Role Of Phospholipase C Research Articles

Phospholipase C: Diverse functions in plant biotic stress resistance and fungal pathogenicity.

Phospholipase C (PLC) generates various second messenger molecules and mediates phospholipid hydrolysis. In recent years, the important roles of plant and fungal PLC in disease resistance and pathogenicity, respectively, have been determined. However, the roles of PLC in plants and fungi are unintegrated and relevant literature is disorganized. This makes it difficult for researchers to implement PLC-based strategies to improve disease resistance in plants. In this comprehensive review, we summarize the structure, classification, and phylogeny of the PLCs involved in plant biotic stress resistance and fungal pathogenicity. PLCs can be divided into two groups, nonspecific PLC (NPC) and phosphatidylinositol-specific PLC (PI-PLC), which present marked differences in phylogenetic evolution. The products of PLC genes in fungi play significant roles in physiological activity and pathogenesis, whereas those encoded by plant PLC genes mediate the immune response to fungi. This review provides a perspective for the future control of plant fungal diseases.

Role of Phospholipase C in the Dopamine D2 Signaling Pathway in the Control of Lateral Cilia in Gill of Crassostrea virginica

Lateral cell cilia of gill of Crassostrea virginica are controlled by serotonergic‐dopaminergic innervations from their ganglia. Dopamine is the neurotransmitter causing cilio‐inhibition, serotonin cilio‐excitation. Previous work of our lab showed the post‐synaptic dopamine receptors present in lateral cells are D2‐type. Dopamine D2 receptors (D2R) are G protein‐coupled metabotropic receptors. Ligand binding to D2R splits its Gi/o protein into the Gβγ and Gαi/o subunits. Gαi/o inhibits adenylyl cyclase; while Gβγ opens GIRK channels, closes L‐type Ca2+ channels and activates phospholipase C (PLC). The inhibition of adenylyl cyclase and subsequent lowering of cAMP is known to slow lateral cilia; however, the role of PLC in the signaling pathway has not been well studied with respect to control of lateral cilia. Using immunohistofluorescence, our lab visualized PLC in lateral cell. In this study we hypothesized that the PLC component of the D2 pathway also plays a role in the cilio‐inhibition. To test this we utilized PLC activators and inhibitors on excised gill to determine their effects on lateral cilia. Cilia beating rates were measured using stroboscopic microscopy. In control gill, applying a dose response of dopamine (10−6 – 10−3M) is cilio‐inhibitory, decreasing beating rates of lateral cilia to 0. Applying m‐3M3FBS (10−5 – 10−4M), a PLC activator, caused a moderate decrease in cilia beating rates. Applying U73122 (10−4M), a phosphatidylinositol 4,5‐bisphosphate specific PLC inhibitor, did not alter cilia beating rates by itself, but did effect the dose response to dopamine (10−5 – 10−3M). In the presence of the inhibitor, while the 10−5M dose of dopamine caused the expected decrease in cilia beating rates, the addition of higher doses of dopamine was unable to reduce beating rates any further. As expected, the drug D609, a PLC inhibitor but specific for phosphatidylcholine, had no effects on cilia beating rates or the actions of dopamine. The results of this study supports our hypothesis that the PLC component of the D2R signal transduction pathway plays a role in the inhibitory control of gill lateral cilia. The results show that both components of the D2R pathway, the adenylyl cyclase component and the PLC component are involved in controlling lateral cilia beating. In humans the D2R signal transduction pathway is significantly involved in the actions of antidepressants, neuroleptics, and drugs of abuse, as well as implicated in neuropsychiatric and neurodegenerative disorders. This physiology study is helpful in furthering the understanding of the pathway and should help to generate further investigations.Support or Funding InformationThis work was supported in part by grant 2R25GM06003 of the Bridge Program of NIGMS, NIH grant K12GM093854‐07A1 IRACDA Program of Rutgers University and 604060048 of PSC‐CUNY.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arabidopsis PLC2 is involved in auxin‐modulated reproductive development

PhospholipaseC (PLC) is an enzyme that plays crucial roles in various signal transduction pathways in mammalian cells. However, the role of PLC in plant development is poorly understood. Here we report involvement of PLC2 in auxin-mediated reproductive development in Arabidopsis. Disruption of PLC2 led to sterility, indicating a significant role for PLC2 in reproductive development. Development of both male and female gametophytes was severely perturbed in plc2 mutants. Moreover, elevated auxin levels were observed in plc2 floral tissues, suggesting that the infertility of plc2 plants may be associated with increased auxin concentrations in the reproductive organs. We show that expression levels of the auxin reporters DR5:GUS and DR5:GFP were elevated in plc2 anthers and ovules. In addition, we found that expression of the auxin biosynthetic YUCCA genes was increased in plc2 plants. We conclude that PLC2 is involved in auxin biosynthesis and signaling, thus modulating development of both male and female gametophytes in Arabidopsis.

Dopamine D1 receptor signaling: does GαQ-phospholipase C actually play a role?

Despite numerous studies showing therapeutic potential, no central dopamine D1 receptor ligand has ever been approved, because of potential limitations, such as hypotension, seizures, and tolerance. Functional selectivity has been widely recognized as providing a potential mechanism to develop novel therapeutics from existing targets, and a highly biased, functionally selective D1 ligand might overcome some of the past limitations. SKF-83959 [6-chloro-3-methyl-1-(m-tolyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-7,8-diol] is reported to be a highly biased D1 ligand, having full agonism at D1-mediated activation of phospholipase C (PLC) signaling (via GαQ) and antagonism at D1-mediated adenylate cyclase signaling (via GαOLF/S). For this reason, numerous studies have used this compound to elucidate the physiologic role of D1-PLC signaling, including a novel molecular mechanism (GαQ-PLC activation via D1-D2 heterodimers). There is, however, contradictory literature that suggests that SKF-83959 is actually a partial agonist at both D1-mediated adenylate cyclase and β-arrestin recruitment. Moreover, the D1-mediated PLC stimulation has also been questioned. This Minireview examines 30 years of relevant literature and proposes that the data strongly favor alternate hypotheses: first, that SKF-83959 is a typical D1 partial agonist; and second, that the reported activation of PLC by SKF-83959 and related benzazepines likely is due to off-target effects, not actions at D1 receptors. If these hypotheses are supported by future studies, it would suggest that caution should be used regarding the role of PLC and downstream pathways in D1 signaling.

Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2–3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

Phospholipase C Activator m-3M3FBS Protects against Morbidity and Mortality Associated with Sepsis

Although phospholipase C (PLC) is a crucial enzyme required for effective signal transduction and leukocyte activation, the role of PLC in polymicrobial sepsis remains unclear. In this study, we show that the direct PLC activator m-3M3FBS treatment significantly attenuates vital organ inflammation, widespread immune cell apoptosis, and mortality in a mouse sepsis model induced by lethal cecal ligation and puncture challenge. Mechanistically, m-3M3FBS-dependent protection was largely abolished by pretreatment of mice with the PLC-selective inhibitor U-73122, thus confirming PLC agonism by m-3M3FBS in vivo. PLC activation enhanced the bactericidal activity and hydrogen peroxide production of mouse neutrophils, and it also enhanced the production of IFN-γ and IL-12 while inhibiting proseptic TNF-α and IL-1β production in cecal ligation and puncture mice. In a second model of sepsis, PLC activation also inhibited the production of TNF-α and IL-1β following systemic LPS challenge. In conclusion, we show that agonizing the central signal transducing enzyme PLC by m-3M3FBS can reverse the progression of toxic shock by triggering multiple protective downstream signaling pathways to maintain organ function, leukocyte survival, and to enhance microbial killing.

Roles of Phospholipase C Isozymes in Organogenesis and Embryonic Development

Phosphoinositide metabolism is an important intracellular signaling system that regulates a variety of cellular functions. Phospholipase C (PLC) is a key enzyme in this system. Recent studies on genetically manipulated mice have clarified the functions of PLC in vivo. This review focuses on the roles of PLC in organogenesis and embryonic development.

Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.

In Skeletal Muscle Advanced Glycation End Products (AGEs) Inhibit Insulin Action and Induce the Formation of Multimolecular Complexes Including the Receptor for AGEs

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.

The variety of cytosolic calcium responses and possible roles of PLC and PKC

A model of ligand-induced intracellular calcium (Ca2+) responses incorporating phospholipase C (PLC) and protein kinase C (PKC) is developed for the purpose of understanding the mechanisms underlying the observed temporal patterns of intracellular calcium (Ca2+i) under sustained agonist stimulation. Some studies have suggested that inhibition of ligand receptors and PLC by PKC could generate sinusoidal Ca2+ oscillations, while PKC-independent Ca2+-induced Ca2+ release (CICR) via IP3-gated Ca2+ channels on the endoplasmic reticulum (ER) is believed to be responsible for baseline spiking. However, some evidence also indicates that baseline spiking can be observed under high-PKC activity, or under low-PKC activity with low agonist stimulus, as well. Insight into the basis of these observations regarding the role of PKC in Ca2+i response patterns can be gained by developing and analyzing a mathematical model of Ca2+i responses. We do this herein and find that (1) interaction of CICR and the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump is enough to generate both types of Ca2+i oscillations, (2) there exist four possible Ca2+i response patterns under sustained agonist stimulus: a sub-threshold response (SR), baseline spiking, sinusoidal oscillations (SO) and transient with plateau, and (3) the IP3 concentration, which is controlled by the strength of the interaction between PKC and PLC, can be used to predict the Ca2+i response patterns. From this analysis we conclude that the different patterns of Ca2+i oscillations can be understood as a generic consequence of the interactions between CICR via the IP3-gated Ca2+ channels in response to changes in the level of IP3, and re-uptake into the ER/SR via the SERCA pump. PKC, in conjunction with PLC, can act as a switch between different Ca2+i response patterns by modulating the cytosolic IP3 level, which determines the Ca2+i patterns.

Does Phospholipase C Mediate Muscarinic Receptor-Induced Rat Urinary Bladder Contraction?

Muscarinic acetylcholine receptors, particularly M(3) receptors, are physiologically the most important mechanism to induced urinary bladder smooth muscle contraction. Their prototypical signaling response is a stimulation of phospholipase C (PLC), and this also has been shown in the urinary bladder. Nevertheless, it has remained controversial whether PLC signaling mediates bladder contraction induced by muscarinic receptor agonists. Studies in favor and against a role for PLC differed in their experimental protocol (single versus repeated concentration-response curves within a single preparation) and in the PLC inhibitors that have been used. We have now tested whether previous differential conclusions regarding a role for PLC are related to inhibitors and/or experimental protocols. In a single curve protocol, U-73,122 [1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione] did not attenuate carbachol responses. In a repeated curve protocol, ET-18-OCH(3) (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine) lacked significant inhibition relative to vehicle time controls. In contrast, D609 (O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt) depressed maximal carbachol effects but also nonspecifically inhibited contraction induced by KCl. Neomycin did not affect the carbachol-induced rat urinary bladder contraction. We conclude that previously reported differences relate to the use of inhibitors rather than experimental protocols and that the overall data do not support a role for PLC in M(3) muscarinic receptor-mediated rat bladder contraction.

Involvement of phospholipase C in endothelin 1–induced stimulation of Ca++ channels and basilar artery contraction in rabbits

Endothelin 1 (ET-1) is a major cause of cerebral vasospasm after subarachnoid hemorrhage (SAH), and extracellular Cal++ influx plays an essential role in ET-1-induced vasospasm. The authors recently demonstrated that ET-1 activates two types of Ca"-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Cal++ channel (SOCC) in vascular smooth-muscle cells located in the basilar arteries (BAs) of rabbits. In the present study, they investigate the effects of phospholipase C (PLC) on ET-1-induced activation of these Ca++ channels and BA contraction by using the PLC inhibitor U73122. Methods. To determine which Cal++ channels are activated via a PLC-dependent pathway, these investigators monitored the intracellular free Cal++ concentration ([Ca++]i). The role of PLC in ET-1-induced vascular contraction was examined by performing a tension study of rabbit BA rings. The U73122 inhibited the ET-1-induced transient increase in [Ca++]i, which resulted from mobilization of Ca++ from the intracellular store. Phospholipase C also inhibited ET-1-induced extracellular Ca++ influx through the SOCC and NSCC-2, but not through the NSCC-1. The U73122 inhibited the ET-1-induced contraction of the rabbit BA rings, which depended on extracellular Cal++ influx through the SOCC and NSCC-2. Conclusions. These results indicate the following. (1) The SOCC and NSCC-2 are stimulated by ET-1 via a PLC-dependent cascade whereas NSCC-1 is stimulated via a PLC-independent cascade. (2) The PLC is involved in the ET-1-induced contraction of rabbit BA rings, which depends on extracellular Ca++ influx through the SOCC and NSCC-2.

Permselective Dysfunction of Podocyte-Podocyte Contact upon Angiotensin II Unravels the Molecular Target for Renoprotective Intervention

Ameliorating the function of the glomerular barrier to circulating proteins by blocking angiotensin II (Ang II) translates into less risk of progression toward end-stage renal failure in diabetic and nondiabetic nephropathies. However, the mechanisms underlying this barrier protection are not clear. Specialized contacts between adjacent podocytes are major candidate targets, and the actin cytoskeleton is emerging as a regulatory element. Here, we present data demonstrating that Ang II induced reorganization of F-actin fibers and redistribution of zonula occludens-1 (ZO-1) that is physically associated with actin in murine podocytes. These effects were paralleled by increased albumin permeability across podocyte monolayers. The F-actin stabilizer jasplakinolide prevented both ZO-1 redistribution and albumin leakage, suggesting that actin cytoskeleton rearrangement is instrumental to podocyte permselective dysfunction induced by Ang II. Changes in both F-actin and ZO-1 patterns were confirmed in glomeruli of rat isolated perfused kidneys on short infusion of Ang II, leading to increased protein excretion. Podocyte dysfunction was mediated by Ang II type 1 receptor and was partly dependent on Src kinase-phospholipase C activation. These data demonstrate that strategies aimed at stabilizing podocyte-podocyte contacts and targeting the relevant intracellular signal transduction are crucial to renoprotection.

Dual Phospholipase C/Diacylglycerol Requirement for Protein Kinase D1 Activation in Lymphocytes

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.

Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation.

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

Nod factor‐induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation

SummaryRhizobium‐secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [32P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

A novel phospholipase C- and cAMP-independent positive inotropic mechanism via a P2 purinoceptor.

Although ATP, acting through a P2 purinoceptor, can stimulate a pronounced positive inotropic effect in cardiac ventricular myocytes, the receptor-effector mechanism that underlies this stimulatory cardiac action is not well understood. The objectives of the present study were to develop the cultured chick embryo ventricular myocytes as a novel model for the cardiac P2 purinoceptor and to determine the mechanism underlying its positive inotropic effect. ATP caused an 89 +/- 8.9% (n = 14 cells) increase in the myocyte contractility, with an efficacy and potency order of ATP > ADP > AMP >> adenosine. 2-Methylthio-ATP (2-MeS-ATP) but not alpha,beta-methylene-ATP was able to stimulate myocyte contractility, with a maximal increase of 54 +/- 2.6% (n = 11 cells). Although UTP potently stimulates phosphoinositide hydrolysis, it had an only modest positive inotropic effect (27 +/- 7% maximal increase; n = 8 cells). In contrast to previous suggestions, the 2-MeS-ATP-stimulated positive inotropic response does not require the action of phospholipase C (PLC), such as that of the inositol phosphates; the UTP effect on contractility appears to be mediated via the 2-MeS-ATP-sensitive P2 receptor. The PLC inhibitor U-73122 had no effect on the 2-MeS-ATP-stimulated increase in contractility, providing further evidence against a role for PLC in the inotropic effect of 2-MeS-ATP. An adenosine 3',5'-cyclic monophosphate-independent Ca2+ entry-stimulating mechanism appears to underlie a direct coupling of the receptor to stimulation of the myocyte contractility. This new PLC- and adenosine 3',5'-cyclic monophosphate-independent positive inotropic mechanism represents a target for developing novel positive inotropic therapeutics.

Rapid Ca2+-mediated activation of Rap1 in human platelets.

Rap1 is a small, Ras-like GTPase whose function and regulation are still largely unknown. We have developed a novel assay to monitor the active, GTP-bound form of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of RalGDS (RBD). Stimulation of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RBD in vitro. Binding to RBD increased from undetectable levels in resting platelets to >50% of total Rap1 within 30 s after stimulation. An increase in the intracellular Ca2+ concentration is both necessary and sufficient for Rap1 activation since it was induced by agents that increase intracellular Ca2+ and inhibited by a Ca2+-chelating agent. Neither inhibition of translocation of Rap1 to the cytoskeleton nor inhibition of platelet aggregation affected thrombin-induced activation of Rap1. In contrast, prostaglandin I2 (PGI2), a strong negative regulator of platelet function, inhibited agonist-induced as well as Ca2+-induced activation of Rap1. From our results, we conclude that Rap1 activation in platelets is an important common event in early agonist-induced signalling, and that this activation is mediated by an increased intracellular Ca2+ concentration.

Activity of phospholipase C and release of prostaglandin F2 by endometrial tissue from ewes during the oestrous cycle and early pregnancy

Oxytocin appears to play an important role in regulating uterine secretion of prostaglandin F2 alpha (PGF2 alpha) in sheep. Changes in uterine secretion of PGF2 alpha throughout the oestrous cycle and early pregnancy may be due to changes in the intracellular regulatory pathways that control synthesis of PGF2 alpha in response to oxytocin. In this experiment, caruncular endometrial tissue was collected from ewes throughout the oestrous cycle and early pregnancy. Endometrial tissue was incubated in vitro to assess release of PGF2 alpha and activity of phospholipase C (PLC) in response to oxytocin. Release of PGF2 alpha in the presence of arachidonic acid was used to assess the activity of prostaglandin H2 endoperoxide synthase (PGS). In non-pregnant ewes, oxytocin stimulated release of PGF2 alpha from endometrial tissue collected on days 14 and 16, but not on days 4-7, 10 or 12 after oestrus. This coincided with times when oxytocin stimulated the activity of PLC. Release of PGF2 alpha was enhanced by the addition of arachidonic acid to tissues collected on days 12, 14 and 16 after oestrus. As with tissue from nonpregnant ewes, oxytocin could stimulate release of PGF2 alpha on days 14 and 16 of early pregnancy. Yet, oxytocin had no effect on activity of PLC in tissue from pregnant ewes. Release of PGF2 alpha in the presence of arachidonic acid by tissue from pregnant ewes was similar to that in nonpregnant ewes at comparable times after oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)

A suggested role for phospholipase C in the immune response of Galleria mellonella

The immune response of Galleria mellonella is characterized by a variety of biological activities, including the production of antibacterial products (J. S. Chadwick and G. B. Dunphy, 1986, in “Hemocytic and Humoral Immunity in Arthropods,” A. G. Gupta, Ed., Wiley, New York). Recently, a novel inducible hemolytic activity (D. J. Phipps, R. G. Leeder, J. S. Chadwick, and W. P. Aston, Dev. Comp. Immunol. 13, 103-l 11, 1989) and a novel antitumor activity (J. K. C. Chiu, W. P. Aston, and J. S. Chadwick, Abstr. 116, “XXII Ann. Mtg. Sot. Invertebr. Pathol.,” College Park, Maryland, 1989) have been demonstrated. In addition, phospholipase C (PLC) activity has been found to increase in the cell-free hemolymph (CFH) of G. mellonella following injection of an immunizing dose (2.5 pg/larva) (P. J. De Verno, M.Sc. Thesis, Queen’s University, Kingston, Ontario, Canada, 1983) of lipopolysaccharide (LPS) derived from Salmonella typhimurium. The PLC activity was determined by a calorimetric assay using p-nitrophenylphosphorylcholine (NPPC) as the specific substrate (S. Kurioka, J. Biochem. 63, 678-680,1968; S. Kurioka and M. Matsuda, Anal. Biochem. 75, 281-289, 1976; R. G. Sheikhnejad and P. N. Srivastava, J. Biol. Chem. 261, 7544-7549, 1986; T. P. Yoshino, J. Invertebr. Pathol. 51, 32-40, 1988). The injection of endotoxin-free water did not increase the PLC activity over a 72-h period. However, LPS caused a significant increase in PLC activity, apparently reaching the maximum at 24 h postinjection. The increase and subsequent decline of PLC activity in the CFH of G. mellonella following LPS injection was found to parallel the profile of the protective immune response (Fig. 1). The isolated hemolytically active material (HAM), which can be induced by either formalized Pseudomonas aeruginosa or LPS, did not possess any PLC activity (D. J. Phipps, R. G. Leeder, J. S. Chadwick, and W. P. Aston, Dev. Comp. Zmmunol. 13, 103-111, 1989). This suggests that the PLC enzyme is a different entity from the HAM. It also differs from an LPS-induced hemolymph-derived cytotoxic activity (for human tumor cell lines), which peaks at 2 h postinjection of LPS, as described previously (J. K. C. Chiu, W. P. Aston, and J. S. Chadwick, Abstr. 116, “XXII Ann. Mtg. Sot. Invertbr. Pathol.,” College Park, Maryland, 1989). The mechanism for the LPS-induced increase in PLC activity in the hemoiymph of G. mellonella is not clearly understood. The presence of a high level of such an enzyme in the hemolymph during the height of the protective immune response suggests a role for PLC in this process. It has been shown that total hemocyte counts in G. mellonella declined rapidly upon injection of LPS (G. B. Dunphy, D. B. Morton, and J. M. Chadwick, J. Invertebr. Pathol. 47, 56-64, 1986; G. B. Dunphy and J. M. Webster, J. Gen. Microbial. 134, 1017-1028, 1988). This may be due to the damage and eventual lysis of hemocytes caused by LPS or autolysis. The membraneand/or cytosol-associated PLC enzyme might then be released into the hemolymph via cell lysis or secretion. Several investigators have reported earlier that the granular contents may be released from isolated hemocytes via exocytosis following LPS stimulation (M. W. Johansson and K. Soderhall, J. Comp. Physiol. B, 156, 175-181, 1985; K. Soderhall, V. J. Smith, and M. W. Johansson, Cell Tissue Res. 245, 43-49) 1986. It has also been shown that treatment of mammalian tissues and cells with exogenous PLC (external to cells) led to the sub-