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Inorganic polyphosphates (polyP) are polymers composed of phosphate residues linked by energy-rich phosphoanhydride bonds. As polyP can bind calcium, the hypothesis of this study is that polyP enters chondrocytes and exerts its anabolic effect by calcium influx through calcium channels. PolyP treatment of cartilage tissue formed in 3D culture by bovine chondrocytes showed an increase in proteoglycan accumulation but only when calcium was also present at a concentration of 1.5 mM. This anabolic effect could be prevented by treatment with either ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid or the calcium channel inhibitors gadolinium and nifedipine. Calcium and polyP cotreatment of chondrocytes in monolayer culture resulted in calcium oscillations that were polyP chain length specific and were inhibited by gadolinium and nifedipine. The calcium influx resulted in increased gene expression of sox9, collagen type II, and aggrecan which was prevented by treatment with either calphostin, an inhibitor of protein kinase C, and W7, an inhibitor of calmodulin; suggesting activation of the protein kinase C-calmodulin pathway. Tracing studies using 4',6-diamidino-2-phenylindole, Mitotracker Red, and/or Fura-AM staining showed that polyP was detected in the nucleus, mitochondria, and intracellular vacuoles suggesting that polyP may also enter the cell. PolyP colocalizes with calcium in mitochondria. This study demonstrates that polyP requires the influx of calcium to regulate chondrocyte matrix production, likely via activating calcium signaling. These findings identify the mechanism regulating the anabolic effect of polyP in chondrocytes which will help in its clinical translation into a therapeutic agent for cartilage repair.

Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.

We previously showed that impaired vasodilation in systemic and renal vessels contributes to salt-sensitive hypertension in a mouse model of impaired PPARγ (peroxisome proliferator-activated receptor gamma) function. We determined the mechanisms mediating impaired salt-induced vasodilation and whether improved vasodilation attenuates augmented hypertension in response to salt. Mice selectively expressing a PPARγ dominant negative mutation in vascular smooth muscle (S-P467L) exhibited salt-sensitive hypertension and severely impaired vasodilation in systemic and renal vessels. High-salt diet–fed S-P467L and control mice displayed comparable levels of renal oxidative stress markers. Preincubation with Tempol, a superoxide dismutase mimetic, or calphostin C, a PKC (protein kinase C) inhibitor, failed to improve salt-induced impairment of vasodilation in S-P467L mice, arguing against a role of oxidative stress or PKC activity. Inhibition of Rho kinase partially rescued impaired vasodilation in high-salt diet–fed S-P467L mice suggesting a contribution of the Ras homolog family member A (RhoA)/Rho kinase pathway. High-salt diet selectively increased synthesis of PGE2 (prostaglandin E2) in S-P467L aorta. Expression of EP3 (E-prostanoid 3) receptor mRNA was increased in aorta from chow-fed and high salt–fed S-P467L mice. Pharmacological inhibition of COX (cyclooxygenase) 2 or blockade of EP3 completely normalized the impaired vasodilation, and EP3 antagonism induced larger decreases in systolic blood pressure in high-salt diet–fed S-P467L mice. In conclusion, interference with PPARγ in vascular smooth muscle causes activation of the PGE2/EP3 signaling pathway in systemic and renal vasculature resulting in salt-induced impairment of vasodilation and salt-sensitive hypertension. PGE2/EP3 axis maybe a druggable target to prevent salt-sensitive hypertension in chronic conditions associated with decreased PPARγ activity.

Phosphatidylserine (PS) exposure is increased in red cells from sickle cell anaemia (SCA) patients. Externalised PS is prothrombotic and attractive to phagocytes and activated endothelial cells and thus contributes to the anaemic and ischaemic complications of SCA. The mechanism of PS exposure remains uncertain but it can follow increased intracellular Ca2+ concentration ([Ca2+]i). Normally, [Ca2+]i is maintained at very low levels but in sickle cells, Ca2+ permeability is increased, especially following deoxygenation and sickling, mediated by a pathway sometimes called Psickle. The molecular identity of Psickle is also unclear but recent work has implicated the mechanosensitive channel, PIEZO1. We used Yoda1, an PIEZO1 agonist, to investigate its role in sickle cells. Yoda1 caused an increase in [Ca2+]i and PS exposure, which was inhibited by its antagonist Dooku1 and the PIEZO1 inhibitor GsMTx4, consistent with functional PIEZO1. However, PS exposure did not necessitate an increase in [Ca2+]i. Two PKC inhibitors were also tested, chelerytherine chloride and calphostin C. Both reduced PS exposure whilst chelerytherine chloride also reduced Yoda1-induced increases in [Ca2+]i. Findings are therefore consistent with the presence of PIEZO1 in sickle cells, able to mediate Ca2+ entry but that PKC was also involved in both Ca2+ entry and PS exposure.

Bronchial asthma is a chronic inflammatory airway disease that can be aggravated by cold air. However, its mechanism remains largely unknown. As a thermo-sensing cation channel, transient receptor potential melastatin 8 (TRPM8) can be activated by cold stimuli (8-22 °C) and cooling agents. Whereas TRPM8 activation leads to enhanced expression of inflammatory cytokines and mucus hypersecretion in human bronchial epithelial cell lines, no previous study has examined its role in regulating the cold-induced inflammatory responses and its mechanism in asthmatic airway epithelium. Airway epithelial cells were isolated from asthma model mice and exposed to low temperature (18 °C). The TRPM8 overexpression plasmid and siRNA lentivirus were transfected to up- or downregulate the TRPM8 level. The expression of mRNAs of inflammatory cytokines was tested using real-time reverse transcription-polymerase chain reaction (RT-PCR). The activities of phosphorylated protein kinase C (PKC) and phosphorylated inhibitor of nuclear factor kappa B (IκB) were measured using the immunofluorescence assay. The expression of mRNAs of inflammatory cytokines [interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-13, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α] increased significantly under cold conditions, which was boosted after TRPM8 overexpression and augmented further in the presence of PKC inhibitor, calphostin C. However, the downregulation of TRPM8 and nuclear factor kappa B (NF-κB) impaired the transcription of these cytokine genes. In addition, the phosphorylated PKC and phosphorylated IκB were activated by cold stimuli. Moreover, the expression of phosphorylated IκB protein improved in the presence of TRPM8, while disruption with the TRPM8 gene or TRPM8 antagonist prohibited the activation of IκB. Cold air could induce inflammatory responses through the TRPM8-mediated PKC/NF-κB signal pathway in primary airway epithelial cells of asthmatic mice.

As potential protein kinase C (PKC) inhibitors and photodynamic agents, the novel amino-functionalized calphostin analog 1,12-bis((benzoyl-amino)methyl)-3,10-perylenequinone was successfully prepared by dimerization of the key intermediate 3-(benzoylamino)methyl-1,2-naphthoquinone (9), which was synthesized by an efficient and relatively short synthetic sequence (eight steps) with satisfactory overall yield. The naturally occurring form of perylenequinone 12 was prepared by consecutive methylation and demethylation reactions. In our synthetic strategy, it was beneficial that the amino functionality of 1,2-naphthoquinone 9 could be easily introduced at an early synthetic stage and subsequently dimerized to prepare various potentially bioactive perylenequinones.

Sphingosine-1-phosphate (S1P) was found previously to inhibit Na+–K+ATPase in HepG2 cells. Whether fingolimod (FTY720), a S1P receptor (S1PR) agonist, similarly inhibits the ATPase is a question that needs to be addressed. The aim of this work was to study the effect of FTY720P, the active form of the drug, on the activity of Na+–K+ATPase in HepG2 cells and determine its mechanism of action. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and the absence of ouabain. FTY720-P (7.5 nmol/L, 15 min) significantly reduced the activity of the ATPase. This effect disappeared completely in the presence of JTE-013, which is a specific blocker of sphingosine-1-phosphate receptor 2 (S1PR2), as well as in the presence of calphostin and indomethacin, which are inhibitors of protein kinase C (PKC) and COX-2, respectively. The effect of FTY720P was mimicked by prostaglandin E2 (PGE2) and PMA, but abrogated by NF-κB inhibition. When NF-κB was inhibited, the effect of exogenous PGE2 still appeared, but that of PMA did not manifest, suggesting that NF-κB is upstream of PGE2 and downstream of PKC. It was concluded that FTY720P activates via S1PR2, PKC, and NF-κB. The latter induces PGE2 generation and inhibits Na+–K+ATPase.

Tetrodotoxin-sensitive Na(+) currents have been extensively studied because they play a major role in neuronal firing and bursting. In this study, we showed that voltage-dependent Na(+) currents are regulated in a slow manner by oxotremorine (oxo-M) and angiotensin II in rat sympathetic neurons. We found that these currents can be readily inhibited through a signaling pathway mediated by G proteins and phospholipase C (PLC) β1. This inhibition is slowly established, pertussis toxin-insensitive, partially reversed within tens of seconds after oxo-M washout, and not relieved by a strong depolarization, suggesting a voltage-insensitive mechanism of inhibition. Specificity of the M1 receptor was tested by the MT-7 toxin. Activation and inactivation curves showed no shift in the voltage dependency under the inhibition by oxo-M. This inhibition is blocked by a PLC inhibitor (U73122, 1-(6-{[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione), and recovery from inhibition is prevented by wortmannin, a PI3/4 kinase inhibitor. Hence, the pathway involves Gq/11 and is mediated by a diffusible second messenger. Oxo-M inhibition is occluded by screening phosphatidylinositol 4,5-bisphosphate (PIP2)-negative charges with poly-l-lysine and prevented by intracellular dialysis with a PIP2 analog. In addition, bisindolylmaleimide I, a specific ATP-competitive protein kinase C (PKC) inhibitor, rules out that this inhibition may be mediated by this protein kinase. Furthermore, oxo-M-induced suppression of Na(+) currents remains unchanged when neurons are treated with calphostin C, a PKC inhibitor that targets the diacylglycerol-binding site of the kinase. These results support a general mechanism of Na(+) current inhibition that is widely present in excitable cells through modulation of ion channels by specific G protein-coupled receptors.

Background In physiological and pathological conditions activated protein kinace C (PKC) has been observed in the erythrocytes. Externalization of ankyrin followed by Arg–Gly–Asp (RGD)/integrin recognition also triggers erythrophagocytosis. In the present study, to test whether activated PKC is associated with ankyrin exposure in erythrophagocytosis. Methods Phorbol 12-myristate-13-acetate (PMA)-induced PKC activation and ankyrin phosphorylation were tested, and under different treatment conditions the subpopulation of erythrocytes with ankyrin exposure and the levels of intracellular calcium were analyzed by flow cytometry. Results Results showed that treatment of erythrocytes with PMA in a calcium-containing buffer led to ankyrin exposure. In the absence of extracellular calcium, no ankyrin exposure was observed. PKC inhibition with calphostin C, a blocker of the PMA binding site, completely prevented the calcium entry, protein phosphorylation and ankyrin exposure. PKC inhibition with chelerythrine chloride, an inhibitor of the active site, diminished the level of ankyrin-exposing cells and ankyrin phosphorylation; however it even led to a higher percentage of cells with increased levels of calcium than with PMA treatment alone. Although PKC was activated and ankyrin phosphorylation occurred, no ankyrin exposure was observed in the absence of extracellular calcium. Conclusion Analyses of results suggested that PMA induces calcium influx into the erythrocytes, leading to the activation of calcium-dependent enzymes and the phosphorylation of membrane proteins, ultimately inducing ankyrin exposure and erythrophagocytosis. This study may provide insights into the molecular mechanisms of removing aged or diseased erythrocytes.

In contrast to the negative feedback of angiotensin II (ANG II) on juxtaglomerular renin, ANG II stimulates renin in the principal cells of the collecting duct (CD) in rats and mice via ANG II type 1 (AT1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT1R activation through protein kinase C (PKC), but the exact mechanisms are unknown. We hypothesize that ANG II stimulates CD renin synthesis through AT1R via PKC and the subsequent activation of cAMP/PKA/CREB pathway. In M-1 cells, ANG II increased cAMP, renin mRNA (3.5-fold), prorenin, and renin proteins, as well as renin activity in culture media (2-fold). These effects were prevented by PKC inhibition with calphostin C, PKC-α dominant negative, and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca2+depletion impaired ANG II-mediated CREB phosphorylation and upregulation of renin. Adenylate cyclase 6 (AC) siRNA remarkably attenuated the ANG II-dependent upregulation of renin mRNA. Physiological activation of AC with vasopressin increased renin expression in M-1 cells. The results suggest that the ANG II-dependent upregulation of renin in the CD depends on PKC-α, which allows the augmentation of cAMP production and activation of PKA/CREB pathway via AC6. This study defines the intracellular signaling pathway involved in the ANG II-mediated stimulation of renin in the CD. This is a novel mechanism responsible for the regulation of local renin-angiotensin system in the distal nephron.

The primary cause of death of hemodialysis (HD) patients is cardiovascular disease, and increased oxidative stress has been proposed to be involved in the disease pathogenesis. In this study, we examined the effect of olmesartan on oxidative stress induced by angiotensin II, lipopolysaccharide, indoxyl sulfate, advanced oxidation protein products (AOPP) or hydrogen peroxide (H2O2), which are known to be present at higher concentrations in the blood of HD patients, using human umbilical vein endothelial cells (HUVECs). Oxidative stress was evaluated by measuring the mean fluorescence intensity of CM-H2DCFCA, an ROS-sensitive fluorescent dye, in HUVECs. HUVECs were incubated with each of the above compounds in the presence or absence of olmesartan. Moreover, these oxidant-stimulated cells were also treated with the reactive oxygen species (ROS) inhibitor N-acetyl-cysteine (NAC), NADPH oxidase inhibitor diphenylene iodonium (DPI) or PKC inhibitor calphostin C. In addition, we investigated the effects of olmesartan on cytotoxicity and vascular endothelial growth factor (VEGF) secretion, which is involved in vascular inflammation in HUVECs induced by AOPP or H2O2. The treatment of these oxidant-stimulated cells with olmesartan resulted in a significant reduction in intracellular ROS production to an extent that was nearly equivalent to that of NAC, DPI or calphostin C. Furthermore, olmesartan reduced the cytotoxicity and VEGF secretion induced by AOPP or H2O2. These results demonstrated that the antioxidant activity of olmesartan might contribute to both its vasculoprotective and anti-hypertensive effects.

Studies revealed that the use of renin-angiotensin-aldosterone system antagonism is not associated with a statistically significant reduction in the risk of cardiovascular events in patients with chronic kidney disease (CKD) compared with that in the general population. We tested the hypothesis that indoxyl sulfate (IS) can interfere with the protective effect of valsartan-mediated on endothelial function in vitro and neovascularization in mice underwent subtotal nephrectomy. In human aortic endothelial cells, we first demonstrated that IS impaired the valsartan-mediated phosphorylation of eNOSThr495, nitric oxide production and tube formation via NADPH oxidase (NOX) and protein kinase C (PKC) phosphorylation, but this effect was suppressed by cotreatment with apocynin and calphostin C. In vivo, IS attenuated valsartan-induced angiogenesis in Matrigel plugs in mice. Moreover, in subtotal nephrectomy mice who underwent hindlimb ischemic surgery, valsartan significantly increased the mobilization of endothelial progenitor cells in circulation as well as the reperfusion of blood flow and density of CD31+ capillaries in ischemic limbs. However, IS attenuated the protective effect of valsartan-induced neovascularization and increased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Cotreatment of apocynin and calphostin C reversed the IS impaired-neovascularization and decreased the expression of p-PKCαSer657 and p-eNOSThr497 in ischemic limbs. Our study suggests that the NOX/PKC/eNOS signaling pathway plays a pivotal role in the IS-mediated inhibition of valsartan-conferred beneficial effects on endothelial function in vitro and neovascularization in subtotal nephrectomy mice. We proposed a novel causative role for IS in cardiovascular complications in CKD patients., Highlights • The use of renin-angiotensin-aldosterone system antagonism fails to reduce in the risk cardiovascular events in patients with CKD. • Indoxyl sulfate interferes with the protective effect of angiotensin II receptor blocker-mediated neovascularization in CKD mice. • Indoxyl sulfate interferes with the beneficial effect of of valsartan on endothelial function by activating the NOX/PKC signaling pathway. • This article proposed a novel causative role for indoxyl sulfate in cardiovascular complications in CKD patients.

Increases in intracellular calcium (Ca(2+)) inhibit renal sodium (Na(+)) absorption in cortical collecting ducts, but the precise mechanism is unclear. We, therefore, studied the effects of raising intracellular Ca(2+) (using 10 µmol/L A23187, a Ca(2+) ionophore) on wild-type and Liddle-mutated human epithelial Na(+) channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. A23187 decreased amiloride-sensitive Na(+) current by 55% in oocytes expressing wild-type hENaC, an effect prevented by co-exposure to 50 μmol/L W-7 (to inhibit the Ca(2+)/calmodulin complex). By contrast, co-exposure to 50 μmol/L calphostin (to inhibit protein kinase C) or 5 μmol/L KN-62 (to inhibit Ca(2+)/calmodulin-dependent protein kinase II) had no effect on the decrease in amiloride-sensitive Na(+) current elicited by A23187 alone. Whereas A23187 reduced amiloride-sensitive Na(+) current in oocytes expressing wild-type hENaC, it had no similar effect in those expressing Liddle-mutated hENaCs, suggesting that the activity of individual Na(+) channels in situ was unchanged by the rise in intracellular Ca(2+). These data suggest that the A23187-induced rise in intracellular Ca(2+) inhibited wild-type hENaC through a W-7-sensitive mechanism, which likely reflected enhanced removal of Na(+) channels from the cell membrane by endocytosis. We, therefore, propose that Na(+) absorption in cortical collecting duct cells is inhibited by Ca(2+), possibly when complexed with calmodulin.

We used the patch-clamp technique to examine the effect of angiotensin II (ANG II) on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. Application of ANG II increased the channel activity and the current amplitude of the basolateral 50-pS K channel. The stimulatory effect of ANG II on the K channels was completely abolished by losartan, an inhibitor of type 1 angiotensin receptor (AT1R), but not by PD123319, an AT2R antagonist. Moreover, inhibition of phospholipase C (PLC) and protein kinase C (PKC) also abrogated the stimulatory effect of ANG II on the basolateral K channels in the TAL. This suggests that the stimulatory effect of ANG II on the K channels was induced by activating PLC and PKC pathways. Western blotting demonstrated that ANG II increased the phosphorylation of c-Src at tyrosine residue 416, an indication of c-Src activation. This effect was mimicked by PKC stimulator but abolished by calphostin C. Moreover, inhibition of NADPH oxidase (NOX) also blocked the effect of ANG II on c-Src tyrosine phosphorylation. The role of Src-family protein tyrosine kinase (SFK) in mediating the effect of ANG II on the basolateral K channel was further suggested by the experiments in which inhibition of SFK abrogated the stimulatory effect of ANG II on the basolateral 50-pS K channel. We conclude that ANG II increases basolateral 50-pS K channel activity via AT1R and that activation of AT1R stimulates SFK by a PLC-PKC-NOX-dependent mechanism.

Background: Evidences have shown that local anaesthetics are clinically useful compounds that exert a pharmacological effect by blocking nerve impulse propagation and also it is able to provoke proliferation and cell growth. Aims: The aim of this study was to investigate the proliferation and cell growth capacity of lidocaine on human gingival fibroblast cells and the different signal pathways involved in its effect. Method: For this purpose in vitro cultures of human gingival fibroblasts were assayed and the effects of lidocaine on proliferation and cell DNA synthesis, Na+-K+-ATPase and PKC activities and K+ efflux were also evaluated. Results: Lidocaine stimulated in a concentration-dependent manner proliferation and cell growth of human gingival cells and the mechanism involve an increment in Na+-K+-ATPase and PKC activities, which led to an increase in K+ release. All of these effects were blocked by tetrodotoxin, ouabain and calphostin C. In addition, PMA (activator of PKC) increased per se the DNA synthesis of human gingival fibroblast cells. Conclusions: This work demonstrates that lidocaine increase human gingival fibroblasts DNA synthesis and proliferation through an activation of PKC pathway accompanied by the stimulation of Na+-K+-ATPase activity with an increase in K+ efflux. These results contribute to showing another action of lidocaine different to its general use as a drug that relieves odontologic pain or acts as an anti-arrithmogenic agent.

Objectives: Shengmaisan (SMS) is a traditional Chinese medicine prescription widely used for the treatment of diverse organs in Korea. The interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the generation of coordinated gastrointestinal (GI) motility. We have aimed to investigate the effects of SMS in the ICCs in the mouse small intestine. Methods: To dissociate the ICCs, we used enzymatic digestions from the small intestine in a mouse. After that, the ICCs were identified immunologically by using the anti-c-kit antibody. In the ICCs, the electrophysiological whole-cell patch-clamp configuration was used to record pacemaker potentials in the cultured ICCs. Results: The ICCs generated pacemaker potentials in the mouse small intestine. SMS produced membrane depolarization with concentration-dependent manners in the current clamp mode. Pretreatment with a Ca 2+ free solution and thapsigargin, a Ca 2+ -ATPase inhibitor in the endoplasmic reticulum, stopped the generation of the pacemaker potentials. In the case of Ca 2+ -free solutions, SMS induced membrane depolarizations. However, when thapsigargin in a bath solution was applied, the membrane depolarization was not produced by SMS. The membrane depolarizations produced by SMS were inhibited by U-73122, an active phospholipase C (PLC) inhibitors. Furthermore, chelerythrine and calphostin C, a protein kinase C (PKC) inhibitors had no effects on SMS-induced membrane depolari zations. Conclusions: These results suggest that SMS might affect GI motility by modulating the pacemaker activ ity through an internal Ca2+- and PLC-dependent and PKC-independent pathway in the ICCs.

Several receptor systems in the bladder causing detrusor smooth muscle contraction stimulate phospholipase C (PLC). PLC inhibition abolishes bladder contraction via P2Y6 but not that via M3 muscarinic receptors, indicating a receptor-dependent role of PLC. Therefore, we explored the role of PLC in rat bladder contraction by bradykinin. The PLC inhibitor U 73,122 [1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione] did not affect the bradykinin response to a significantly greater degree than its inactive analog U 73,343 [10 μM each; 1-(6-[-([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-2,5-pyrrolidinedione], whereas the phospholipase D inhibitor butan-1-ol relative to its inactive control butan-2-ol caused a weak but significant inhibition (0.3% each). The cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (300 μM) and the cyclooxygenase inhibitor indomethacin (10 μM) caused strong inhibition of the bradykinin response. The L-type Ca(2+) channel blocker nifedipine (10-100 nM) concentration-dependently caused strong inhibition, whereas only a small but significant inhibition was seen with SKF 96,365 [10 μM; 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl], an inhibitor of receptor-operated Ca(2+) channels. Several protein kinase C inhibitors yielded an equivocal picture (inhibition by 10 μM bisindolylmaleimide I and 1 μM calphostin but not by 10 μM chelerythrine). The rho kinase inhibitor Y 27,632 [1-10 μM; trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide] caused a strong and concentration-dependent inhibition of the bradykinin response. Our data support that not only M3 but also bradykinin receptors cause bladder contraction by a largely PLC-independent mechanism. Both responses strongly involve L-type Ca(2+) channels and rho kinase, whereas only the bradykinin response additionally involves the phospholipase A2/cyclooxygenase pathway.

Nitric oxide (NO) is an important molecule involved in nociceptive processing in the central nervous system. The release of NO within the spinal cord has long been implicated in the mechanisms underlying exaggerated pain sensitivity, and administration of NO donors can induce hyperalgesia. To elucidate the supraspinal mechanism responsible for NO-induced nociceptive hypersensitivity, we investigated the modulation of protein kinase C (PKC) and downstream effectors following treatment with the NO donors nitroglycerin and sodium nitroprusside. Both compounds induced a prolonged cold allodynia and heat hyperalgesia, increased levels of c-Fos and IL-1β, and activated NF-κB within periaqueductal grey matter and thalamus. Simultaneously, an increased expression and phosphorylation of PKC γ and ε were detected. To clarify the cellular mechanism involved in the NO-induced hypernociception, we examined the expression of transcription factors that act as PKC downstream effectors. A dramatic hyperphosphorylation of CREB and STAT1 was observed. The i.c.v. administration of the PKC blocker calphostin C prevented the NO-induced hypernociception, the hyperphosphorylation of CREB and STAT1, and partially reduced NF-κB activation. Conversely, the increase of IL-1β was unmodified by calphostin C. These results suggest the relevance of cerebral PKC-mediated CREB and STAT1 activation in the NO donor-induced nociceptive behavior.

We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca2+]iproduced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca2+]i, augmented the KCl-induced increase in [Ca2+]i, and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-l-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca2+], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca2+]i. Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca2+]i, whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na+–K+ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca2+-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca2+]iin response to ouabain.

Hyperglycemia of diabetes is associated with increase in the generation of reactive oxygen species (ROS) and vascular complications. Resveratrol (RSV) has been proposed as a therapeutic resource for human diseases such as type 2 diabetes mellitus (T2DM). However, the role of hyperglycemia on resveratrol action in cells from innate immunity is poorly studied.The aim of the present study was, to evaluate the effects of RSV on ROS production by granulocytes from T2DM patients “primed” by chronic hyperglycemia “in vivo”. The effects of RSV on ROS production in granulocytes were quantified using a luminol assay. The granulocyte activators were phorbol 12,13-dibutyrate (PDB;), andLPS or zymosan (TRL4- and TRL2-activators, respectively). In some experiments, calphostin C were compared with those of RSV. RSV suppressed ROS generation in granulocytes from T2DM patients and non- diabetic (ND) controls in a similar manner (p>0.05). Percentage inhibition values was 85% and 64% inresting cells, 69 and 77%in PDB-stimulated cells, 69 and 67%in TRL4-activated cells, and 76 and 59%in TRL2-activated cells in T2DM patients and ND controls, respectively. The profile of inhibition of ROS production by calphostin C was similar to that obtained with RSV. The effect of RSV is not affected by hyperglycemia. ROS production in granulocytes from T2DM patients and ND controls wasinhibited by the polyphenol in a similar manner (p>0.05) The inhibition of ROS generation by RSV was comparable with that observed with calphostin C. PKC and/or NADPH-oxidase are targets of RSV action. RSV could be considered a therapeutic option to control innate immune oxidizing response and inflammation in diabetic complications.

Activation of protein kinase C (PKC), a serine/threonine protein kinase, ubiquitously influences cellular signal transduction and has been shown to play a role in viral entry. In this study, we explored a role for PKC in human adenovirus type 37 infection of primary human corneal fibroblasts, a major target cell for infection. We sought evidence for an interaction between PKC activation and two potential downstream targets: cSrc kinase, shown previously to play a critical role in adenovirus signaling in these cells, and caveolin-1, reported earlier to be important to entry of adenovirus type 37. Infection of fibroblasts increased PKCα phosphorylation and translocation of PKCα from the cytosol to caveolin-1 containing vesicles. Virus-induced phosphorylation of both cSrc and AKT was abolished in cell lysates pretreated with calphostin C, a chemical inhibitor of PKC. Inhibition of PKC also reduced virus associated phosphorylation of caveolin-1, while inhibition of cSrc by the chemical inhibitor PP2 reduced only caveolin-1 phosphorylation, but not PKCα phosphorylation, in lipid rafts. These results suggest a role for PKCα upstream to both cSrc and caveolin-1. Phosphorylated PKCα was found in the same endosomal fractions as phosphorylated cSrc, and PKCα was present to a greater degree in caveolin-1 pull downs from virus infected than mock infected cell lysates. Calphostin C also reduced early viral gene expression, indicating that PKCα activity may be required for viral entry. PKCα plays a central role in adenovirus infection of corneal fibroblasts and regulation of downstream molecules, including the important lipid raft component caveolin-1.

Formation of foam cells from macrophages, which are formed by the differentiation of blood-borne monocytes, is a critical early event in atherogenesis. To examine how pre-exposure of monocytes to modified proteins, such as oxLDL, influences their differentiation to macrophages, an in vitro model system using isolated PBMC maintained in culture in the presence of oxLDL was used. Pretreatment of monocytes with oxLDL caused a faster rate of expression of macrophage-specific functions and loss of monocyte-specific functions compared to unmodified LDL. The effect of oxidation of lipid component of LDL by CuSO(4) and its protein component by HOCl, on mo-mϕ differentiation was studied by monitoring the upregulation of macrophage-specific functions, particularly MMP-9. Chloroquine, a lysosomal degradation blocker, significantly reversed the effect mediated by CuSO(4) oxLDL, indicating the involvement of lysosomal degradation products, while no such effect was observed in HOCl oxLDL-treated cells, indicating the existence of a pathway independent of its lysosomal degradation products. Reversal of the effect of oxLDL by NAC and Calphostin C, an inhibitor of PKC, suggested the activation of RO-mediated signaling pathways. Use of inhibitors of signaling pathways showed that CuSO(4) oxLDL upregulated mϕ-specific MMP-9 through p38 MAPK and Akt-dependent pathways, while HOCl oxLDL utilized ERK ½ and Akt. Further analysis showed the activation of PPARγ and AP-1 in CuSO(4) oxLDL, while HOCl-oxLDL-mediated effect involved NFκB and AP-1. These results suggest that lipid oxLDL- and protein oxLDL-mediated upregulation of mo-mϕ-specific functions involve lysosomal degradation-dependent and -independent activation of intracellular signaling pathways.

Kv1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (IKur). The regulatory Kvβ1.3 subunit converts Kv1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by Kvβ1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack Kvβ subunits) transiently cotransfected with Kv1.5+Kvβ1.3 and also rat ventricular and atrial tissue to study native α-β subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that Kv1.5 and Kvβ1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, Kvβ1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed Kvβ1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of Kv1.5 and Kvβ1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between Kv1.5, Kvβ1.3, the receptor for activated C kinase (RACK1), PKCβI, PKCβII, and PKCθ in HEK293 cells. A very similar Kv1.5 channelosome was found in rat ventricular tissue but not in atrial tissue.

Background GLUT2 is translocated to the apical membrane of enterocytes exposed to glucose concentrations >∼50 mM. Mechanisms of GLUT2-mediated glucose uptake in cell culture models of enterocytes have not been studied. Aim To explore mechanism(s) of glucose uptake in 3 enterocyte-like cell lines. Methods Glucose uptake was measured in Caco-2, RIE-1, and IEC-6 cell lines using varying concentrations of glucose (0.5–50 mM). Effects of phlorizin (SGLT1 inhibitor), phloretin (GLUT2 inhibitor), nocodazole and cytochalasin B (disrupters of cytoskeleton), calphostin C and chelerythrine (PKC inhibitors), and phorbol 12-myristate 13-acetate (PKC activator) were evaluated. Results Phlorizin inhibited glucose uptake in all 3 cell lines. Phloretin inhibited glucose uptake in Caco-2 and RIE-1 cells. Starving cells decreased glucose uptake in Caco-2 and RIE-1 cells. Glucose uptake was saturated at >10 mM glucose in all 3 cell lines when exposed briefly ( 5 min in Caco-2 and RIE-1 cells, glucose uptake did not saturate and Km and Vmax increased. This increase in glucose uptake was inhibited by phloretin, nocodazole, cytochalasin B, calphostin C, and chelerythrine. PMA enhanced glucose uptake by 20%. Inhibitors and PMA had little or no effect in the IEC-6 cells. Conclusion Constitutive expression of GLUT2 in the apical membrane along with additional translocation of cytoplasmic GLUT2 to the apical membrane via an intact cytoskeleton and activated PKC appears responsible for enhanced carrier-mediated glucose uptake at greater glucose concentrations (>20 mM) in Caco-2 and RIE-1 cells. IEC-6 cells do not appear to express functional GLUT2.

Endothelin-1 (ET-1) is produced in unusually large amounts by the renal collecting duct and acts locally to control renal salt and water excretion and arterial pressure; disorders of collecting duct ET-1 activity can cause marked hypertension. The mechanisms regulating collecting duct ET-1 synthesis are, however, poorly understood. In this study, we investigated the role of protein kinase C (PKC), a known regulator of ET-1 production in endothelial cells, in (1) the control of collecting duct ET-1 production; and (2) the modulation of ET-1 promoter region activity. Cultured rat inner medullary collecting duct (IMCD) cells were studied. Calphostin C, a PKC inhibitor, greatly reduced IMCD ET-1 release. Sustained exposure to phorbol myristate acetate (PMA) also decreased ET-1 secretion. PKC inhibition decreased steady-state ET-1 mRNA content. A brief exposure (15 min) to PMA augmented ET-1 mRNA levels, while prolonged PMA exposure (120 min) reduced ET-1 mRNA content, PKC inhibition did not affect ET-1 mRNA stability. Transfection of ET-1 promoter-luciferase reporter constructs into IMCD cells demonstrated that PKC inhibition reduced activity of only the larger promoter fragments (containing at least 1,725 bp 5' to the ET-1 gene transcription start site). Mutation of a previously identified AP-1 site at -186 in the ET-1 promoter greatly reduced activity of transfected ET-1 promoter-reporter constructs (containing 366 or 1,725 bp 5' to the transcription start site); however, this region appears not to be regulated by PKC in IMCD cells. In summary, PKC stimulates collecting duct ET-1 synthesis via transcriptional activation of the ET-1 promoter. Such transcriptional activation occurs at a heretofore undescribed PKC-regulated region of the ET-1 promoter.

Although great advances have recently been made in the study of signal transduction, the pathogenesis of affective disorders is still unknown. There is mounting evidence suggesting that elevated phosphoinositide–protein kinase C (PI–PKC) signal transduction pathway may be a pathophysiological feature of bipolar and major depressive disorders. The aim of the present study was to further investigated the phospholipase C–protein kinase C (PLC–PKC) cascade by evaluating the effect produced by an acute blockade of this intracellular pathway at PLC and PKC level. Adult male mice were administered with pharmacological inhibitors of PLC or PKC and then subjected to the forced swim test (FST), an animal model which emulates the behavioural despair paradigm of depression. In this study we also tested the hypothesis that it might be possible to selectively modulate depressive behaviour by inhibiting the expression of specific PLC and PKC isoforms by means of specific antisense oligonucleotides (aODNs). Administration of the PLC inhibitors neomycin and U73122 as well as of the PKC inhibitors calphostin C and chelerytrine dose-dependently reduced the immobility time in the FST producing an antidepressant-like behaviour. Selective knockdown of the PLCβ 1 and PKCγ isoforms also induced an antidepressant phenotype. Conversely, the inhibition of the expression of PLCβ 3 was unable to modify the immobility time values. The PLC and PKC modulators used, at the highest effective doses, altered neither locomotor activity nor motor coordination. We demonstrate that selective blockade of PLCβ 1 –PKCγ signalling pathway produces an antidepressant-like phenotype in mice.

UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of a wide variety of xeno/endobiotics. Previous studies have reported that human UGT enzymes are phosphorylated and that treatment of cells with protein kinase C (PKC) inhibitors results in decreased UGT activities without affecting the UGT protein levels. In this study, we investigated the effects of PKC inhibitors on human UGT1A protein levels and activities in detail. When UGT1A-expressing HEK293 cells and LS180 cells were treated with curcumin or calphostin C, the exogenous and endogenous UGT1A protein levels in homogenates prepared with Tris-buffered saline were significantly decreased. Enzyme activity levels mirrored the changes in UGT protein levels. When the curcumin- or calphostin C-treated cells were lysed with buffer containing a detergent, the UGT protein levels did not decrease. We found that curcumin or calphostin C treatment facilitated the degradation of UGT protein after the cells were collected in the absence of a detergent. Finally, by in cellulo evaluation, we found that curcumin decreased UGT activity by the direct inhibitory effect, but calphostin C did not affect UGT activity. Thus, this study suggests that we should evaluate the data carefully when interpreting the effects of PKC inhibitors on UGT activity.

In Oncopeltus fasciatus, evidence shown here indicates it is calmodulin (CaM) that activates phospholipase-C (PLC), beginning a signalling pathway necessary for endocytic uptake of yolk precursor molecules. Epithelial cell-produced CaM, transported to oocytes via gap junctions, has been shown to be required for receptor-mediated endocytic uptake of vitellogenins (Vgs, the protein precursors of yolk). To determine if CaM was directly or indirectly stimulating the phospholipase-C (PLC) signalling cascade and thus controlling Vg endocytosis we used a series of molecules known to inactivate various elements of the pathway. W-7 prevents CaM from interacting with other molecules. Neomycin isolates PIP(2) from PLC. U-73122 directly inactivates PLC. 2-APB blocks IP(3) receptors which would otherwise cause release of Ca(2+). Verapamil and CdCl(2) block Ca(2+) release channels. Staurosporin and calphostin are inhibitors of PK-C. 1-Hexadecyl-2-acetyl glycerol (HAG) binds to diacylglycerol (DAG). Through the use of these antagonists we show here that: (1) the activation of phospholipase-C in this system requires CaM. (2) Stimulated phospholipase-C converts PIP(2) into IP(3) and DAG. (3) IP(3) causes increase in cytosolic Ca(2+). (4) DAG and Ca(2+) each stimulate phosphokinase-C, resulting in endocytosis of Vgs.

Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP(3)Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP(3)Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP(3)Rs requires binding of IP(3), generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP(3)R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP(3)R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells.

Protein kinase C (PKC) is a multigene family of serine/threonine protein kinases involved in cell signaling pathways of proliferation and motility. PKC interacts with Rho GTPases in the regulation of the actin cytoskeleton. The PKC-alpha isozyme binds the Rho GTPase cdc42, and both are coordinated with the Rac-phosphatidylinositol-3 kinase (PI3K) signaling pathway in melanoma cell invasion and migration on extracellular matrix proteins. To further define the role of PKC-alpha in melanoma cell migration, we tested the effect of PDBu and Ca dependent activation of PKC-alpha as well as treatment with the PKC-alpha inhibitors calphostin C and Go6976. Furthermore, we transfected siRNA targeted against PKC-alpha into human melanoma cells and performed time-lapse analysis of cell migration followed by western immunoblotting. We found that significant enhancement of cell migration at 0.5 h after PDBu treatment directly correlated with Ca dependent activation of PKC-alpha and was inhibited by the PKC-alpha inhibitor calphostin C. PKC-alpha siRNA transfection nearly abrogated PKC-alpha expression and significantly reduced melanoma cell migration compared with siRNA controls. These findings provide further evidence that PKC-alpha plays an important role in melanoma cell migration and may have implications in therapies designed to disrupt melanoma cell motility by alteration of PKC-alpha signaling.

In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na(+)+K(+))ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH(3) and U73122, specific inhibitors of PI-PLC. (Na(+)+K(+))ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50nM. This effect was completely reversed by 10nM calphostin C, an inhibitor of PKC. Thus, the effect of 50nM heme on (Na(+)+K(+))ATPase activity is completely abolished by ET-18-OCH(3) and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na(+)+K(+))ATPase activity through a PI-PLC/PKC signaling pathway.

Gelsolin plays an important role in the regulation of amyloid beta-protein fibrillogenesis. We report here that calcium ionophore A23187 induces the expression of cytoplasmic gelsolin (c-gelsolin), and that protein kinase C (PKC) is involved in the up-regulation of c-gelsolin. In the presence of calcium, both SH-SY5Y and HEK-293 cells upon treatment with A23187 showed an increase in c-gelsolin expression in a concentration-dependent manner. Calcium-mediated up-regulation of c-gelsolin was inhibited by cycloheximide (a general inhibitor of protein synthesis). When cells were pretreated with staurosporine (an inhibitor of a variety of protein kinases including PKC), the up-regulation of c-gelsolin induced by A23187 was inhibited. Calphostin C, an inhibitor of PKC, blocked the up-regulation of c-gelsolin induced by A23187, while inhibitors of mitogen-activated protein kinases had no effect on c-gelsolin expression. In addition, phorbol-12-myristate-13-acetate, an activator of PKC, up-regulated c-gelsolin expression. These results suggest that calcium mediates up-regulation of c-gelsolin in a PKC-dependent manner.

We have previously reported that gelsolin, an actin binding protein, regulates the fibrillization of amyloid-beta protein. We report here that the expression of cytoplasmic gelsolin (cgelsolin) was upregulated in a concentration-dependent manner when SH-SY5Y, PC-12, and HEK-293 cells were subjected to oxidative stress by treatment with hydrogen peroxide (H(2)O(2). Further studies were done to elucidate the mechanism involved in the regulation of c-gelsolin expression in cells. Pretreatment of cells with cycloheximide (an inhibitor of protein synthesis) resulted in significant inhibition of H(2)O(2)induced c-gelsolin expression, suggesting the possible de novo synthesis of c-gelsolin in cells. Staurosporine, a potent inhibitor of a variety of protein kinases including protein kinase C (PKC), also blocked the H(2)O(2)induced expression of cgelsolin. However, both H(2)O(2) and staurosporine activated the mitogen-activated protein kinases (MAPKs), i.e., c-Jun N-terminal kinase, P38, and extracellular signal-regulated kinase. Pretreatment of cells with Calphostin C, an inhibitor of PKC, blocked the upregulation of cgelsolin induced by H(2)O(2), while specific inhibitors of MAPKs had no effect on c-gelsolin expression, suggesting that MAPKs may not be involved in H(2)O(2)mediated upregulation of cgelsolin. On the other hand, phorbol-12-myristate-13-acetate, an activator of PKC, induced the expression of c-gelsolin. Our studies indicate that c-gelsolin is upregulated in cells under oxidative stress, and PKC is involved in its upregulation. It is suggested that activators of PKC that induce gelsolin expression may have therapeutic significance in Alzheimer's disease.

The invasiveness of glioma cells, a major cause of mortality in malignant brain tumors, is mediated in part by the cellular microenvironment. We have reported that in a three-dimensional matrix of type 1 collagen (3D-CL) gel, the extracellular matrix protein tenascin-C (TN) increased the invasiveness of glioma cells through the downstream production of matrix metalloproteinase (MMP)-12. In the present study, we have investigated the signaling mechanisms involved in the TN-stimulated glioma invasiveness. We found that the pan protein kinase C (PKC) inhibitor, bisindolylmaleimide I, decreased TN-enhanced glioma invasion in 3D-CL. Calphostin C, an inhibitor of conventional and novel PKC isozymes, and the relatively selective PKCdelta inhibitor rottlerin decreased TN-stimulated glioma invasiveness in a concentration- and time-dependent manner. These findings of the possible involvement of PKCdelta was supported by its translocation from the cytosol to membrane fraction in 3D-CL gel supplemented with TN as detected by western blot assays and immunofluorescence microscopy and by elevation of PKCdelta enzyme activity. Moreover, pharmacological blockade of PKCdelta decreased MMP-12 levels and glioma invasiveness. Finally, small interfering RNA to PKCdelta reduced TN-stimulated glioma invasiveness concurrent with decreased MMP-12 production. Our results implicate PKCdelta as a therapeutic target to reduce MMP-12 expression and glioma invasiveness when tumor cells are stimulated by the TN-enriched glioma microenvironment.

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.

Background and purpose: We have previously demonstrated that stimulation of imidazoline receptors in the CNS prevented halothane–adrenaline arrhythmias during halothane anaesthesia and that stimulation of the vagus nerve may be critical to this effect. However, details of the mechanism(s) involved are not yet available. The present study was designed to examine the role of muscarinic receptors, protein kinase C (PKC), ATP-sensitive potassium channels (KATP) and the mitochondrial permeability transition pore (MPTP) in the antiarrhythmic effect of rilmenidine, an imidazoline receptor agonist. Experimental approach: Rats were anaesthetized with halothane and monitored continuously for arterial blood pressure and premature ventricular contractions. The arrhythmogenic dose of adrenaline was defined as the lowest dose producing three or more premature ventricular contractions within a 15-s period. We confirmed that centrally administered rilmenidine prevented halothane–adrenaline arrhythmias and then examined the antiarrhythmic effect of rilmenidine in the presence of atropine methylnitrate, a muscarinic receptor antagonist, calphostin C, a PKC inhibitor, HMR-1098, a sarcolemmal KATP inhibitor, 5-hydroxydecanoic acid, a mitochondrial KATP inhibitor or atractyloside, an MPTP opener. Key results: The antiarrhythmic effect of rilmenidine was significantly inhibited by atropine methylnitrate, calphostin C, 5-hydroxydecanoic acid and atractyloside, but the effects of HMR-1098 in our model were not clear. Conclusions and implications: The present results suggest that muscarinic receptors, PKC, mitochondrial KATP channels and MPTP may be crucial components of the mechanism involved in the antiarrhythmic effect of rilmenidine given into the CNS.

In a previous study, we observed that angiotensin(1–7) (Ang(1–7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1–7) on Na+-ATPase activity through AT1R involves a Gq protein–phosphatidyl inositol-phospholipase Cβ(PI-PLCβ) pathway because: (1) the effect was reversed by GDPβS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPγS, a non-hydrolysable GTP analogue; (3) Ang(1–7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCβ substrate; and (4) U73122, a specific inhibitor of PI-PLCβ, abolished Ang(1–7)-induced stimulation of Na+-ATPase activity. Angiotensin(1–7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1–7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1–7) stimulates Na+-ATPase activity through the AT1R–Gq protein–PI-PLCβ–PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.

Alpha-secretase (alpha-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence, resulting in the release of a secreted fragment of APP (alphaAPPs) and precluding Abeta generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Abeta generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0-10 microM) increased alphaAPPs release. Therefore, we evaluated two alpha-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-alpha converting enzyme (TACE)/ADAM17 inhibited the Hup A-induced rise in alphaAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic alpha-secretase pathway. Hup A had no effect on Abeta generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A-induced alphaAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer's disease (AD).

The vascular effects of glucose-intolerance were investigated using the neonatal streptozotocin-treated (nSTZ) rat model. Glucose-intolerance was initiated by administration of STZ (90 mg/kg, IP) into 2-day-old male rats. Aortic reactivity was assessed in vitro at 3 and 6 months of age. Both the 3- and 6-month-old nSTZ rats displayed higher blood glucose levels in response to a glucose challenge. At 3 months of age, aortic responsiveness to both norepinephrine and acetylcholine was not altered. However, at 6 months of age, the responses of endothelium-denuded aortas from nSTZ rats to norepinephrine and serotonin were enhanced compared to controls. Endothelium-mediated relaxation of aortas from these animals to acetylcholine was also augmented, and this effect was linked to NO release. Although norepinephrine did not elicit enhancement of aortic contraction in calcium-free medium in 6-month-old nSTZ rats, the responses to both maximum and submaximum concentrations of the agonist after readdition of calcium were greater in these tissues than in control preparations. Pretreatment of aortas with calphostin C eliminated the difference in NE-induced contraction between the control and experimental groups. Although the concentration-response curves for phorbol 12,13-dibutyrate were not different between the 2 groups, the responses of the aortas from 6-month-old nSTZ rats to a submaximum concentration of the phorbol ester were enhanced relative to controls, and this enhancement was normalized with calphostin C. Overall, the data suggest that glucose-intolerance of sufficient duration causes increases in vascular reactivity to agonists. While these findings warrant further investigations, such vascular alterations during the prediabetes stage of glucose intolerance can be a predisposing factor for the eventual development of cardiovascular complications.

Cardiomyocyte sodium accumulation after burn injury precedes the development of myocardial contractile dysfunction. The present study examined the effects of burn injury on Na-K-ATPase activity in adult rat hearts after major burn injury and explored the hypothesis that burn-related changes in myocardial Na-K-ATPase activity are PKC dependent. A third-degree burn injury (or sham burn) was given over 40% total body surface area, and rats received lactated Ringer solution (4 ml·kg−1·% burn−1). Subgroups of rats were killed 2, 4, or 24 h after burn ( n = 6 rats/time period), hearts were homogenized, and Na-K-ATPase activity was determined from ouabain-sensitive phosphate generation from ATP by cardiac sarcolemmal vesicles. Additional groups of rats were studied at several times after burn to determine the time course of myocyte sodium loading and the time course of myocardial dysfunction. Additional groups of sham burn-injured and burn-injured rats were given calphostin, an inhibitor of PKC, and Na-K-ATPase activity, cell Na+, and myocardial function were measured. Burn injury caused a progressive rise in cardiomyocyte Na+, and myocardial Na-K-ATPase activity progressively decreased after burn, while PKC activity progressively rose. Administration of calphostin to inhibit PKC activity prevented both the burn-related decrease in myocardial Na-K-ATPase and the rise in intracellular Na+and improved postburn myocardial contractile performance. We conclude that burn-related inhibition of Na-K-ATPase likely contributes to the cardiomyocyte accumulation of intracellular Na+. Since intracellular Na+is one determinant of electrical-mechanical recovery after insults such as burn injury, burn-related inhibition of Na-K-ATPase may be critical in postburn recovery of myocardial contractile function.

Vasodilator-stimulated phosphoprotein (VASP) is a cAMP-dependent protein kinase A (PKA) substrate, which links cellular signaling to cytoskeletal organization and cellular movement. VASP is phosphorylated by PKA on serine 157 (Ser 157), which is required for VASP function in platelet adhesion and fibroblast motility. Our hy- pothesis is that PKA regulates neutrophil migration through VASP Ser 157 phosphorylation. The ob- jective of this study was to characterize VASP Ser 157 phosphorylation in chemoattractant-stimu- lated neutrophils. fMLF, IL-8, leukotriene B4 ,o r platelet-activating factor stimulation resulted in an initial increase in VASP Ser 157 phosphorylation, which was maximal by 30 s and was followed by a return to baseline Ser 157 phosphorylation by 10 min. In contrast, stimulation with the nonchemoat- tractant, proinflammatory cytokine TNF- did not affect Ser 157 phosphorylation. The kinetics of fMLF-induced VASP Ser 157 phosphorylation lev- els closely matched the kinetics of the fold-change in F-actin levels in fMLF-stimulated neutrophils. fMLF-induced Ser 157 phosphorylation was abol- ished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced Ser 157 phosphorylation was unaffected by the PKC inhibitors calphostin and staurosporine, the PKG inhibitors Rp-8-pCPT- cGMP and KT5823, and the calmodulin-depen- dent protein kinase II inhibitor KN-62. Inhibition of adhesion with EDTA or the anti-2-integrin an- tibody IB4 did not alter fMLF-induced VASP phos- phorylation or dephosphorylation. These data show that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA- dependent VASP Ser 157 phosphorylation. Adhe- sion does not appear to be an important regulator of the state of VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils. J. Leu- koc. Biol. 82: 1311-1321; 2007.

Angiotensin II (ANG II) inhibits bTREK-1 (bovine KCNK2) K+channels in bovine adrenocortical cells through a Gq-coupled AT1receptor by activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways. Whole cell patch-clamp recording from bovine adrenal zona fasciculata (AZF) cells was used to characterize the ATP-dependent signaling mechanism for inhibition of bTREK-1 by ANG II. We discovered that ATP-dependent inhibition of bTREK-1 by ANG II occurred through a novel mechanism that was independent of PLC and its established downstream effectors. The ATP-dependent inhibition of bTREK-1 by ANG II was not reduced by the PLC antagonists edelfosine and U73122, or by the PKC antagonists bisindolylmaleimide I (BIM) or calphostin C. bTREK-1 was partially inhibited (∼25%) by the PKC activator phorbol 12,13 dibutyrate (PDBu) through an ATP-dependent mechanism that was blocked by BIM. Addition of Phosphatidylinositol( 4 , 5 ) bisphosphate diC8 [DiC8PI( 4 , 5 )P2], a water-soluble derivative of phosphotidyl inositol 4,5 bisphosphate (PIP2) to the pipette solution failed to alter inhibition by ANG II. bTREK-1 inhibition by ANG II was also insensitive to antagonists of other protein kinases activated by ANG II in adrenocortical cells but was completely blocked by inorganic polytriphosphate PPPi. DiC8PI( 4 , 5 )P2was a weak activator of bTREK-1 channels, compared with the high-affinity ATP analog N6-(2-phenylethyl)adenosine-5′-O-triphosphate (6-PhEt-ATP). These results demonstrate that the modulation of bTREK-1 channels in bovine AZF cells is distinctive with respect to activation by phosphoinositides and nucleotides and inhibition by Gq-coupled receptors. Importantly, ANG II inhibits bTREK-1 channels through a novel pathway that is different from that described for inhibition of native TREK-1 channels in neurons, or cloned channels expressed in cell lines. They also indicate that, under physiological conditions, ANG II inhibits bTREK-1 and depolarizes AZF cells by two, novel, independent pathways that diverge proximal to the activation of PLC.

Agonists of protease-activated receptor 2 (PAR(2)) evoke hyperexcitability of dorsal root ganglia (DRG) neurons by unknown mechanisms. We examined the cellular mechanisms underlying PAR(2)-evoked hyperexcitability of mouse colonic DRG neurons to determine their potential role in pain syndromes such as visceral hyperalgesia. Colonic DRG neurons were identified by injecting Fast Blue and DiI retrograde tracers into the mouse colon. Using immunofluorescence, we found that DiI-labelled neurons contained PAR(2) immunoreactivity, confirming the presence of receptors on colonic neurons. Whole-cell current-clamp recordings of acutely dissociated neurons demonstrated that PAR(2) activation with a brief application (3 min) of PAR(2) agonists, SLIGRL-NH(2) and trypsin, evoked sustained depolarizations (up to 60 min) which were associated with increased input resistance and a marked reduction in rheobase (50% at 30 min). In voltage clamp, SLIGRL-NH(2) markedly suppressed delayed rectifier I(K) currents (55% at 10 min), but had no effect on the transient I(A) current or TTX-resistant Na(+) currents. In whole-cell current-clamp recordings, the sustained excitability evoked by PAR(2) activation was blocked by the PKC inhibitor, calphostin, and the ERK(1/2) inhibitor PD98059. Studies of ERK(1/2) phosphorylation using confocal microscopy demonstrated that SLIGRL-NH(2) increased levels of immunoreactive pERK(1/2) in DRG neurons, particularly in proximity to the plasma membrane. Thus, activation of PAR(2) receptors on colonic nociceptive neurons causes sustained hyperexcitability that is related, at least in part, to suppression of delayed rectifier I(K) currents. Both PKC and ERK(1/2) mediate the PAR(2)-induced hyperexcitability. These studies describe a novel mechanism of sensitization of colonic nociceptive neurons that may be implicated in conditions of visceral hyperalgesia such as irritable bowel syndrome.