8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Evidence for the involvement of a phospholipase C--protein kinase C signaling pathway in insulin stimulated glucose transport in skeletal muscle

The primary purpose of this investigation was to determine the relationship between phospholipase C (PLC) and diacylglycerol (DAG) sensitive protein kinase C isoforms in insulin signaling in skeletal muscle. Using an in vitro preparation of rat soleus muscle we found that insulin (0.6 nM) stimulated glucose transport was inhibited approximately 20 and 25% by the PKC inhibitor GF109203X and the phospholipase C inhibitor U73122 respectively (p<0.05). The combined effects of these inhibitors were no greater than the inhibitory effects of either compound alone. Western blot analysis revealed that insulin induced a redistribution of PKC beta II from the cytosol to the membrane that was reversed in the presence of GF109203X (1 microM) and U73122 (20 microM). Similarly, U73122 and GF109203X reversed the insulin induced increase in membrane associated phosphorylated (ser 660) PKC beta II. The novel finding of this investigation is that insulin induces an increase in PKC beta II translocation and phosphorylation through a U73122 sensitive pathway in quantatively the most important insulin responsive tissue, skeletal muscle. Furthermore, these results imply that PKC beta II may be one of the DAG sensitive isoforms involved in glucose transport.     

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.     

Modulation of ANP-C receptor signaling by arginine-vasopressin in A-10 vascular smooth muscle cells: role of protein kinase C

We have previously shown that pretreatment of A-10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering [125I]ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by arginine-vasopressin (AVP). Pretreatment of A-10 VSMC with AVP for 24h resulted in a reduction in ANP receptor binding activity by about 50% (B(max); control cells, 22.9+/-2.5 fmol/mg protein, AVP-treated cells, 11.4+/-1.2 fmol/mg protein). In addition, the expression of ANP-C receptor as determined by immunoblotting was also decreased by about 50% by AVP treatment, which was prevented by GF109203X, an inhibitor of protein kinase C (PKC). The decreased expression of ANP-C receptor was reflected in an attenuation of ANP-C receptor-mediated inhibition of adenylyl cyclase. C-ANP(4-23) [des(Gln(18),Ser(19),Gln(20),Leu(21),Gly(22))ANP(4-23)-NH(2)], a ring deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity by about 30% in control cells, which was completely attenuated in AVP-treated cells. This attenuated inhibition was significantly restored by GF 109203X. In addition, AVP treatment augmented the levels of Gialpha-2 and Gialpha-3 proteins; however, the Gi functions were completely attenuated. The increased expression of Gialpha proteins induced by AVP was inhibited by GF109203X as well as by actinomycin D treatments. In addition, AVP treatment also enhanced the expression of Gsalpha protein and Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, N-ethylcarboxamide adenosine (NECA), and forskolin (FSK), whereas the levels of Gbeta were not altered by AVP treatment. These results indicate that AVP-induced PKC signaling may be responsible for the down-regulation of ANP-C receptor that results in the attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase activity, and suggest a cross-talk between vasopressin V(1) and ANP-C receptor-mediated signaling pathways.     

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K_M. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition ( N -[2-( p -bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4-β-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.     

Regulation of ERK1/2 by the C. elegans muscarinic acetylcholine receptor GAR-3 in Chinese hamster ovary cells

Three G-protein-linked acetylcholine receptors (GARs) exist in the nematode C. elegans. GAR-3 is pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). We observed that carbachol stimulated ERK1/2 activation in Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the predominant alternatively spliced isoform of GAR-3. This effect was substantially reduced by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor GF109203X, implying that PLC and PKC are involved in this process. On the other hand, GAR-3b-mediated ERK1/2 activation was inhibited by treatment with forskolin, an adenylate cyclase (AC) activator. This inhibitory effect was blocked by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These results suggest that GAR-3b-mediated ERK1/2 activation is negatively regulated by cAMP through PKA. Together our data show that GAR-3b mediates ERK1/2 activation in CHO cells and that GAR-3b can couple to both stimulatory and inhibitory pathways to modulate ERK1/2.     

Characterization of the protease-activated receptor-1-mediated contraction and relaxation in the rat duodenal smooth muscle

Activation of protease-activated receptor-1 (PAR-1) produces a dual action, apamin-sensitive relaxation followed by contraction, in the rat duodenal smooth muscle, which is partially dependent on activation of L-type Ca2+ channels, protein kinase C (PKC) or tyrosine kinase (TK), and resistant to tetrodotoxin. The present study further characterized the PAR-1-mediated duodenal responses. Removal of extracellular Ca2+ as well as SK&F96365 reduced the contraction due to the PAR-1 agonist TFLLR-NH2 (TFp-NH2) by 60-80% that was similar to the extent of the inhibition by nifedipine. Lowering of the extracellular Na+ concentration, but not IAA-94, a Cl- channel inhibitor, reduced both the PAR-1-mediated contraction and relaxation by about 50%. U73122, a phospholipase C (PLC) inhibitor, or wortmannin, a phosphatidyl inositol 3'-kinase (PI3K) inhibitor, significantly reduced the PAR-1-mediated contraction, but not the relaxation, by itself, as the PKC inhibitor GF109203X and the TK inhibitor genistein did. U73122 or wortmannin, like GF109203X, when applied in combination with genistein, significantly reduced the PAR-1-mediated relaxation. The relaxation was resistant to antagonists of PACAP receptors, VIP receptors and P2 purinoceptors. Thus, the PAR-1-mediated contraction is considered to be dependent on intracellular and extracellular Ca2+, the influx of the latter being induced through activation of L-type Ca2+ channels triggered by the enhanced Na+ permeability, and that PLC and PI3K, in addition to PKC and TK, are involved in the PAR-1-mediated dual responses. Furthermore, non-adrenergic, non-cholinergic nerve neurotransmitter candidates that may modulate K+ channels do not appear to contribute to the relaxation by PAR-1 activation.     

Modulatory effect of endothelin-1 and -3 on neuronal norepinephrine release in the rat posterior hypothalamus

Based upon the existence of high density of ET-receptors on catecholaminergic neurons of the hypothalamus, we studied the effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on neuronal norepinephrine (NE) release in the rat posterior hypothalamus. The intracellular pathways and receptors involved were also investigated. Neuronal NE release was enhanced by ET-1 and ET-3 (10 etaM). The selective antagonists of subtype A and B ET receptors (ETA, ETB) (100 etaM BQ-610 and 100 etaM BQ-788, respectively) abolished the increase induced by ET-1 but not by ET-3. The PLC inhibitor, U73122 (10 microM), abolished ET-1 and ET-3 response. GF-109203X (100 etaM) (PKC inhibitor) blocked the increase in NE release produced by ET-3 and partially blocked ET-1 response. The inositol 1,4,5-trisphosphate-induced calcium release inhibitor, 42 microM 2-APB, inhibited the stimulatory effect induced by ET-3 but not by ET-1. The PKA inhibitor, 500 etaM H-89, blocked the increase in neuronal NE release evoked by ET-1 but not by ET-3. Our results showed that ET-1 as well as ET-3 displayed an excitatory neuromodulatory effect on neuronal NE release in the rat posterior hypothalamus. ET-1 through an atypical ETA or ETB receptor activated the PLC/PKC signalling pathway as well as the cAMP pathway, whereas ET-3 through a non-ETA/non-ETB receptor activated the phosphoinositide pathway. Both ETs would enhance the sympathoexcitatory response elicited by the posterior hypothalamus and thus participate in cardiovascular regulation.     

The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C

Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.     

Signals in the activation of opioid mu-receptors by loperamide to enhance glucose uptake into cultured C2C12 cells.

In an attempt to understand the signal pathways of opioid mu-receptors for glucose metabolism, we used loperamide to investigate the glucose uptake into the myoblast C2C12 cells. Loperamide enhanced the uptake of radioactive deoxyglucose into C2C12 cells in a concentration-dependent manner that was abolished in cells pre-incubated with naloxone or naloxonazine at concentrations sufficient to block opioid mu-receptors. Pharmacological inhibition of phospholipase C (PLC) by U73122 resulted in a concentration-dependent decrease in loperamide-stimulated uptake of radioactive deoxyglucose into C2C12 cells. This inhibition of glucose uptake by U73122 was specific since the inactive congener, U73343, failed to modify loperamide-stimulated glucose uptake. Moreover, both chelerythrine and GF 109203X diminished the action of loperamide at concentrations sufficient to inhibit protein kinase C (PKC). The obtained data suggest that an activation of opioid mu-receptors in C2C12 cells by loperamide may increase glucose uptake via the PLC-PKC pathway.     

Olfactory transduction pathways in the Senegalese sole Solea senegalensis

This study tested whether differences in sensitivity between the upper and lower olfactory epithelia of Solea senegalensis are associated with different odorant receptors and transduction pathways, using the electro‐olfactogram. Receptor mechanisms were assessed by cross‐adaptation with amino acids (L‐cysteine, L‐phenylalanine and 1‐methyl‐L‐tryptophan) and bile acids (taurocholic acid and cholic acid). This suggested that relatively specific receptors exist for 1‐methyl‐L‐tryptophan and L‐phenylalanine (food‐related odorants) in the lower epithelium, and for taurocholic acid (conspecific‐derived odorant) in the upper. Inhibition by U73122 [a phospholipase C (PLC) inhibitor] suggested that olfactory responses to amino acids were mediated mostly, but not entirely, by PLC‐mediated transduction (IC₅₀; 15–55 nM), whereas bile acid responses were mediated by both PLC and adenylate cyclase–cyclic adenosine monophosphate (AC–cAMP) (using SQ‐22536; an AC inhibitor). Simultaneous application of both drugs rarely inhibited responses completely, suggesting possible involvement of non‐PLC and non‐AC mediated mechanisms. For aromatic amino acids and bile acids, there were differences in the contribution of each transduction pathway (PLC, AC and non‐PLC and non‐AC) between the two epithelia. These results suggest that differences in sensitivity of the two epithelia are associated with differences in odorant receptors and transduction mechanisms.     

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used G?6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.     

Activation of G-Proteins with AlF4Induces LFA-1-Mediated Adhesion of T-Cell Hybridoma Cells to ICAM-1 by Signal Pathways That Differ from Phorbol Ester- and Manganese-Induced Adhesion

T-cell hybridomas metastasize widely, and the extent of dissemination correlates with invasiveness in fibroblast cultures. Previously, we provided evidence that both metastasis andin vitroinvasion require activation of LFA-1, induced by G-protein-transduced signals triggered by as yet unidentified factors. We show here that LFA-1-mediated adhesion of TAM2D2 T-cell hybridoma cells to ICAM-1 can in fact be induced by direct activation of G-proteins using AlF⁻₄, to the same extent as by using PMA or Mn²⁺. We assessed effects of protein kinase C (PKC), tyrosine kinase (TK), PI3-kinase (PI3K), and phospholipase C (PLC) inhibitors. Both AlF⁻₄-induced adhesion and invasion were completely blocked by the TK inhibitor genistein and partially blocked by the PI3K inhibitor wortmannin, but not influenced by PKC inhibitor GF109203X. Downregulation of PKC did not affect invasion or adhesion induced by AlF⁻₄either. In contrast, GF109203X and PKC downregulation blocked PMA-induced adhesion, but genistein and wortmannin had no effect. Invasion and both AlF⁻₄- and PMA-induced adhesion were completely blocked by the PLC inhibitor U73122. Mn²⁺-induced adhesion, which was not or was only partially blocked by the other inhibitors, was delayed by U73122, and spreading of Mn²⁺-treated cells was completely prevented by U73122. However, PLC activity during adhesion was not detected. We conclude that signals required for invasion and G-protein-induced adhesion are similar and are distinct from PKC-induced adhesion, and that in all cases PLC is likely to be activated, but is probably too local and/or transient to be detected.     

Inhibition by Rho-kinase and protein kinase C of myosin phosphatase is involved in thrombin-induced shape change of megakaryocytic leukemia cell line UT-7/TPO

Thrombin induced a shape change of UT-7/TPO, a thrombopoietin-dependent human megakaryocytic cell line. Expression of myosin light chain (MLC) kinase was negligible in UT-7/TPO cells, while Rho-kinase and protein kinase C (PKC) were detected. Thrombin stimulated both monophosphorylation at Ser19 and diphosphorylation at Thr18 and Ser19 of 20 kDa MLC, as well as phosphorylation of myosin-binding subunit (MBS) and PKC-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI). The Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-(1-aminoethyl)-N-(4-phynidyl) cyclohexane-carboxamide dihydrochloride, monohydrade] strongly inhibited thrombin-induced shape change, MBS phosphorylation, and mono- and diphosphorylation of MLC. The PKC inhibitor GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) partially inhibited thrombin-induced shape change and MLC diphosphorylation even at the concentration that completely inhibited thrombin-induced CPI phosphorylation. In shape-changed UT-7/TPO cells induced by thrombin, phosphorylated MBS and CPI were colocalized with diphosphorylated MLC at pseudopods, whereas monophosphorylated MLC was mainly located in the cortical region. The accumulation of diphosphorylated MLC was blocked by preincubation with either Y-27632 or GF109203X. These results suggest that Rho-kinase is responsible for the induction of MLC phosphorylation in thrombin-induced shape change of UT-7/TPO cells and that myosin phosphatase inactivation through Rho-kinase-MBS and PKC-CPI pathways could be necessary for enhancement of MLC diphosphorylation which promote the pseudopod formation.     

The arachidonic acid effect on platelet nitric oxide level

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A₂ mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A₂ receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.     

Dopamine stimulates 45Ca2+ uptake through cAMP, PLC/PKC, and MAPKs in renal proximal tubule cells

We have examined the effect of dopamine on Ca(2+) uptake and its related signaling pathways in primary renal proximal tubule cells (PTCs). Dopamine increased Ca(2+) uptake in a concentration (>10(-10) M) and time- (>8 h) dependent manner. Dopamine-induced increase in Ca(2+) uptake was prevented by SCH 23390 (a DA(1) antagonist) rather than spiperone (a DA(2) antagonist). SKF 38393 (a DA(1) agonist) increased Ca(2+) uptake unlike the case with quinpirole (a DA(2) agonist). Dopamine-induced increase in Ca(2+) uptake was blocked by nifedipine and methoxyverapamil (L-type Ca(2+) channel blockers). Moreover, dopamine-induced increase in Ca(2+) uptake was blocked by pertussis toxin (a G(i) protein inhibitor), protein kinase A (PKA) inhibitor amide 14/22 (a PKA inhibitor), and SQ 22536 (an adenylate cyclase inhibitor). Subsequently, dopamine increased cAMP level. The PLC inhibitors (U 73122 and neomycin), the PKC inhibitors (staurosporine and bisindolylmaleimide I) suppressed the dopamine-induced increase of Ca(2+) uptake. SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a MAPKK inhibitor) also inhibited the dopamine-induced increase of Ca(2+) uptake. Dopamine-induced p38 and p42/44 MAPK phosphorylation was blocked by SQ 22536, neomycin, and staurosporine. The stimulatory effect of dopamine on Ca(2+) uptake was significantly inhibited by the NF-kappaB inhibitors SN50, TLCK, and Bay 11-7082. In addition, dopamine significantly increased the level of NF-kappaB p65, which was prevented by either SQ 22536, neomycin, staurosporine, PD 98059, or SB 203580. Thus, dopamine stimulates Ca(2+) uptake in PTCs, initially through by G(s) coupled dopamine receptors, PLC/PKC, followed by MAPK, and ultimately by NF-kappaB activation.     

Mechanical hyperalgesia induced by endothelin-1 in rats is mediated via phospholipase C, protein kinase C, and MAP kinases

In addition to causing overt nociception, intraplantar (ipl) endothelin (ET)-1 injection into the rat hind paw induces hyperalgesia to mechanical stimuli, mediated via local ET(B) receptors coupled to protein kinase (PK) C, but not PKA. The present study further examines the intracellular signaling mechanisms underlying this effect of ET-1. ET-1 (30 pmol) or phospate-buffered saline (PBS) was injected ipl in rats and the threshold of responsiveness to mechanical stimulation was assessed repeatedly each hour up to 8 hrs and 24 hrs, using the dynamic plantar aesthesiometer test, which detects the minimal pressure required to evoke paw withdrawal. Different groups were treated, 15 mins before ET-1 administration, with ipsilateral injection of selective inhibitors of either phospholipase (PL) A2 (1 nmol PACOCF3), PLC (30 pmol U73122), PKC (1 nmol GF109203X), p38 mitogen-activated protein kinase (MAPK; 30 nmol SB203580), extracellular signal-regulated kinase (ERK1/2; 30 nmol PD98059), c-Jun N-terminal kinase (JNK; 30 nmol SP600125), or vehicle, to assess their influence on the hyperalgesic response. The mechanical hyperalgesia caused by ET-1 started 2 hrs after injection, peaked at 5 hrs (PBS, 29 +/- 0.5 g; ET-1, 17 +/- 1.3 g) and lasted up to 8 hrs. The inhibitors of PLC, PKC, p38 MAPK, ERK1/2, and JNK caused long-lasting reductions of the mechanical hyperalgesia (inhibitions at 4 hrs of 100%, 90%, 97%, 90%, and 100%, respectively), but the PLA2 inhibitor reduced hyperalgesia only at 4 hrs (by 58%). Thus, mechanical hyperalgesia triggered by ET-1 in the rat hind paw depends importantly on signaling pathways involving PLC, PKC, p38 MAPK, ERK1/2, and JNK, whereas the contribution of PLA2 is relatively minor.     

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.     

Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.