Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

Regulation of cadmium-induced apoptosis by PKCδ in U937 human promonocytic cells

Pulse treatment with cadmium chloride followed by recovery caused apoptosis in U937 human promonocytic cells. In addition, the treatment-induced PKCδ translocation from cytosol to membrane fraction, which was already detected at 30 min of treatment; and also caused PKCδ cleavage to give a 41-kDa fragment, which was detected at 3–6 h of recovery, concomitantly with the execution of apoptosis. All these effects were reduced by the PKCδ-specific inhibitor rottlerin. By contrast, rottlerin did not prevent the cadmium-provoked stimulation of the stress response (as measured by HSP70 expression), nor inhibited the generation of apoptosis by heat-shock, which failed to cause PKCδ translocation. Cadmium chloride rapidly induced p38^MAPK activation, which was not affected by rottlerin. By contrast, the p38^MAPK inhibitor SB203580 reduced PKCδ translocation and cleavage, indicating that p38^MAPK activation precedes and regulates PKCδ activation. It is concluded that PKCδ mediates apoptosis induction by cadmium ions via early membrane translocation, and also possibly through late kinase proteolytic cleavage and phosphorylation on tyrosine residues.     

Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

Treatment of bovine pulmonary artery smooth muscle with the O₂^•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O₂^•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O₂^•− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O₂^•− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G_iα in the microsomes. Treatment of the smooth muscle tissue with the O₂^•− generating system causes phosphorylation of G_iα in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na⁺ dependent Ca²⁺ uptake in microsomes of bovine pulmonary artery smooth muscle under O₂^•− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)     

Hydrogen Peroxide-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells: Possible Involvement of Ca2+-Dependent Protein Tyrosine Kinase

Abstract: The mechanism for hydrogen peroxide (H₂O₂)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled PC12 cells. In the presence of butanol, H₂O₂ caused a great accumulation of [³H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H₂O₂ of cell lysates exerted no effect on PLD activity. Treatment with H₂O₂ had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H₂O₂-induced PLD activity. Thus, H₂O₂-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H₂O₂-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H₂O₂ treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca²⁺ abolished H₂O₂-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca²⁺ potentiated H₂O₂-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca²⁺-dependent protein tyrosine kinase(s) somehow participates in H₂O₂-induced PLD activation in PC12 cells.     

Propofol Protects Against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Oxidative Injury in Differentiated PC12 Cells via Inhibition of Ca<Superscript>2+</Superscript>-Dependent NADPH Oxidase

Propofol (2,6-diisopropylphenol) is a widely used general anesthetic with anti-oxidant activities. This study aims to investigate protective capacity of propofol against hydrogen peroxide (H₂O₂)-induced oxidative injury in neural cells and whether the anti-oxidative effects of propofol occur through a mechanism involving the modulation of NADPH oxidase (NOX) in a manner of calcium-dependent. The rat differentiated PC12 cell was subjected to H₂O₂ exposure for 24 h to mimic a neuronal in vitro model of oxidative injury. Our data demonstrated that pretreatment of PC12 cells with propofol significantly reversed the H₂O₂-induced decrease in cell viability, prevented H₂O₂-induced morphological changes, and reduced the ratio of apoptotic cells. We further found that propofol attenuated the accumulation of malondialdehyde (biomarker of oxidative stress), counteracted the overexpression of NOX core subunit gp91^phox (NOX2) as well as the NOX activity following H₂O₂ exposure in PC12 cells. In addition, blocking of L-type Ca²⁺ channels with nimodipine reduced H₂O₂-induced overexpression of NOX2 and caspase-3 activation in PC12 cells. Moreover, NOX inhibitor apocynin alone or plus propofol neither induces a significant downregulation of NOX activity nor increases cell viability compared with propofol alone in the PC12 cells exposed to H₂O₂. These results demonstrate that the protective effects of propofol against oxidative injury in PC12 cells are mediated, at least in part, through inhibition of Ca²⁺-dependent NADPH oxidase.     

Activation of PKCδ and ERK1/2 in the sensitivity to UV-induced apoptosis of human cells harboring 4977 bp deletion of mitochondrial DNA

The 4977 bp deletion of mitochondrial DNA (mtDNA), often found in patients with chronic progressive external ophthalmoplegia (CPEO), has been demonstrated to increase the susceptibility to apoptosis of human cells. We investigated the mechanism underlying the apoptotic susceptibility of the Δ4977 cybrid harboring about 80% 4977 bp-deleted mtDNA. The production of hydrogen peroxide (H₂O₂) and phosphorylation of PKCδ and ERK1/2 were increased in the Δ4977 cybrid, which was more susceptible to UV-induced apoptosis. Moreover, treatment with N-acetyl-l-cysteine (NAC) or blocking of activation of PKCδ by rottlerin or PKCδ-siRNA, and inhibition of ERK1/2 by PD98059 or ERK1/2-siRNA significantly attenuated the susceptibility of the Δ4977 cybrid to apoptosis. Furthermore, the increase of PKCδ expression in the Δ4977 cybrid also amplified the apoptotic signal through caspase 3-mediated proteolytic activation of PKCδ. In addition, PKCδ and ERK1/2 were hyperphosphorylated in skin fibroblasts of CPEO patients harboring 4977 bp-deleted mtDNA. We suggest that the activation of PKCδ and ERK1/2 elicited by 4977 bp-deleted mtDNA-induced oxidative stress plays a role in the susceptibility of the mutant cells to apoptosis. This may explain, at least in part, the degenerative manifestation of brain and muscle in patients with mitochondrial encephalomyopathies such as CPEO syndrome.     

A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species-induced delayed cell death

Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn²⁺ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H₂O₂ induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn²⁺]_i, and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H₂O₂ increased concentration-dependently the [Zn²⁺]_i and caused lysosomal dysfunction and Zn²⁺ loss and, furthermore, mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn²⁺ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn²⁺ uptake in isolated mitochondria, which was prevented by TPEN. H₂O₂-induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H₂O₂-induced ROS generation, lysosomal dysfunction and Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.     

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H₂O₂) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H₂O₂ evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H₂O₂ resulted in a increase in [Ca²⁺] _i , indicating the release of calcium from intracellular stores. H₂O₂ abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H₂O₂-induced calcium release. We conclude that H₂O₂ induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.     

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y₂ receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y₂ receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca²⁺ mobilization indicating that nucleotides cause Ca²⁺ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca²⁺ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca²⁺ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca²⁺ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca²⁺ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca²⁺ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y₂ receptor can cause Ca²⁺ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.     

The dynamics of PKC‐induced phosphorylation triggered by Ca2+ oscillations in mouse eggs

Fertilization of mammalian eggs is characterized by a series of Ca²⁺ oscillations triggered by a phospholipase C activity. These Ca²⁺ increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC‐induced phosphorylation in mouse eggs, alongside Ca²⁺ oscillations, using fluorescent C‐kinase activity reporter (CKAR) probes. Ca²⁺ oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr²⁺ all caused repetitive increases in PKC‐induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca²⁺ transients. High frequency oscillations caused by PLCζ led to an integration of PKC‐induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca²⁺ oscillations using an isoform‐specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ‐specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC‐induced phosphorylation outlasts each Ca²⁺ increase in mouse eggs but that signal integration only occurs at a non‐physiological, high Ca²⁺ oscillation frequency. The results also suggest that Ca²⁺‐induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization. J. Cell. Physiol. 228: 110–119, 2013. © 2012 Wiley Periodicals, Inc.     

Hydrogen peroxide activates calcium influx in human neutrophils

The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback. This work shows that exogenous H₂O₂ produced a rapid influx of Mn²⁺ and an increase of intracellular calcium. The H₂O₂ was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca²⁺ to H₂O₂-treated cells suspended in a free Ca²⁺/EGTA buffer resulted in a great increase in [Ca²⁺]_i reflecting influx of Ca²⁺ across the cell membrane. The increase of intracellular calcium was inhibited by Ni²⁺, La³⁺, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H₂O₂ by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry.     

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells

In this study we investigated the responses of intracellular calcium ([Ca²⁺]_i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [³H]PDBu binding approximately 2–3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca²⁺]_i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca²⁺]_i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.Special issue dedicated to Dr. Leon S. Wolfe.     

Trpm2 enhances physiological bioenergetics and protects against pathological oxidative cardiac injury: Role of Pyk2 phosphorylation

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H₂O₂ resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca²⁺ increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca²⁺ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca²⁺ uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca²⁺ influx phosphorylated Ca²⁺-calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H₂O₂-treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca²⁺ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Hydrogen Peroxide Elicits Constriction of Skeletal Muscle Arterioles by Activating the Arachidonic Acid Pathway

Aims

The molecular mechanisms of the vasoconstrictor responses evoked by hydrogen peroxide (H₂O₂) have not been clearly elucidated in skeletal muscle arterioles.

Methods and Results

Changes in diameter of isolated, cannulated and pressurized gracilis muscle arterioles (GAs) of Wistar-Kyoto rats were determined under various test conditions. H₂O₂ (10–100 µM) evoked concentration-dependent constrictions in the GAs, which were inhibited by endothelium removal, or by antagonists of phospholipase A (PLA; 100 µM 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid), protein kinase C (PKC; 10 µM chelerythrine), phospholipase C (PLC; 10 µM U-73122), or Src family tyrosine kinase (Src kinase; 1 µM Src Inhibitor-1). Antagonists of thromboxane A2 (TXA2; 1 µM SQ-29548) or the non-specific cyclooxygenase (COX) inhibitor indomethacin (10 µM) converted constrictions to dilations. The COX-1 inhibitor (SC-560, 1 µM) demonstrated a greater reduction in constriction and conversion to dilation than that of COX-2 (celecoxib, 3 µM). H₂O₂ did not elicit significant changes in arteriolar Ca²⁺ levels measured with Fura-2.

Conclusions

These data suggest that H₂O₂ activates the endothelial Src kinase/PLC/PKC/PLA pathway, ultimately leading to the synthesis and release of TXA2 by COX-1, thereby increasing the Ca²⁺ sensitivity of the vascular smooth muscle cells and eliciting constriction in rat skeletal muscle arterioles.     

PKC-δ controls the fMLF-induced overproduction of superoxide by neutrophils

Antimicrobial defense by neutrophils implicates the production of reactive oxygen species. Neutrophil responses can be modulated by agonists such as bacterial peptides and proinflammatory factors that modulate neutrophil activity and survival. We evaluated the production of superoxide anions (O₂^?) in response to fMLF by normal human neutrophils after 3 days of preincubation with GM-CSF + IL-4 + TNF-α (survival medium). After 3 days of incubation in survival medium, long-lived neutrophils produced up to six times more O₂^? relative to control neutrophils in response to fMLF and WKYMVM. This augmented response to fMLF was preferentially linked to formyl peptide receptor (FPR), whereas the response to WKYMVM was dependent on formyl peptide receptor-like 1 (FPRL-1). Real-time RT-PCR revealed a diminution of FPR and FPRL-1 expression in neutrophils incubated in survival medium. fMLF-induced overproduction of O₂^? by long-lived neutrophils was independent of intracellular calcium mobilization. The protein tyrosine phosphorylation profile of long-lived neutrophils was modified with respect to control cells. Pharmacological inhibitors of intracellular signals indicated that mechanisms of the excessive fMLF-induced production of O₂^? by long-lived neutrophils implicated the protein kinase C (PKC) pathway, preferentially the PKC-δ isoform, whose protein was augmented in these cells. Thus, long-term cytokine exposure modifies molecular pathways and functional characteristics of the neutrophil.     

An inhibitor of cyclic AMP-dependent protein kinase enhances the superoxide production of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

Intact human neutrophils produced superoxide (O₂ ^–) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca²⁺ was absent (0.56±0.13 nmol/min per 10⁶ cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca²⁺. Moreover, the O₂ ^– production by fMLP in the presence of extracellular Ca²⁺ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca²⁺ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O₂ ^– production of electropermeabilized neutrophils in which the intracellular Ca²⁺ concentration was fixed to about 100 nM. These observations suggest that not only Ca²⁺ influx but the inhibition of PKA is necessary for the maximum O₂ ^– production by fMLP and that the O₂ ^– production is partially suppressed by the activation of PKA induced by fMLP.     

Congruence between PM H<Superscript>+</Superscript>-ATPase and NADPH oxidase during root growth: a necessary probability

Plasma membrane (PM) H⁺-ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O₂˙^?, respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H⁺-ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H⁺-ATPase inhibitor). Conversely, H⁺-ATPase activity retarded in response to different ROS scavengers [CuCl₂, N, N’ –dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl₂ and diphenyleneiodonium (DPI)], while H₂O₂ promoted PM H⁺-ATPase activity at lower concentrations. Repressing effects of Ca⁺² antagonists (La⁺³ and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H⁺-ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H⁺-ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October