phosphatidylinositol-specific PLC inhibitor), respectively. 2-APB (2-aminoethyldiphenyl borate) (a putative IP 3 receptor antagonist) also abolished the U-46619-induced inhibition of noradrenaline release, but Ro 31-8220 [2-{1-[3-(amidinothio)propyl]-1 H-indol-3-yl}-3-(1-methylindol-3-yl)-maleimide] (a protein kinase C inhibitor) had no effect. Furthermore, a small dose of tetraethylammonium and charybdotoxin [blockers of BK type Ca 2+-activated K + channel] abolished the U-46619-induced inhibition, but apamin (a blocker of SK-type Ca 2+-activated K + channel) had no effect. These results suggest that BK type Ca 2+-activated K + channels are involved in prostanoid TP receptor-mediated inhibition of electrically evoked noradrenaline release from the gastric sympathetic nerve terminals in rats.

M1965 Phosphatidylinositol-Specific Phospholipase C (PI-PLC) Mediates Bile Acid-Induced NADPH Oxidase NOX5-S Expression in Barrett's Esophageal Adenocarcinoma Cells

We have shown that NADPH oxidase NOX5-S (S = short, i.e. lacking EF-hand motifs at its N-terminal) is overexpressed in SEG1 esophageal adenocarcinoma cells associated with Barrett's esophagus (BE) and in BE with high-grade dysplasia. NOX5-S also contributes to increased cell proliferation and decreased apoptosis in SEG1 cells. We have also shown that low dose of bile acid increases cell proliferation through upregulation of NOX5-S. In the present study we examined the role of PI-PLC in bile acid-induced NOX5-S expression in SEG1-esophageal adenocarcinoma cells. In SEG1 cells, low dose of taurodeoxycholic acid (TDCA 10-11M, 24h) significantly increased NOX5-S expression and hydrogen peroxide (H2O2) production. TDCA-induced increase in NOX5-S expression and H2O2 production was significantly reduced by the PI-PLC inhibitor U73122, and the MAP kinase kinase inhibitor PD98059. In SEG1 cells transfected with NOX5-S luciferase reporter plasmid NOX5LP containing NOX5-S promoter fragment (-2501 to -1 from ATG), TDCA significantly increased the luciferase activity, an increase which was significantly decreased by U73122. In addition, TDCA significantly increased ERK2 phosporylation, which was significantly reduced by U73122. The data suggest that TDCA-induced NOX5-S expression may depend on sequential activation of PI-PLC and ERK MAP kinases. Isoforms of PI-PLC β1, β3, γ1 and γ2 were detectable by Western blot analysis, but not β2, β4, δ1 and δ2. Knockdown of PI-PLC γ2 with siRNA significantly decreased TDCA-induced NOX5-S protein expression and H2O2 production. The data suggest that PI-PLC γ2 may be involved in TDCA-induced NOX5-S expression. We conclude that in SEG1 cells low dose of TDCA increases NOX5S expression and H2O2 production. This increase in NOX5-S expression may depend on sequential activation of PI-PLC and ERK MAP kinases. Supported by NIDDK R21 DK073327-01.

Roles of brain phosphatidylinositol-specific phospholipase C and diacylglycerol lipase in centrally administered histamine-induced adrenomedullary outflow in rats

Recently, we reported that intracerebroventricularly (i.c.v.) administered histamine evokes the secretion of noradrenaline and adrenaline from adrenal medulla by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid cascade in the histamine-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by phospholipase A2 (PLA2)-dependent pathway or phospholipase C (PLC)/diacylglycerol lipase-dependent pathway. In the present study, histamine (27 nmol/animal, i.c.v.) -induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by U-73122 (PLC inhibitor) (10 and 100 nmol/animal, i.c.v.), ET-18-OCH3 (phosphatidylinositol-specific PLC inhibitor) (10 and 30 nmol/animal, i.c.v.) and RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 micromol/animal, i.c.v.). However, mepacrine (PLA2 inhibitor) (1.1 and 2.2 micromol/animal, i.c.v.) and D609 (phosphatidylcholine-specific PLC inhibitor) (30, 100 and 300 nmol/animal, i.c.v.) had no effect. These results suggest the involvement of brain phosphatidylinositol-specific PLC and diacylglycerol lipase in the centrally administered histamine-induced activation of the adrenomedullary outflow in rats.

Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G 1 phase of the cell cycle

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 min and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCβ 1 was detected with no change in the amount of PI-PLCβ 1b in nuclei isolated at 30 min and 11 h after the addition of serum. PI-PLC inhibitor ET-18-OCH 3 and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCβ 1b activity occur in serum-stimulated cells during G 1 phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.

Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

Soluble ULBP suppresses natural killer cell activity via down-regulating NKG2D expression

NKG2D is an activating receptor that is expressed on most natural killer (NK) cells and CD8 + T cells. MHC class I-related chain A(MICA) and UL16-binding protein (ULBP) 1, 2, and 3 are well-known ligands for NKG2D. Human gastric cancer cell lines, SNU216 and SNU638 cells which expressed UL16-binding protein (ULBP) were susceptible to NK cells in a NKG2D-dependent manner. However, SNU484 and SNU620 cells which had no ULBP on their surface were resistant to NK cells. ULBP 1, 2, and 3 are glycosylphosphatidylinositol (GPI)-anchored proteins which are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC). When SNU620 cells were treated with U73122, an inhibitor of PI-PLC, the surface expression of ULBP was elevated with increased NK susceptibility. Pre-incubating NK cells with culture supernatants of SNU620 or SNU638 cells, which contained soluble ULBP protein, reduced NK cell activity by decreasing surface expression of NKG2D in NK cells. Furthermore, recombinant ULBP-Fc induced the down-regulation of NKG2D expression in NK cells. Taken together, down-regulation of NKG2D by soluble ULBP provides a potential mechanism by which gastric cancer cells escape NKG2D-mediated attack by the immune cells.

Characterization of Phosphatidylinositol-Specific Phospholipase C (PI-PLC) from Liliumdaviddi Pollen

The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of [Ca(2+)](cyt) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.

Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca 2+-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T m for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 °C (well below the T m for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure.

Cysteine-less glycosylphosphatidylinositol-specific phospholipase C is inhibited competitively by a thiol reagent: evidence for glyco-mimicry by p-chloromercuriphenylsulphonate.

Glycosylphosphatidylinositol (GPI)-specific phospholipases are highly valuable for studying the structure and function of GPIs. GPI-specific phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus are the most widely studied of this class of phospholipases C. Inhibition of protein activity by thiol reagents is indicative of the participation of cysteine residues in biochemical events. The thiol reagent p-chloromercuriphenylsulphonate (pCMPS) inhibits T. brucei GPI-PLC, which has eight cysteine residues. Surprisingly, we found that the activity of B. cereus PI-PLC is also blocked by pCMPS, although the protein does not contain cysteine residues. Inhibition of B. cereus PI-PLC was reversed when pCMPS was size-separated from a preformed pCMPS.PI-PLC complex. In contrast, no activity was recovered when T. brucei GPI-PLC was subjected to a similar protocol. Equimolar beta-mercaptoethanol (beta-ME) reversed the inhibition of PI-PLC activity in a pCMPS.PI-PLC complex. For T. brucei GPI-PLC, however, ultrafiltration of the pCMPS.GI-PLC complex and addition of a large excess of beta-ME was necessary for partial recovery of enzyme activity. Thus T. brucei GPI-PLC is susceptible to inactivation by covalent modification with pCMPS, whereas PI-PLC is not. Kinetic analysis indicated that pCMPS was a competitive inhibitor of PI-PLC when a GPI was a substrate. Curiously, with phosphatidylinositol as substrate, inhibition was no longer competitive. These data suggest that pCMPS is a glyco-mimetic that occupies the glycan binding site of PI-PLC, from where, depending on the substrate, it inhibits catalysis allosterically or competitively.

Protective effects of protein kinase C during myocardial ischemia require activation of phosphatidyl-inositol specific phospholipase C

Background. Protein kinase C (PKC) activation during myocardial ischemia is thought to be cardioprotective. However, the mechanism of ischemia-induced PKC activation remains unclear. We hypothesized that ischemic PKC activation occurs through activation of phosphatidyl-inositol specific phospholipase C (PI-PLC) and protects the heart from ischemic injury.Methods. Isolated rabbit hearts were subjected to 20 minutes of normothermic ischemia and reperfusion. The PI-PLC inhibitor U73122 (0.5 μmol/L), its inactive analogue U73343 (0.5 μmol/L), or the PKC inhibitor chelerythrine (2 μmol/L) were given just before ischemia. Another group received U73122 plus the direct PKC activator phorbol 12-myristate-13-acetate (PMA, 10 pmol/L). Measurements included contractile function, intracellular calcium, PI-PLC activity, and translocation of PKC isoforms.Results. PI-PLC activity increased during myocardial ischemia and was inhibited by U73122. PI-PLC inhibition prevented the ischemic translocation of PKC-α, PKC-ε, and PKC-η, and impaired cardiac recovery and cytosolic calcium regulation without significant changes in energy metabolism. PMA restored both contractile function and PKC translocation pattern in U73122-treated hearts. Direct PKC inhibition with chelerythrine mimicked the effects of U73122.Conclusions. PI-PLC mediates PKC translocation during myocardial ischemia. Inhibition of PI-PLC or PKC activation, or both, during ischemia significantly impairs postischemic myocardial recovery.

Invited commentary

It is a privilege to review and comment on this interesting article. The experimental study presented by Dr Munakata and coworkers demonstrates the role of phosphatidyl-inositol specific phospholipase C (PI-PLC) in the myocardium exposed to a short period of normothermic ischemia using an isolated rabbit heart model. The authors used a specific inhibitor of PI-PLC, U73122 and its inactive analogue U73343 to modify the effect of PI-PLC to activate protein kinase C (PKC). The authors also were able to show reversal of the U73122 induced post-ischemic cardiac dysfunction by direct PKC activation with phorbol myristate acetate (PMA). This suggests that the myocardial protective effect of PI-PLC after ischemia occurs through PKC activation. Many studies have shown a significant myocardial protective effect of ischemic preconditioning [1Murry C.E. Richard V.J. Reimer K.A. Jennings R.B. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode.Circ Res. 1990; 66: 913-931Crossref PubMed Scopus (746) Google Scholar]. However, the mechanism and its clinical significance have been discussed for more than 10 years without a definitive answer. One promising candidate for the mechanism of ischemic preconditioning is activation of PKC by PI-PLC [2De Jonge H.W. Van Heugten H.A. Lamers J.M. Signal transduction by the phosphatidylinositol cycle in myocardium.J Mol Cell Cardiol. 1995; 27: 93-106Abstract Full Text PDF PubMed Scopus (62) Google Scholar] to protect the heart against impairment in intracellular calcium flux. The authors’ observation in this article that PI-PLC inhibition impairs diastolic calcium flux without a significant decrease in cellular ATP level is new and important. Further investigation using modifications of the cellular calcium transport system, eg, sarcoplasmic reticulum inhibition and membrane L-type calcium channel inhibition would be interesting and may clarify the mechanism underlying the intrinsic, antiischemic property of myocardium [3Liu D.M. Katnik C. Stafford M. Adams D.J. P2Y purinoceptor activation mobilizes intracellular Ca2+ and induces a membrane current in rat intracardiac neurones.J Physiol. 2000; 526: 287-298Crossref PubMed Scopus (25) Google Scholar]. Although the myocardial protective effect related to PKC activation during ischemia attracts much interest, many controversies remain. These controversies may due to the difference between species, experimental models, ischemic durations, and degree of ischemic insult. In addition, differences in the role of PKC-ε and PKC-α during short periods of ischemia is not fully understood. Researchers and clinicians must resolve these problems before this mechanism can be used to improve clinical myocardial protection. Further investigation is required. I congratulate these investigators for stimulating our thinking about the mechanism of the intrinsic protective property of the myocardium against ischemic reperfusion damage.

Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.

In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholine-specific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin alpha4beta1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC.

Synthesis of tritiated 1-octadecyl-phosphothiolyl-myo-[1-3H]-inositol. A new inhibitor of phosphatidylinositol-specific phospholipase-C

The title myo-[1-3H]-Inositol derivative was synthesized and characterized by tritium-N.M.R. The unlabelled compound proved a potent inhibitor of the cellular key enzyme phosphatidylinositol-specific phospholipase-C (PI-PLC) involved in the production of intracellular second messengers and its tritium derivative was necessary in order to fully understand the mechanism of inhibition. © 1997 John Wiley & Sons, Ltd.