Phospholipase C Inhibitor Research Articles

Mechanisms of vasorelaxation induced by oleoylethanolamide in the rat small mesenteric artery

The actions of the anandamide-like mono-unsaturated fatty acid oleoylethanolamide (OEA) were first linked to satiety and control of food intake and recently reported to relax resistance vessels. This study characterizes its vasorelaxant mechanisms. Vasorelaxation to OEA were assessed in third order branches of rat superior mesenteric artery using a wire myograph. The roles of the endothelium, KCa channels, perivascular sensory nerves, NO, cannabinoid receptors, and the phospholipase C (PLC)/inositol trisphosphate (InsP3) and RhoA/ROCK signalling pathways, were assessed. OEA caused concentration- and endothelium-dependent vasorelaxation (pEC50=6.7±0.1, Rmax=93.1±2.5%). L-NAME greatly reduced the response (residual relaxation of only 24.6±12.8%). Capsaicin and pertussis toxin significantly reduced the vasorelaxation. Precontraction with KCl abolished the response. TRAM-34 had no effect, but both iberiotoxin and apamin+charybdotoxin markedly shifted the OEA concentration–response curve to the right (∼5-fold). O-1918 but not rimonabant attenuated the vasorelaxation. Both the CB1 receptor antagonist, AM251 and the CB2 receptor antagonist, AM630, given alone or in combination, reduced the response to OEA. Inhibition of PLC by U73122, ROCK by Y-27632 and antagonism of inositol trisphosphate (InsP3) receptors by 2-APB abolished OEA vasorelaxation. OEA vasorelaxation involves an endothelial site of action but not the known cannabinoid receptors. It involves Ca2+ released from InsP3-sensitive endothelial stores by mechanisms involving RhoA kinase and phospholipase C. It is likely that the released Ca2+ causes NO generation and opening of mainly large-conductance KCa channels. This study demonstrates a possible novel endothelial target that might be important in the control of regional blood flow induced by this lipid molecule.

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.

Lysophosphatidylcholine-induced cytotoxicity in osteoblast-like MG-63 cells: Involvement of transient receptor potential vanilloid 2 (TRPV2) channels

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Accordingly, atherogenic determinants such as oxidized low density lipoprotein (OxLDL) particles have been shown to alter bone cell functions. In this work, we investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells. Cell viability was reduced by lysoPC in a concentration-dependent manner with a LC50 of 18.7±0.7 μM. LysoPC-induced cell death was attributed to induction of both apoptosis and necrosis. Since impairment of intracellular calcium homeostasis is often involved in mechanism of cell death, we determined the involvement of calcium in lysoPC-induced cytotoxicity. LysoPC promoted a rapid and transient increase in intracellular calcium attributed to mobilization from calcium stores, followed by a sustained influx. Intracellular calcium mobilization was associated to phospholipase C (PLC)-dependent mobilization of calcium from the endoplasmic reticulum since inhibition of PLC or calcium depletion of reticulum endoplasmic with thapsigargin prevented the calcium mobilization. The calcium influx induced by lysoPC was abolished by inhibition of transient receptor potential vanilloid (TRPV) channels with ruthenium red whereas gadolinium, which inhibits canonical TRP (TRPC) channels, was without effect. Accordingly, expression of TRPV2 and TRPV4 were shown in MG-63 cells. The addition of TRPV2 inhibitor Tranilast in the incubation medium prevent the calcium influx triggered by lysoPC and reduced lysoPC-induced cytotoxicity whereas TRPV4 inhibitor RN 1734 was without effect, which confirms the involvement of TRPV2 activation in lysoPC-induced cell death.

Lipid peroxidation is essential for phospholipase C activity and IP3 related calcium signal

Reactive oxygen species (ROS) are produced in enzymatic and non-enzymatic reactions and have important roles in cell signalling but also detrimental effects. ROS-induced damage has been implicated in a number of neurological diseases; however, antioxidant therapies targeting brain diseases have been unsuccessful. Such failure might be related to inhibition of ROS-induced signalling in the brain. Using direct kinetic measures of lipid peroxidation in astrocytes and measurements of lipid peroxidation products in brain tissue, we here show that phospholipase C (PLC) preferentially cleaves oxidised lipids. Because of this, an increase in the rate of lipid peroxidation leads to increased Ca(2+) release from endoplasmic reticulum (ER) stores in response to physiological activation of purinoreceptors with ATP. Both vitamin E and its water-soluble analogue Trolox, potent ROS scavengers, were able to suppress PLC activity, therefore dampening intracellular Ca(2+) signalling. This implies that antioxidants can compromise intracellular Ca(2+) signalling through inhibition of PLC, and that PLC plays a dual role - signalling and antioxidant defence.

Depletion of PtdIns(4,5)P2 underlies retinal degeneration in Drosophila trp mutants

The prototypical transient receptor potential (TRP) channel is the major light-sensitive, and Ca(2+)-permeable channel in the microvillar photoreceptors of Drosophila. TRP channels are activated following hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] by the key effector enzyme phospholipase C (PLC). Mutants lacking TRP channels undergo light-dependent retinal degeneration, as a consequence of the reduced Ca(2+) influx. It has been proposed that degeneration is caused by defects in the Ca(2+)-dependent visual pigment cycle, which result in accumulation of toxic phosphorylated metarhodopsin-arrestin complexes (MPP-Arr2). Here we show that two interventions, which prevent accumulation of MPP-Arr2, namely rearing under red light or eliminating the C-terminal rhodopsin phosphorylation sites, failed to rescue degeneration in trp mutants. Instead, degeneration in trp mutants reared under red light was rescued by mutation of PLC. Degeneration correlated closely with the light-induced depletion of PtdIns(4,5)P₂ that occurs in trp mutants due to failure of Ca(2+)-dependent inhibition of PLC. Severe retinal degeneration was also induced in the dark in otherwise wild-type flies by overexpression of a bacterial PtdInsPn phosphatase (SigD) to deplete PtdIns(4,5)P₂. In degenerating trp photoreceptors, phosphorylated Moesin, a PtdIns(4,5)P₂-regulated membrane-cytoskeleton linker essential for normal microvillar morphology, was found to delocalize from the rhabdomere and there was extensive microvillar actin depolymerisation. The results suggest that compromised light-induced Ca(2+) influx, due to loss of TRP channels, leads to PtdIns(4,5)P₂ depletion, resulting in dephosphorylation of Moesin, actin depolymerisation and disintegration of photoreceptor structure.

Characteristics of the Cholecystokinin-Induced Depolarization of Pacemaking Activity in Cultured Interstitial Cells of Cajal from Murine Small Intestine

Background/Aims: In this study, we studied the effects of cholecystokinin (CCK) on pacemaker potentials in cultured interstitial cells of Cajal (ICCs) from mouse small intestine using the whole cell patch clamp technique. Methods: ICCs are pacemaker cells that exhibit periodic spontaneous depolarization, which is responsible for the production of slow waves in gastrointestinal smooth muscle, and generate periodic pacemaker potentials in current-clamp mode. Results: Exposure to CCK (100 nM-5 µM) decreased the amplitudes of pacemaker potentials and depolarized resting membrane potentials. To identify the type of CCK receptors involved in ICCs, we examined the effects of CCK agonists and found that the addition of CCK₁ agonist (A-71323, 1 µM) depolarized resting membrane potentials, whereas exposure to CCK₂ agonist (gastrin , 1 µM) had no effect on pacemaker potentials. To confirm these results, we examined the effects of CCK antagonists and found that pretreatment with CCK₁ antagonist (SR 27897, 1 µM) blocked CCK-induced effects. However, pretreatment with CCK₂ antagonist (LY 225910, 1 µM) did not. Furthermore, intracellular GDP_βS suppressed CCK-induced effects. To investigate the involvements of phospholipase C (PLC), protein kinase C (PKC), and protein kinase A (PKA) in the effects of CCK in cultured ICCs, we used U-73122 (an active PLC inhibitor), chelerythrine (a PKC inhibitor), SQ-22536 (an inhibitor of adenylate cyclase), or mPKAI (an inhibitor of myristoylated PKA). All inhibitors blocked the CCK-mediated effects on pacemaker potentials. In addition, we found that transient receptor potential classical 5 (TRPC5) channel was involved in CCK-activated currents in cultured ICCs. Conclusion: These results suggest that the CCK induced depolarization of pacemaking activity occurs in a G-protein-, PLC-, PKC-, and PKA-dependent manner via CCK₁ receptor and TRPC5 channel is a candidate for CCK-activated currents in cultured ICCs in murine small intestine. Therefore, the ICCs are targets for CCK and their interaction can affect intestinal motility.

Phospholipase C is Required for GαQ Protein-Coupled Receptor Mediated Task Channel Inhibition

TWIK-related acid sensitive K+ (TASK) channels belong to the K2P channel family and contribute significantly to the background conductance in various cell types, e.g. to IK,SO in cerebellar granule cells. It is known that stimulation of Gq-protein coupled receptors (GqPCRs) causes strong and reversible inhibition of TASK channels. Yet, the underlying signaling cascade is still controversial: Both, the inhibition of TASK by Gαq-protein via a direct molecular interaction, or, alternatively, by signals downstream of phospholipase C (PLC) activation have been proposed. PLC mediates the hydrolysis of phosphoinositide-4,5-bisphosphate (PI(4,5)P2), thereby producing the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). Here, we examine the requirement of PLC in GqPCR-signaling for TASK-inhibition.Whole cell patch clamp experiments were performed on CHO cells expressing TASK3 channels while PLC activity was modulated pharmacologically. PLC was activated through the application of the PLC-activator m-3M3FBS causing TASK3 current suppression. In contrast, Gq-mediated TASK3 inhibition by activation of muscarinic M1 receptor was abolished by the PLC inhibitor U73122. The efficiency of both pharmacological manipulations of PLC was verified with the DAG-sensor PKCγ-C1 and the PI(4,5)P2-sensor PLCδ1-PH, monitored in total internal reflection fluorescence microscopy.These data suggest that activation of PLC is an indispensable step in GqPCR – TASK signaling and are inconsistent with the hypothesis that direct Gαq-interaction mediates TASK current inhibition.This work was supported by DFG grant OL 240/3-1 (FOR 1086) to DO.Chen, X. et al., 2006, PNAS 103, 3422-7.Chemin, J. et al., 2003, EMBO J. 22, 5403-11.Lindner, M. et al., 2011, J. Physiol. 589, 3149-62.

Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP3pathway and the ryanodine receptor

The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P < 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P < 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 ± 8 to 118 ± 22% of control (P < 0.01). Combining inhibition of IP(3) and RyR was not additive. G(i) inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 ± 12 to 41 ± 8% of control, P < 0.001). We conclude stimulating JG cell CaSR activates G(q), initiating the PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

Important role of PLC-γ1 in hypoxic increase in intracellular calcium in pulmonary arterial smooth muscle cells

An increase in intracellular calcium concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-γ1 protein in PAs and PASMCs, respectively. The activity of PLC-γ1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-γ1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates the hypoxic vasoconstriction in PAs and hypoxic increase in [Ca(2+)](i) in PASMCs. PLC-γ1 knockdown suppresses its protein expression and the hypoxic increase in [Ca(2+)](i). Hypoxia remarkably increases inositol 1,4,5-trisphosphate (IP(3)) production, which is blocked by U73122. The IP(3) receptor (IP(3)R) antagonist 2-aminoethoxydiphenyl borate (2-APB) or xestospongin-C inhibits the hypoxic increase in [Ca(2+)](i). PLC-γ1 knockdown or U73122 reduces H(2)O(2)-induced increase in [Ca(2+)](i) in PASMCs and contraction in PAs. 2-APB and xestospongin-C produce similar inhibitory effects. In conclusion, our findings provide novel evidence that hypoxia activates PLC-γ1 by increasing RISP-dependent mitochondrial ROS production in the complex III, which causes IP(3) production, IP(3)R opening, and Ca(2+) release, playing an important role in hypoxic Ca(2+) and contractile responses in PASMCs.

Phospholipase C mediates (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-, but not lysergic acid diethylamide (LSD)-elicited head bobs in rabbit medial prefrontal cortex

The phenethylamine and indoleamine classes of hallucinogens demonstrate distinct pharmacological properties, although they share a serotonin2A (5-HT2A) receptor mechanism of action (MOA). The 5-HT2A receptor signals through phosphatidylinositol (PI) hydrolysis, which is initiated upon activation of phospholipase C (PLC). The role of PI hydrolysis in the effects of hallucinogens remains unclear. In order to better understand the role of PI hydrolysis in the MOA of hallucinogens, the PLC inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to study the effects of two hallucinogens, the phenethylamine, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and the indoleamine, lysergic acid diethylamide (LSD). PI hydrolysis was quantified through release of [3H]inositol-4-phosphate from living rabbit frontocortical tissue prisms. Head bobs were counted after hallucinogens were infused into the medial prefrontal cortex (mPFC) of rabbits. Both DOI and LSD stimulated PI hydrolysis in frontocortical tissue through activation of PLC. DOI-stimulated PI hydrolysis was blocked by 5-HT2A/2C receptor antagonist, ketanserin, whereas the LSD signal was blocked by 5-HT2B/2C receptor antagonist, SB206553. When infused into the mPFC, both DOI- and LSD-elicited head bobs. Pretreatment with U73122 blocked DOI-, but not LSD-elicited head bobs. The two hallucinogens investigated were distinct in their activation of the PI hydrolysis signaling pathway. The serotonergic receptors involved with DOI and LSD signals in frontocortical tissue were different. Furthermore, PLC activation in mPFC was necessary for DOI-elicited head bobs, whereas LSD-elicited head bobs were independent of this pathway. These novel findings urge closer investigation into the intracellular mechanism of action of these unique compounds.

Involvement of presynaptic voltage-dependent Kv3 channel in endothelin-1-induced inhibition of noradrenaline release from rat gastric sympathetic nerves

We previously reported that two types of K+ channels, the BK type Ca2+-activated K+ channel coupled with phospholipase C (PLC) and the voltage-dependent K+ channel (Kv channel), are, respectively, involved in the prostanoid TP receptor- and muscarinic M2 receptor-mediated inhibition of noradrenaline (NA) release from rat gastric sympathetic nerves. In the present study, therefore, we examined whether these K+ channels are involved in endothelin-1-induced inhibition of NA release, using an isolated, vascularly perfused rat stomach. The gastric sympathetic postganglionic nerves around the left gastric artery were electrically stimulated twice at 2.5Hz for 1min, and endothelin-1 was added during the second stimulation. Endothelin-1 (1, 2 and 10nM) dose-dependently inhibited gastric NA release. Endothelin-1 (2nM)-induced inhibition of NA release was neither attenuated by PLC inhibitors [U-73122 (3μM) and ET-18-OCH3 (3μM)] nor by Ca2+-activated K+ channel blockers [charybdotoxin (0.1μM) (a blocker of BK type K+ channel) and apamin (0.3μM) (a blocker of SK type K+ channel)]. The endothelin-1-induced inhibitory response was also not attenuated by α-dendrotoxin (0.1μM) (a selective inhibitor of Kv1 channel), but abolished by 4-aminopyridine (20μM) (a selectively inhibitory dose for Kv3 channel). These results suggest the involvement of a voltage-dependent Kv3 channel in the endothelin-1-induced inhibition of NA release from the gastric sympathetic nerves in rats.

Adenosine A3 receptor-induced proliferation of primary human coronary smooth muscle cells involving the induction of early growth response genes

In human coronary smooth muscle cells adenosine A2B receptors mediate the inhibition of platelet-derived growth factor (PDGF)-induced proliferation via induction of the transcription factor nuclear receptor subfamily 4, group A, member 1 (NR4A1). In the absence of PDGF, adenosine analogues increased proliferation. In the present study we characterised the adenosine receptor mediating the increase in proliferation of these cells and identified involved transcription factors. Cultured human coronary smooth muscle cells were treated with selective A3 receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The A3 receptor agonist 2-chloro-IB-MECA (2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide) enhanced the number of human coronary smooth muscle cells with a half-maximal concentration of only 1nM. 2-chloro-IB-MECA also increased the expression of the transcription factors early growth response protein (EGR)2 and EGR3, but not of EGR1, NR4A1, NR4A2 and NR4A3. The responses to 2-chloro-IB-MECA were blocked by two A3 receptor antagonists, MRS1523 (3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate; 10–300μM) and VUF 5574 (N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea; 1–100nM, as well as by the phospholipase C-inhibitor U73343 (0.2μM). Small interfering RNA directed against EGR2 and EGR3 abolished the increases in proliferation induced by 2-chloro-IB-MECA. In summary, this is the first report demonstrating a coupling of smooth muscle adenosine A3 receptors to increases in proliferation of human coronary smooth cells by the activation of phospholipase C and an induction of the transcriptions factors EGR2 and EGR3. The results facilitate the understanding of the role of adenosine A3 receptors in the cardiovascular system.

Characterization of preptin-induced insulin secretion in pancreatic β-cells

We aimed to characterize the effects of preptin on insulin secretion at the single-cell level, as well as the mechanisms underlying these changes, with respect to regulation by intracellular Ca(2+) [Ca(2+)](i) mobilization. This study assessed the effect of preptin on insulin secretion and investigated the link between preptin and the phospholipase C (PLC)/protein kinase C (PKC) pathway at the cellular level using fura-2 pentakis(acetoxymethyl) ester-loaded insulin-producing cells (Min 6 cells). Our results demonstrate that preptin promotes insulin secretion in a concentration-dependent manner. Using a PLC inhibitor (chelerythrine) or a PKC inhibitor (U73122) resulted in a concentration-dependent decrease in insulin secretion. Also, preptin mixed with IGF2 receptor (IGF2R) antibodies suppressed insulin secretion in a dose-dependent manner, which indicates that activation of IGF2R is mediated probably because preptin is a type of proIGF2. In addition, preptin stimulated insulin secretion to a similar level as did glibenclamide. The activation of PKC/PLC by preptin stimulation is highly relevant to the potential mechanisms for increase in insulin secretion. Our results provide new insight into the insulin secretion of preptin, a secreted proIGF2-derived peptide that can induce greater efficacy of signal transduction resulting from PLC and PKC activation through the IGF2R.

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.

Involvement of phospholipases C and D in the defence responses of riboflavin-treated tobacco cells

Riboflavin is an activator of defence responses in plants that increases resistance against diseases caused by fungal, oomycete, bacterial and viral pathogens. However, the mechanisms driving defence activation by riboflavin are poorly understood. We investigated the signal transduction pathways of phospholipase C (PLC) and phospholipase D (PLD) in tobacco (Nicotiana tabacum) suspension cells using a pharmacological approach to confirm whether riboflavin-mediated activation of the defence response is dependent on both PLC and PLD. The expression patterns analysed by quantitative reverse transcription-polymerase chain reaction demonstrated that the tobacco PLC and PLD gene families were differentially expressed in riboflavin-treated tobacco cells. PLC and PLD expression accompanied defence responses including the expression of defence response genes (PAL, PR-1a and PR-1b), the production of hydrogen peroxide and the accumulation of the phytoalexin scopoletin in tobacco cells treated with riboflavin. These defence responses were significantly inhibited in the presence of the PLC inhibitor U73122 and the PLD inhibitor 1-butanol; however, inhibitor analogues had no effect. Moreover, treating tobacco cells with phosphatidic acid, a signalling molecule produced by phospholipase catalysis, induced the accumulation of the phytoalexin scopoletin and compensated for the suppressive effects of U73122 and 1-butanol on riboflavin-induced accumulation of the phytoalexin. These results offer pharmacological evidence that PLC and PLD play a role in riboflavin-induced defences of tobacco.

Abstract 4328: Specific epidermal growth factor receptor autophosphorylation sites promote epithelial cell chemotaxis and restitution

Abstract BACKGROUND: Cell migration is required for the physiological processes of wound repair, resolution of inflammation, and organogenesis, but is also involved in the pathology of tumor invasion. Thus, investigation of the regulatory mechanisms of cell migration is important for both understanding basic intestinal biology and identifying potential therapeutic targets for disorders such as inflammatory bowel diseases and colon cancer. HYPOTHESIS: Upon ligand binding, epidermal growth factor (EGF) receptor (R) autophosphorylates on c-terminal tyrosines, generating docking sites for signaling partners that stimulate proliferation, restitution, and chemotaxis. Specificity for individual EGFR tyrosines in cellular responses has been hypothesized but not well documented. Here we tested the requirement for particular tyrosines, and associated downstream pathways, in mouse colon epithelial cell chemotactic migration. We compared these requirements to those for the phenotypically distinct restitution (wound healing) migration. METHODS: Wild-type, Y992/1173F, Y1045F, Y1068F, and Y1086F EGFR constructs were expressed in EGFR-/- cells; EGF-induced chemotaxis or restitution were determined by Boyden chamber or modified scratch wound assay, respectively. Pharmacological inhibitors of p38, phospholipase C (PLC), Src, MEK, JNK/SAPK, phosphatidylinositol 3-kinase (PI 3-kinase), and protein kinase C (PKC) were used to block EGF-stimulated signaling. Pathway activation was determined by immunoblot analysis. RESULTS: Unlike wild-type EGFR, Y992/1173F and Y1086F EGFR did not stimulate colon epithelial cell chemotaxis towards EGF; Y1045F and Y1068F EGFR partially stimulated chemotaxis. Only wt EGFR promoted colonocyte restitution. Inhibition of p38, PLC, and Src blocked chemotaxis; JNK, PI 3-kinase, and PKC inhibitors blocked restitution but not chemotaxis. All four EGFR mutants stimulated downstream signaling in response to EGF, but Y992/1173F EGFR was partially defective in PLCγ activation while both Y1068F and Y1086F EGFR failed to activate Src. CONCLUSIONS: Specific EGFR tyrosines play key roles in determining cellular responses to ligand. Chemotaxis and restitution, which have different migration phenotypes and physiological consequences, have overlapping but not identical EGFR signaling requirements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4328. doi:1538-7445.AM2012-4328

VEGFR2 activation during the early onset of flow is ligand‐dependent and mediated by the release of autocrine VEGF

Previous studies have shown that VEGFR2 is part of a mechanosensory complex in endothelial cells (ECs) and is activated in response to fluid shear stress in a ligand‐independent manner. Here, we tested the hypothesis that shear stress induces a rapid release of heparan sulfate (HS)‐sequestered VEGF, which subsequently activates VEGFR2 in a ligand‐dependent manner. Our results showed that VEGFR2 is phosphorylated at Y1175 and Y1214 within 15 sec of flow in human coronary artery ECs. Removal of HS by heparinase III digestion led to a decrease in VEGFR2 phosphorylation. Transfection of siRNA against VEGF and blockade of VEGFR2 with a neutralizing antibody were also performed to demonstrate that early activation of VEGFR2 is ligand‐dependent. To determine whether VEGFR2 activation is regulated via the phospholipase C (PLC) pathway, we sheared ECs after pre‐incubation with two structurally different PLC inhibitors, U73122 and Edelfosine. Phosphorylation of VEGFR2 in response to flow was diminished by both drugs, suggesting that VEGFR2 activation may be downstream of PLC activation. Together, these studies aim to identify the role(s) and mechanism(s) by which HSPGs participate in flow‐induced VEGFR2 activation.

Crucial role of phospholipase C gamma 2 in hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) may ensure the physiological regional alveolar ventilation‐pulmonary perfusion matching, but can also serve as a key pathological mechanism leading to pulmonary hypertension and associated right ventricular failure. An increase in [Ca2+]i in pulmonary artery smooth muscle cells (PASMCs) is vital for HPV. However, the underlying molecular mechanisms remain incompletely understood. We have found that acute hypoxia significantly increases the activity of total phospholipase C (PLC) in isolated, endothelium‐denuded mouse resistance pulmonary arteries. The activity of PLCγ2 is largely augmented as well following hypoxic exposure. The PLC inhibitor U73122 (1 μM) greatly diminishes the hypoxia‐induced increase in [Ca2+]i in PASMCs, whereas U73433 (1 μM), an inactive analog of U73122, has no effect. 2‐aminoethoxydiphenyl borate (2‐APB) and xestospongin C to block inositol trisphosphate receptors (IP3Rs) also inhibit the hypoxic Ca2+ response. Excitingly, shRNA‐mediated PLCγ2 gene silencing diminishes the hypoxia‐induced increase in [Ca2+]i. Using myxothiazol to inhibit mitochondrial ROS production blocks the hypoxic activation of PLCγ2. In conclusion, hypoxia can specifically activate PLCγ2 and then IP3Rs to increase [Ca2+]i in PASMCs by enhancing mitochondrial ROS production, which may play an important role in HPV and hypoxic pulmonary hypertension. This study was supported by AHA EIA 0340160N and NIH R01 HL64043, HL064043‐S1, and HL075190.

Endothelin‐1 Signaling in Coronary Arteriolar Constriction

Endothelin‐1 (ET‐1) is a potent vasoconstrictor implicated in coronary heart diseases, but its signaling for vasoconstriction remains elusive. Herein, we probed the role of voltage‐gated Ca2+ channels (VGCCs), phospholipase C (PLC), myosin light chain kinase (MLCK), protein kinase C (PKC) and Rho kinase (ROCK) in the porcine coronary arteriolar response to a pathologic dose of ET‐1 (0.1 nM). Isolated coronary arterioles (~70 μm) constricted to ET‐1 (from 52±3% to 32±3% max. diameter) in a manner sensitive to ETA receptor blocker BQ123, zero‐extraluminal Ca2+, and PLC inhibitor U73122. Inhibiting VGCCs by nifedipine or MLCK by ML‐9 abolished basal tone but did not affect ET‐1 response. ROCK inhibitor H1152 blocked vasoconstrictions to ET‐1 but not PKC activator PDBu. Broad‐spectrum PKC inhibitor Gö6983 blocked vasoconstriction to PDBu but not ET‐1. The PKC‐activated CPI‐17 protein was undetectable in arterioles. Moreover, ET‐1 elicited phosphorylation of myosin‐binding subunit (MBS) in H1152‐sensitive manner. ROCK2, but not ROCK1, siRNA inhibited vasoconstriction to ET‐1. We conclude that VGCCs and MLCK contribute to the generation of basal tone rather than the constriction to ET‐1. The sequential activation of ETA receptors, PLC and the VGCC‐independent Ca2+ entry leads to vasoconstriction to ET‐1 via a pathway involved in ROCK2‐associated MBS phosphorylation independent of PKC signaling.

Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP3 pathway

Activation of the calcium (Ca+2)‐sensing receptor (CaSR) on juxtaglomerular (JG) cells results in inhibition of adenylyl cyclase V, decreases in cAMP content and attenuation of renin release. However, the post‐receptor pathway is unknown. In parathyroid glands, the CaSR is coupled to Gq, activating phospholipase C (PLC)/inositol 1,4,5‐trisphosphate (IP3). Thus, we hypothesized JG cell CaSR activation inhibits renin via the PLC/IP3 pathway. Renin release was measured by radioimmunoassay for AngI in the incubation media from isolated mouse JG cells in primary cultures (n = 10 for each). As expected, the CaSR agonist cinacalcet decreased renin release to 56 ± 7% of control (p&lt; 0.001), while the PLC inhibitor U73122 reversed this CaSR‐mediated inhibition of renin by cinacalcet (104 ±11 % of control). The IP3 inhibitor 2‐APB also reversed CaSR‐mediated inhibition of renin from 56 ± 6 to 104 ±11% of control (p&lt; 0.001). We conclude that stimulating JG cell CaSR acts via its coupling with Gq, leading to activation of the PLC/IP3 pathway, inhibiting adenylyl cyclase V, and decreasing renin release. (NIH 5PO1HL090550‐03)