Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and cPLA2 signaling pathways

Exposure of renal proximal tubule cells to oxalate may play an important role in cell proliferation, but the signaling pathways involved in this effect have not been elucidated. Thus the present study was performed to examine the effect of oxalate on ³H-labeled thymidine incorporation and its related signal pathway in primary cultured rabbit renal proximal tubule cells (PTCs). The effects of oxalate on [³H]thymidine incorporation, lactate dehydrogenase (LDH) release, Trypan blue exclusion, H₂O₂ release, activation of mitogen-activated protein kinases (MAPKs), and ³H-labeled arachidonic acid (AA) release were examined in primary cultured renal PTCs. Oxalate inhibited [³H]thymidine incorporation in a time- and dose-dependent manner. However, its analogs did not affect [³H]thymidine incorporation. Oxalate (1 mM) significantly increased H₂O₂ release, which was blocked by N-acetyl-L-cysteine (NAC) and catalase (antioxidants). Oxalate significantly increased p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun NH₂-terminal kinase (JNK) activity, not p44/42 MAPK. Oxalate stimulated [³H]AA release and translocation of cytosolic phospholipase A₂ (cPLA₂) from the cytosolic fraction to the membrane fraction. Indeed, oxalate significantly increased prostaglandin E₂ (PGE₂) production compared with control. Oxalate-induced inhibition of [³H]thymidine incorporation and increase of [³H]AA release were prevented by antioxidants (NAC), a p38 MAPK inhibitor (SB-203580), a SAPK/JNK inhibitor (SP-600125), or PLA₂ inhibitors [mepacrine and arachidonyl trifluoromethyl ketone (AACOCF₃)], but not by a p44/42 MAPK inhibitor (PD-98059). These findings suggest that oxalate inhibits renal PTC proliferation via oxidative stress, p38 MAPK/JNK, and cPLA₂ signaling pathways. kidney; mitogen-activated protein kinase; phospholipase A₂     

ANG II-induced translocation of cytosolic PLA2 to the nucleus in vascular smooth muscle cells

The accumulation of radiolabeled arachidonicacid (AA), immunoblot analysis of subcellular fractions, andimmunofluorescence tagging of proteins in intact cells were used toexamine the coupling of ANG II receptors with the activity and locationof a cytosolic phospholipase A₂(cPLA₂) in vascular smoothmuscle cells (VSMC). ANG II induced the accumulation of AA, whichpeaked by 10 min and was downregulated by 20 min. A large proportion ofthe AA released in response to ANG II was due to the activation of a Ca²⁺-dependent lipase coupled toan AT₁ receptor. However,regulation of Ca²⁺ availabilityfailed to completely block AA release, and a small but significantreduction in ANG II-mediated AA release was observed in the presence ofan AT₂ antagonist. These findings,coupled with a 25% reduction in the ANG II-induced AA release by aninhibitor specific for aCa²⁺-independentPLA₂, are consistent with thepresence and activation of aCa²⁺-independentPLA₂. In contrast, immunoblotanalysis and immunofluorescence detection showed that the ANGII-mediated translocation of cPLA₂ to a membrane fraction was exclusivelyAT₁ dependent and regulated byCa²⁺ availability. Furthermore,the nucleus was the membrane target. We conclude that ANG II regulatesthe Ca²⁺-dependent activation andtranslocation of cPLA₂ through anAT₁ receptor and that this eventis targeted at the nucleus in VSMC.

     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.     

Agonist-specific cross talk between ERKs and p38(mapk) regulates PGI(2) synthesis in endothelium

We have examined the mechanisms regulatingprostacyclin (PGI₂) synthesis after acute exposure of humanumbilical vein endothelial cells (HUVEC) to interleukin-1 (IL-1).IL-1 evoked an early (30 min) release of PGI₂ and[³H]arachidonate that was blocked by the cytosolicphospholipase A₂ (cPLA₂) inhibitorarachidonyl trifluoromethyl ketone. IL-1-mediated activationof extracellular signal-regulated kinase 1/2 (ERK1/2; p42/p44^mapk) coincided temporally with phosphorylation ofcPLA₂ and with the onset of PGI₂synthesis. The mitogen-activated protein kinase (MAPK) kinase (MEK)inhibitors, PD-98059 and U-0126, blocked IL-1-induced ERKactivation and partially attenuated cPLA₂phosphorylation and PGI₂ release, suggesting thatERK-dependent and -independent pathways regulate cPLA₂phosphorylation. SB-203580 treatment enhanced IL-1-induced MEK,p42/44^mapk, and cPLA₂ phosphorylation butreduced thrombin-stimulated MEK and p42/44^mapk activation.IL-1, but not thrombin, activated Raf-1 as assessed byimmune-complex kinase assay, as did SB-203580 alone. These results showthat IL-1 causes an acute upregulation of PGI₂generation in HUVEC, establish a role for theMEK/ERK/cPLA₂ pathway in this early release, and provideevidence for an agonist-specific cross talk between p38^mapkand p42/44^mapk that may reflect receptor-specificdifferences in the signaling elements proximal to MAPK activation.

     

Mechanisms of MAPK activation by bradykinin in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is a prominentfeature of the atherosclerotic process occurring after endothelial injury. A vascular wall kallikrein-kinin system has been described. Thecontribution of this system to vascular disease is undefined. In thepresent study we characterized the signal transduction pathway leadingto mitogen-activated protein kinase (MAPK) activation in response tobradykinin (BK) in VSMC. Addition of10¹⁰-10⁷M BK to VSMC resulted in a rapid and concentration-dependent increasein tyrosine phosphorylation of several 144- to 40-kDa proteins. Thiseffect of BK was abolished by theB₂-kinin receptor antagonistHOE-140, but not by the B₁-kininreceptor antagonist des-Arg⁹-Leu⁸-BK.Immunoprecipitation with anti-phosphotyrosine antibodies followed byimmunoblot revealed that10⁹ M BK induced tyrosinephosphorylation of focal adhesion kinase (p125^FAK). BK(10⁸ M) promoted theassociation of p60^src with theadapter protein growth factor receptor binding protein-2 and alsoinduced a significant increase in MAPK activity. Pertussis and choleratoxins did not inhibit BK-induced MAPK tyrosine phosphorylation. Protein kinase C downregulation by phorbol 12-myristate 13-acetate and/or inhibitors to protein kinase C,p60^src kinase, and MAPK kinaseinhibited BK-induced MAPK tyrosine phosphorylation. These findingsprovide evidence that activation of theB₂-kinin receptor in VSMC leads togeneration of multiple second messengers that converge to activateMAPK. The activation of this crucial kinase by BK provides a strongrationale to investigate the mitogenic actions of BK on VSMCproliferation in disease states of vascular injury.

     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

     

Immunomodulated signaling in macrophages: Studies on activation of Raf-1, MAPK, cPLA2 and secretion of IL-12

Little is known about the mechanism and signal transduction by LPS-mediated immunomodulation of murine peritoneal macrophages. It is found that the signal molecules of the down-stream of Ras, Raf-1, MAPK p44, and MAPK p42 are phosphorylated, and cPLA₂ is activated with a significant increase of the release of [ H³ ] AA by macrophages in response to LPS and PMA. Compared with the very recent finding that LPS and PMA trigger the activation and translocation of PKC-α and PKC-ε, these findings suggest that there is a connection between PKC signaling pathway and the Raf-1/MAPK pathway and that the activation of these main signaling events may be closely related to the secretion of IL-12 during LPS-induced modulation of macrophages.     

Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries

LocalCa²⁺ transients("Ca²⁺ sparks") caused bythe opening of one or the coordinated opening of a number of tightlyclustered ryanodine-sensitiveCa²⁺-release (RyR) channels in thesarcoplasmic reticulum (SR) activate nearbyCa²⁺-dependentK⁺(K_Ca) channels to cause anoutward current [referred to as a "spontaneous transientoutward current" (STOC)]. TheseK_Ca currents cause membranepotential hyperpolarization of arterial myocytes, which would lead tovasodilation through decreasingCa²⁺ entry throughvoltage-dependent Ca²⁺ channels.Therefore, modulation of Ca²⁺spark frequency should be a means to regulation ofK_Ca channel currents and hencemembrane potential. We examined the frequency modulation ofCa²⁺ sparks and STOCs byactivation of protein kinase C (PKC). The PKC activators, phorbol12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM),decreased Ca²⁺ spark frequency by72% and 60%, respectively, and PMA reduced STOC frequency by 83%.PMA also decreased STOC amplitude by 22%, which could be explained byan observed reduction (29%) inK_Ca channel open probability inthe absence of Ca²⁺ sparks. Thereduction in STOC frequency occurred in the presence of an inorganicblocker (Cd²⁺) ofvoltage-dependent Ca²⁺ channels.The reduction in Ca²⁺ sparkfrequency did not result from SRCa²⁺ depletion, sincecaffeine-induced Ca²⁺ transientsdid not decrease in the presence of PMA. These results suggest thatactivators of PKC can modulate the frequency ofCa²⁺ sparks, through an effect onthe RyR channel, which would decrease STOC frequency (i.e.,K_Ca channel activity).

     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells

The L-type Ca²⁺ channel is the primary voltage-dependent Ca²⁺-influx pathway in many excitable and secretory cells, and direct phosphorylation by different kinases is one of the mechanisms involved in the regulation of its activity. The aim of this study was to evaluate the participation of Ser/Thr kinases and tyrosine kinases (TKs) in depolarization-induced Ca²⁺ influx in the endocrine somatomammotrope cell line GH3. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using a spectrofluorometric method with fura 2-AM, and 12.5 mM KCl (K⁺) was used as a depolarization stimulus. K⁺ induced an abrupt spike (peak) in [Ca²⁺]_i that was abolished in the presence of nifedipine, showing that K⁺ enhances [Ca²⁺]_i, preferably activating L-type Ca²⁺ channels. H89, a selective PKA inhibitor, significantly reduced depolarization-induced Ca²⁺ mobilization in a concentration-related manner when it was applied before or after K⁺, and okadaic acid, an inhibitor of Ser/Thr phosphatases, which has been shown to regulate PKA-stimulated L-type Ca²⁺ channels, increased K⁺-induced Ca²⁺ entry. When PKC was activated by PMA, the K⁺-evoked peak in [Ca²⁺]_i, as well as the plateau phase, was significantly reduced, and chelerythrine (a PKC inhibitor) potentiated the K⁺-induced increase in [Ca²⁺]_i, indicating an inhibitory role of PKC in voltage-dependent Ca²⁺ channel (VDCC) activity. Genistein, a TK inhibitor, reduced the K⁺-evoked increase in [Ca²⁺]_i, but, unexpectedly, the tyrosine phosphatase inhibitor orthovanadate reduced not only basal Ca²⁺ levels but, also, Ca²⁺ influx during the plateau phase. Both results suggest that different TKs may act differentially on VDCC activation. Activation of receptor TKs with epidermal growth factor (EGF) or vascular endothelial growth factor potentiated K⁺-induced Ca²⁺ influx, and AG-1478 (an EGF receptor inhibitor) decreased it. However, inhibition of the non-receptor TK pp60 c-Src enhanced K⁺-induced Ca²⁺ influx. The present study strongly demonstrates that a complex equilibrium among different kinases and phosphatases regulates VDCC activity in the pituitary cell line GH3: PKA and receptor TKs, such as vascular endothelial growth factor receptor and EGF receptor, enhance depolarization-induced Ca²⁺ influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and may therefore participate in the regulation of a plethora of intracellular processes, such as hormone secretion, gene expression, protein synthesis, and cell proliferation, in pituitary cells. phosphatases; protein kinase A; protein kinase C; epidermal growth factor     

Calcium-independent phospholipase A2 is regulated by a novel protein kinase C in human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca²⁺-independent phospholipase A₂ (iPLA₂) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 ± 0.1 vs. 5.3 ± 0.4 nmol PO₄/mg of protein) and plasmenylethanolamine (7.5 ± 1.0 vs. 12.0 ± 0.9 nmol PO₄/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 ± 0.3 nmol PO₄/mg of protein) and plasmenylethanolamine (6.0 ± 1.0 nmol PO₄/mg of protein). Incubation of HCAEC with the selective iPLA₂ inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 ± 0.3 vs. 1.1 ± 0.1%) and increased production of lysoplasmenylcholine (1.4 ± 0.2 vs. 0.6 ± 0.1 nmol PO₄/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 ± 0.6 to 0.5 ± 0.1%) and lysoplasmenylcholine production (2.0 ± 0.1 to 0.2 ± 0.1 nmol PO₄/mg of protein). These data suggest that PKC activates iPLA₂ in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production. lysophospholipids; cell signaling; phospholipid metabolism; arachidonic acid     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C

Localized Ca²⁺ transients resulting from inositoltrisphosphate (IP₃)-dependent Ca²⁺ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa²⁺ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca²⁺([Ca²⁺]_o) reduced localized Ca²⁺transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca²⁺ transients and STOCs showed increasedcoupling strength between Ca²⁺ transients and STOCs when[Ca²⁺]_o was reduced. Gd³⁺ (10 µM) did not affect Ca²⁺ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca²⁺influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca²⁺ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca²⁺ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca²⁺ transients. Single channelstudies indicated that large-conductance Ca²⁺-activatedK⁺ (BK) channels were inhibited by aCa²⁺-dependent PKC. In summary 1)Ca²⁺ release from IP₃ receptor-operated storesactivates Ca²⁺-activated K⁺ channels;2) Ca²⁺ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca²⁺ sensitivity of BK channels, reducing the couplingstrength between localized Ca²⁺ transients and BK channels.

     

Phorbol ester plus calcium ionophore induces release of arachidonic acid from membrane phospholipids of a human B cell line

Binding of LA350, a lymphoblastoid human B cell line, by phorbol myristate acetate (PMA) plus a calcium ionophore, either ionomycin or A23187, produced unique alterations in the release of arachidonic acid (AA) from cellular phospholipids. After equilibrium labeling of cells with radioactive fatty acids, [14C]AA demonstrated a selective enhanced release from the cells in response to the binding of PMA plus calcium ionophore as compared to the release of [14C]stearic acid (STE), [3H]oleic acid (OLE) and [3H]palmitic acid (PAL). The major phospholipid sources of the released [14C]AA were shown to be phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The participation of protein kinase C (PKC) in the enhanced synergistic release of [14C]AA was demonstrated by the inhibition of the release by the PKC inhibitor, staurosporine. Approximately 2-6% of the labeled AA liberated was converted to 5-hydroxyeicosatetraenoic acid by an endogenous 5-lipoxygenase. Therefore during cell activation the B cell is capable of liberating AA via a PKC-dependent mechanism, implicating AA and/or its metabolites in signal transduction.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

EGF stimulates proliferation of mouse embryonic stem cells: involvement of Ca2+ influx and p44/42 MAPKs

We examined the effect of EGF on the proliferation of mouse embryonic stem (ES) cells and their related signal pathways. EGF increased [³H]thymidine and 5-bromo-2'-deoxyuridine incorporation in a time- and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor (EGFR). Inhibition of EGFR tyrosine kinase with AG-1478 or herbimycin A, inhibition of PLC with neomycin or U-73122, inhibition of PKC with bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca²⁺ channels with nifedipine or methoxyverapamil prevented EGF-induced [³H]thymidine incorporation. PKC-, -_I, -, -, and - were translocated to the membrane and intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased in response to EGF. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca²⁺]_i. EGF also increased inositol phosphate levels, which were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H₂O₂, and pretreatment with antioxidant (N-acetyl-L-cysteine) inhibited EGF-induced increase of [Ca²⁺]_i. In addition, we observed that p44/42 MAPK phosphorylation by EGF and inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca²⁺ channels blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs with PD-98059 (MEK inhibitor) attenuated EGF-induced increase of [³H]thymidine incorporation. Finally, inhibition of EGFR tyrosine kinase, PKC, Ca²⁺ channels, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4, respectively. In conclusion, EGF partially stimulates proliferation of mouse ES cells via PLC/PKC, Ca²⁺ influx, and p44/42 MAPK signal pathways through EGFR tyrosine kinase phosphorylation. calcium; epidermal growth factor; mitogen-activated protein kinases; protein kinase C     

Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation

The presentstudy was performed to characterize thrombin-stimulated phospholipaseA₂(PLA₂) activity and theresultant release of lysophospholipids from endothelial cells. Themajority of PLA₂ activity inendothelial cells was membrane associated,Ca²⁺ independent, and arachidonateselective. Incubation with thrombin increased membrane-associatedPLA₂ activity using bothplasmenylcholine and alkylacyl glycerophosphocholine substrates in theabsence of Ca²⁺, with no increasein activity observed with phosphatidylcholine substrate. The increasedPLA₂ activity was accompanied byarachidonic acid and lysoplasmenylcholine (LPlasC) release fromendothelial cells into the surrounding medium. Thrombin-induced changeswere duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with theCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked thrombin-stimulated increases inPLA₂ activity, arachidonic acid,and LPlasC release. Stimulation of protein kinase C (PKC) increasedbasal PLA₂ activity and LPlasCproduction. Thrombin-stimulatedPLA₂ activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cellsactivates a PKC-activated, membrane-associated,Ca²⁺-independentPLA₂ that selectively hydrolyzesarachidonylated, ether-linked phospholipid substrates, resulting inLPlasC and arachidonic acid release.

     

Phosphorylation-dependent stimulation of prostanoid synthesis by nigericin in cerebral endothelial cells

Nigericin decreases intracellular pH(pH_i) and stimulates prostanoid(PG) synthesis in endothelial cells from cerebral microvessels ofnewborn pigs. Nigericin-induced PG production was abolished by proteintyrosine kinase (PTK) inhibitors and amplified by phorbol 12-myristate13-acetate (PMA) or protein tyrosine phosphatase (PTP) inhibitors.Nigericin-induced PG production in PMA-primed cells was potentiated byPTP inhibitors and abrogated by PTK inhibitors. PhospholipaseA₂(PLA₂) activity was stimulatedby nigericin in a phosphorylation-dependent manner. Nigericin'seffects on PG production and PLA₂activity were reproduced by ionomycin, which activates cytosolicPLA₂(cPLA₂).cPLA₂ was immunodetected in endothelial cell lysates. We found no evidence that nigericin's effects are mediated via mitogen-activated protein (MAP) kinase [extracellularly regulated kinase 1 (ERK1) and ERK2]activation: although nigericin stimulateddetergent-soluble MAP kinase, its effects were not amplified by PMA orPTP inhibitors. Phosphorylation-dependent stimulation of PG synthesiswas also observed when pH_i wasdecreased by sodium propionate or a high level ofCO₂. Altogether, our data indicatethat nigericin and decreased pH_istimulate PG synthesis by a protein phosphorylation-dependent mechanisminvolving cross talk between pathways mediated by PTK and PTP and byprotein kinase C; cPLA₂ appears tobe a key enzyme affected by nigericin and decreasedpH_i.

     

Early activation of p160ROCK by pressure overload in rat heart

We investigated the mechanisms underlying regulation of contraction with measurements of isometric force and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in NIH 3T3 fibroblast reconstituted into fibers with the use of a collagen matrix. Treatment with the major phospholipids, neurotransmitters, and growth factors had little effect on baseline isometric force. However, U-46619, a thromboxane A₂ (TxA₂) analog, increased force and [Ca²⁺]_i; EC₅₀ values were 11.0 and 10.0 nM, respectively. The time courses were similar to those induced by calf serum (CS), and the maximal force was 65% of a CS-mediated contraction. The selective TxA₂ receptor antagonist SQ-29548 abolished the U-46619-induced responses. CS-induced contractions are dependent on an intracellular Ca²⁺ store function; however, the U-46619 response depended not only on intracellular Ca²⁺ stores, but also on Ca²⁺ influx from the extracellular medium. Inhibition of Rho kinase suppressed U-46619- and CS-induced responses; in contrast, inhibition of C kinase (PKC) reduced only the U-46619 response. Moreover, addition of U-46619 to a CS contracture enhanced force and [Ca²⁺]_i responses. These results indicate that U-46619-induced responses involve PKC and Rho kinase pathways, in contrast to activation by CS. Thus TxA₂ may have a role in not only the initial step of wound repair as an activator of blood coagulation, but also in fibroblast contractility in later stages. collagen matrix; signal transduction; wound repair