P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε

Abstract: Incubation of Neuro 2A mouse neuroblastoma cells with UTP and UDP results in a concentration-dependent increase in the accumulation of inositol phosphates with equal potency and maximal effect; ATP, ADP, and 2-methylthioadenosine 5′-triphosphate were much less potent, indicating the expression of P2Y receptor in these cells. The effects of UTP and ATP were not affected by pretreatment of cells with pertussis toxin, indicating that the P2Y receptor in Neuro 2A cells is coupled to pertussis toxin-insensitive G_q protein. Short-term (10 min) treatment of cells with 1 µM 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the inhibition of the UTP and ATP effects; this inhibitory effect was gradually attenuated with increased length of TPA treatment (1.5–6 h) and was not seen after long-term (24 h) treatment. Western blot analysis showed the expression of protein kinase C (PKC) α, ε, θ, and ζ in Neuro 2A cells. Translocation of PKCα, ε, and θ from the cytosol to the membrane was seen after 10 min or 1.5 h of treatment with TPA. However, partial and complete down-regulation of both membrane PKCα and θ were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA-induced translocation of PKCε was maintained after 3–6 h of treatment, and almost complete down-regulation occurred only after a 24-h treatment. The observed TPA-induced inhibition of UTP- or ATP-stimulated phosphoinositide hydrolysis, therefore, correlated well with the extent of translocation of PKCε. Phosphoinositide hydrolysis induced by AlF₄^?, but not Ca²⁺ ionophores, was inhibited by a 10-min treatment with TPA. This was not seen after a 24-h treatment, indicating that the site of action of PKCε in the P2Y receptor/G_q protein/phospholipase Cβ pathway might be the G_q protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal PKCε in the regulation of the receptor-mediated phosphoinositide turnover.     

Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway

1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)₂D₃ which have been modified on the A-ring and C,D-ring side chain (1α-(hydroxymethyl)-3β-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YA = 3a) and 1β-(hydroxymethyl)-3α-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)₂D₃. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)₂D₃, 24,25-(OH)₂D₃, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)₂D₃ and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)₂D₃ having a greater effect. 24,25-(OH)₂D₃ caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)₂D₃, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation–dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Biochem. 66:457–470, 1997. © 1997 Wiley-Liss, Inc.     

Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury

Phospholipase A₂ is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A₂ (cPLA₂)-related inflammatory responses after TBI. We found that cPLA₂ and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA₂ expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA₂ inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA₂ in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA₂-related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA₂ activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA₂ activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA₂-related inflammatory response from the PKC pathway.     

Malarial Hemozoin Is a Nalp3 Inflammasome Activating Danger Signal

Background

Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents.

Methodology/Principal Findings

We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1β. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K⁺ efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged.

Significance/Conclusions

The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.     

Cross Talk Between Substance P and Melittin-Activated Cellular Signaling Pathways in Rat Lactotroph-Enriched Cell Cultures

Abstract: We have investigated the possible interaction (cross talk) between the phospholipase A₂ (PLA₂) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [³H]-arachidonic acid ([³H]AA) from [³H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKCα and β immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Mellitin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA₂ inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [³H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [³H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [³H]AA production was not different from control, whereas SP still displayed [³H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [³H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [³H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of ³H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA₂ pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes α and β, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA₂ pathway at a level at or beyond PLA₂, and this effect is mediated, in part, through PKCα and β species and (for SP) intracellular Ca²⁺ recruited from internal stores as well as from external sources; and (3) SP also activates PLA₂ through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.     

The role of protein kinase C in thromboxane A2-induced pulmonary artery vasoconstriction

In order to determine if protein kinase C (PKC) plays a significant role in the stimulant action of thromboxane A₂ (TxA₂) on pulmonary vascular smooth muscle, TxA₂-induced contractile responses were measured following inhibition of PKC. Rabbits were sacrificed and segments of the main trunk of the pulmonary artery were removed and placed within a temperature-controlled (37 °C) organ bath. Contractile responses that were evoked by a TxA₂ mimetic (U46,619, 0.5 µM) decreased by 27 and 35% following treatment with the PKC inhibitors, calphostin C (2 µM) and staurosporine (200 nM), respectively. These results account for the effect of the vehicle, DMSO, which was also found to have a concentration-dependent inhibitory effect on the U46,619-induced contractions. The effects of DMSO alone was subsequently subtracted from the previously measured responses to PKC inhibitors that were dissolved in DMSO to obtain effects attributable to the PKC inhibitor alone. It can therefore be concluded that inhibition of PKC results in partial attenuation of U46,619-induced responses supporting the hypothesis that activation of PKC plays a partial role in TxA₂-induced contraction of pulmonary arterial smooth muscle.     

Lack of evidence for direct phosphorylation of recombinantly expressed P2X2 and P2X3 receptors by protein kinase C

P2X₃ and P2X₂₊₃ receptors are present on sensory neurons, where they contribute not only to transient nociceptive responses, but also to hypersensitivity underlying pathological pain states elicited by nerve injuries. Increased signalling through P2X₃ and P2X₂₊₃ receptors may arise from an increased routing to the plasma membrane and/or gain of function of pre-existing receptors. An obvious effector mechanism for functional modulation is protein kinase C (PKC)-mediated phosphorylation, since all P2X family members share a conserved consensus sequence for PKC, TXR/K, within the intracellularly located N-terminal domain. Contradictory reports have been published regarding the exact role of this motif. In the present study, we confirm that site-directed elimination of the potential phosphor-acceptor threonine or the basic residue in the P+2 position of the TXR/K sequence accelerates desensitization of P2X₂ receptors and abolishes P2X₃ receptor function. Moreover, the PKC activator phorbol 12-myristate 13-acetate increased P2X₃ (but not P2X₂) receptor-mediated currents. Biochemically, however, we were unable to demonstrate by various experimental approaches a direct phosphorylation of wild-type P2X₂ and P2X₃ receptors expressed in both Xenopus laevis oocytes and HEK293 cells. In conclusion, our data support the view that the TXR/K motif plays an important role in P2X function and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes. The underlying mechanism, however, is unlikely to involve direct PKC-mediated P2X receptor phosphorylation.     

Inhibition of protein kinase C βII isoform ameliorates methylglyoxal advanced glycation endproduct-induced cardiomyocyte contractile dysfunction

Aims

Accumulation of advanced glycation endproduct (AGE) contributes to diabetic complication including diabetic cardiomyopathy although the precise underlying mechanism still remains elusive. Recent evidence depicted a pivotal role of protein kinase C (PKC) in diabetic complications. To this end, this study was designed to examine if PKCβII contributes to AGE-induced cardiomyocyte contractile and intracellular Ca^2 + aberrations.

Main methods

Adult rat cardiomyocytes were incubated with methylglyoxal-AGE (MG-AGE) in the absence or presence of the PKCβII inhibitor LY333531 for 12 h. Contractile and intracellular Ca^2 + properties were assessed using an IonOptix system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), rise in intracellular Ca^2 + Fura-2 fluorescence intensity and intracellular Ca^2 + decay. Oxidative stress, O₂⁻ production and mitochondrial integrity were examined using TBARS, fluorescence imaging, aconitase activity and Western blotting.

Key findings

MG-AGE compromised contractile and intracellular Ca^2 + properties including reduced PS, ± dL/dt, prolonged TPS and TR₉₀, decreased electrically stimulated rise in intracellular Ca^2 + and delayed intracellular Ca^2 + clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued MG-AGE-induced oxidative stress, O₂⁻ generation, cell death, apoptosis and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were replicated by the NADPH oxidase inhibitor apocynin and were mitigated by the mitochondrial uncoupler FCCP.

Significance

These findings implicated the therapeutic potential of specific inhibition of PKCβII isoform in the management of AGE accumulation-induced myopathic anomalies.     

Evidence that Prostaglandin E2 Can Block Calcium-Activated 86Rb Efflux from Rat Brain Synaptosomes via a Protein Kinase C-Dependent Mechanism

Abstract: The effects of prostaglandin E₂ (PGE₂) on ⁸⁶Rb efflux from rat brain synaptosomes were studied to explore its role in nerve ending potassium (K⁺) channel modulation. A selective dose-dependent inhibition of the calcium-activated charybdotoxin-sensitive component of efflux was found upon application of PGE₂. No significant effect was seen on basal and voltage-dependent components over the concentration range of 10–⁸ to 10–⁵M. The protein kinase C (PKC) inhibitors H-7 (10 μM) and staurosporine (100 nM), as well as prolonged preincubation (90 min) with 40-phorbol 12, 13-dibutyrate, which has been reported to down-regulate PKC, abolished the PGE₂-in- duced inhibition, whereas HA1004 (10 μM) and Rp-3′,5’cyclic phosphorothioate (100 nM), which are relatively more selective for protein kinase A than PKC, did not. 4β-Phorbol 12, 13-dibutyrate (100 nM), an activator of PKC, produced a similar inhibition of the Ca²⁺-dependent component of ⁸⁶Rb efflux but also had no effect on the basal and voltage-dependent components. These data suggest that PGE₂ can inhibit rat brain nerve ending calcium-activated ⁸⁶Rb efflux, and this inhibition may involve PKC activation.     

ATP7B activity is stimulated by PKCɛ in porcine liver

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)–ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10⁻⁸ M), a PKC activator, increased Cu(I)–ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca²⁺, whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced V_max without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.     

Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

Regulation of Renal Na-HCO3 Cotransporter: VIII. Mechanism of Stimulatory Effect of Respiratory Acidosis

We examined the effect of respiratory acidosis on the Na-HCO₃ cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO₂ for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO₂ showed a significant increase in Na-HCO₃ cotransporter activity (expressed as % of control levels, 5 min: 142 ± 6, 2 hr: 144 ± 13, 4 hr: 145 ± 11, 24 hr: 150 ± 15, 48 hr: 162 ± 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO₂ for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO₂ for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO₂ for 5 min or 4 hr. In cells treated with vehicle, 10% CO₂ significantly increased cotransporter activity as compared to control cells exposed to 5% CO₂. This stimulation by 10% CO₂ was completely prevented by HAC or DHC at 5 min (5% CO₂: 1.8 ± 0.2, 10% CO₂: 2.6 ± 0.2, 10% CO₂+ HAC: 1.6 ± 0.2, 10% CO₂: +DHC: 2.0 ± 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO₂ for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO₃ cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis. Received: 28 March 1997/Revised: 22 December 1997     

The membrane effects of 17β-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

Uptake and degradation of insulin and α2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways

The cell association and degradation of insulin and α₂-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 μM) reduced both the uptake of α₂-macroglobulin · trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2–3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of α₂-macroglobulin · trypsin markedly without affecting that of insulin. Leupeptin (100 μM) increased uptake and reduced degradation of α₂-macroglobulin · trypsin without affecting insulin. Dansylcadaverine (500 μM) almost abolished uptake and degradation of α₂-macroglobulin · trypsin but had little effect on insulin. Moreover, uptake and degradation of α₂-macroglobulin · trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.     

Inactivation of protein kinase C in rat liver during late hypoglycemic phase of sepsis

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat liver during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. Hepatic PKC was extracted and partially purified by ammonium sulfate fractionation and DEAE-cellulose chromatography. PKC activity was assayed based on the rate of incorporation of ³²p from [-³²P]ATP into histone. The results show that during early sepsis, both membrane-associated and cytosolic PKC activities remained relatively unaltered. During late sepsis, membrane-associated PKC was unaffected while cytosolic PKC activity was decreased by 19.5-34.4%. Kinetic analysis of the data on cytosolic PKC during late phase of sepsis reveals that the V_max values for ATP, histone, Ca²⁺, phosphatidylserine, and diacylglycerol were decreased by 23.4, 22.1, 19.5, 25, and 34.4%, respectively, with no changes in their K_m values. These data indicate that cytosolic PKC activity was inactivated in rat liver during late hypoglycemic phase of sepsis. Since PKC-mediated phosphorylation plays an important role in regulating hepatic glucose metabolism, an inactivation of cytosolic PKC may contribute to the development of hypoglycemia during late phase of sepsis.     

Aspirin inhibits lipopolysaccharide-induced COX-2 expression and PGE2 production in porcine alveolar macrophages by modulating protein kinase C and protein tyrosine phosphatase activity

Aspirin has been demonstrated to be effective in inhibiting COX-2 and PGE₂ in Alveolar macrophages (AMs). However, the mechanisms have not been fully understood. In the present study, we found that pretreatment with aspirin inhibited LPS-induced COX-2 and PGE₂ upregulation, IκBα degradation, NFκB activation and the increase of PKC activity, but elevated LPS-induced the decrease of PTP activity. The PKC inhibitor calphostin C dramatically reduced the COX-2 mRNA and PGE₂ levels, but the PTP inhibitor peroxovanadium (POV) significantly increased the COX-2 mRNA and PGE₂ levels. Furthermore, the PTP inhibitor mitigated the inhibitory effect of aspirin on COX-2 and PGE₂ upregulation and NF-κB activation, whereas the PKC inhibitor enhanced the inhibitory effects of aspirin on the production of COX-2 and PGE₂. Our data indicate a novel mechanism by which aspirin acts as a potent anti-inflammatory agent in alveolus macrophages and ALI. [BMB Reports 2014; 47(1): 45-50]     

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 μM or 50 μM OAG for 10 min resulted in meiosis II completion in ∼80% of instances. By contrast, at a lower concentration (25 μM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 μM OAG, the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) increased transiently in all the eggs examined, whereas after the addition of 50 μM OAG, [Ca²⁺]_i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca²⁺ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway. Mol. Reprod. Dev. 48:292–299, 1997. © 1997 Wiley-Liss, Inc.     

P2X7 receptor antagonists display agonist-like effects on cell signaling proteins

Background

The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca²⁺]_i. We report that some agents that can block P2X₇R receptors also promote diverse P2X₇R-independent effects on cell signaling.

Methods

We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X₇R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.

Results

With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated ⁴⁵Ca²⁺ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X₇R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.

Conclusions

Several agents used as P2X₇R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

General significance

Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.