The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation

Challenge of protease-activated receptors induces cytosolic Ca(2+) concentration ([Ca(2+) ](c)) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca(2+) ](c) rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca(2+) ](c) rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca(2+) ](c) response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca(2+) amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O(2) (?-) -release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca(2+) ](c) increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration.     

Involvement of the plasma membrane Ca2+-ATPase in the short-term response of Arabidopsis thaliana cultured cells to oligogalacturonides

Treatment of Arabidopsis thaliana cells with oligogalacturonides (OG) initiates a transient production of reactive oxygen species (ROS), the concentration of which in the medium peaks after about 20 min of treatment. The analysis of OG effects on Ca (2+) fluxes shows that OG influence both Ca (2+) influx and Ca (2+) efflux (measured as (45)Ca (2+) fluxes) in a complex way. During the first 10 - 15 min, OG stimulate Ca (2+) influx and decrease its efflux, while at successive times of treatment, OG cause an increase of Ca (2+) efflux and a slight decrease of its influx. Treatment with sub- micro M concentrations of eosin yellow (EY), which selectively inhibits the Ca (2+)-ATPase of plasma membrane (PM), completely prevents the OG-induced increase in Ca (2+) efflux. EY also suppresses the transient feature of OG-induced ROS accumulation, keeping the level of ROS in the medium high. The biochemical analysis of PM purified from OG-treated cells indicates that treatment with OG for 15 to 45 min induces a significant decrease in Ca (2+)-ATPase activation by exogenous calmodulin (CaM), and markedly increases the amount of CaM associated with the PM. During the same time span, OG do not influence the expression of At-ACA8, the main isoform of PM Ca (2+)-ATPase in suspension-cultured A. thaliana cells, and of CaM genes. Overall, the reported results demonstrate that the PM Ca (2+)-ATPase is involved in the response of plant cells to OG and is essential in regulation of the oxidative burst.     

Osmotically induced cytosolic free Ca(2+) changes in human neutrophils

Cytosolic free Ca(2+) concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300-600 mM) or sucrose (600-1200 mM), caused a rise in cytosolic free Ca(2+) (Delta(max) approximately equal to 600 nM). This was not due to cell lysis as the cytosolic free Ca(2+) concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca(2+) could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca(2+) concentration occurred in the absence of extracellular Ca(2+), demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca(2+) was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca(2+) may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca(2+) (Delta(max) approximately equal to 600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca(2+) to physiologically important levels.     

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.     

Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change.

Platelets undergo shape change upon activation with agonists. During shape change, disc-shaped platelets turn into spiculated spheres with protruding filopodia. When agonist-induced cytosolic Ca(2+) increases were prevented using the cytosolic Ca(2+) chelator, 5, 5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5, 5'-dimethyl-BAPTA), platelets still underwent shape change, although the onset was delayed and the initial rate was dramatically decreased. In the absence of cytosolic Ca(2+), agonist-stimulated myosin light chain phosphorylation was significantly inhibited. The myosin light chain was maximally phosphorylated at 2 s in control platelets compared with 30 s in 5,5'-dimethyl-BAPTA-treated platelets. ADP, thrombin, or U46619-induced Ca(2+)-independent platelet shape change was significantly reduced by staurosporine, a nonselective kinase inhibitor, by the selective p160 Rho-associated coiled-coil-containing protein kinase inhibitor Y-27632, or by HA 1077. Both Y-27632 and HA 1077 reduced peak levels of ADP-induced platelet shape change and myosin light chain phosphorylation in control platelets. In 5,5'-dimethyl-BAPTA-treated platelets, Y-27632 and HA 1077 completely abolished both ADP-induced platelet shape change and myosin light chain phosphorylation. Our results indicate that Ca(2+)/calmodulin-stimulated myosin light chain kinase and p160 Rho-associated coiled-coil-containing protein kinase independently contribute to myosin light chain phosphorylation and platelet shape change, through Ca(2+)-sensitive and Ca(2+)-insensitive pathways, respectively.     

Calcium-dependent oxidation of thioredoxin during cellular growth initiation.

The fraction of cell thiol proteins in the oxidized disulfide form were quantified during mitogen-induced HaCaT keratinocyte growth initiation. Oxidized thioredoxin increased from 11 +/- 1.2% in resting cells to 80 and 61% 2 min after addition of bradykinin or EGF. Thioredoxin oxidation was transient returning toward normal values by 20 min. The disulfide forms of other cellular proteins rose in parallel with thioredoxin oxidation. The oxidation of thioredoxin depended on a rise in cytosolic calcium. It was prevented by preloading cells with BAPTA, a Ca(2+) chelator and induced by addition of Ca(2+)-ionophore A23187 or of thapsigargin. In cell extracts, thioredoxin reductase was inhibited by micromolar calcium. The rise in cytosolic Ca(2+) led to a concomitant burst of H(2)O(2) formation. The oxidizing intracellular milieu suggests that redox regulation actively participates in the growth initiation cascade. The role of peroxiredoxins and ASK 1 cascade activation are discussed in this context.     

Cross-talk between cAMP and calcium signalling in Aspergillus niger

Very little is known about cross-talk between cAMP and calcium signalling in filamentous fungi. The aim of this study was to analyse the influence of cAMP and protein kinase A (PKA)-dependent phosphorylation on calcium signalling in Aspergillus niger. For this purpose, cytosolic free calcium ([Ca2+]c) was measured in living hyphae expressing codon-optimized aequorin. The calcium signature following mechanical perturbation was analysed after applying dibutryl-cAMP or IBMX which increased intracellular cAMP, or H7 which inhibited phosphorylation by PKA. Calcium signatures were also measured in mutant strains in which phosphorylation by PKA was increased or lacking. The results indicated that calcium channels were activated by cAMP-mediated, PKA-dependent phosphorylation. Further evidence for cross-talk between cAMP and calcium signalling came from the analysis of a mutant in which the catalytic subunit of PKA was under the control of an inducible promoter. The consequence of PKA induction was a transient increase in [Ca2+]c which correlated with a polar-apolar transition in hyphal morphology. A transient increase in [Ca2+]c was not observed in this mutant when the morphological shift was in the opposite direction. The [Ca2+]c signatures in response to mechanical perturbation by polarized and unpolarized cells were markedly different indicating that these two cell types possessed different calcium signalling capabilities. These results were consistent with PKA-dependent phosphorylation increasing [Ca2+]c to induce a polar to apolar shift in hyphal morphology.     

Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.     

Shiga toxin activates p38 MAP kinase through cellular Ca(2+) increase in Vero cells

We examined whether the mitogen-activated protein kinase (MAPK) pathway is involved in Shiga toxin (Stx)-induced Vero cell injury. Consonant with cell injury, Stx caused a transient extracellular signal-regulated kinase1/2 (ERK1/2) and a sustained p38 MAPK phosphorylation. p38 MAPK inhibitors (SB 203580 and PD 169316), but not an ERK1/2 kinase inhibitor (PD 98059), partially inhibited the Stx-induced cell death. BAPTA-AM, a Ca(2+) chelator, reduced both cell injury and p38 MAPK phosphorylation. Antioxidants reduced Stx1-induced p38 MAPK phosphorylation. These data indicate that Stx activates p38 MAPK through an increase in intracellular Ca(2+) and reactive oxygen species, and this signaling is involved in Stx-induced cell death.     

TGF-beta1 calcium signaling in osteoblasts

Transforming growth factor-beta1 (TGF-beta1) action is known to be initiated by its binding to multiple cell surface receptors containing serine/threonine kinase domains that act to stimulate a cascade of signaling events in a variety of cell types. We have previously shown that TGF-beta1 and BMP-2 treatment of primary human osteoblasts (HOBs) enhances cell-substrate adhesion. In this report, we demonstrate that TGF-beta1 elicits a rapid, transient, and oscillatory rise in the intracellular Ca(2+) concentration, [Ca(2+)](i), that is necessary for enhancement of cell adhesion in HOBs but does not alter the phosphorylation state of Smad proteins. This rise in [Ca(2+)](i) in HOB is not observed in the absence of extracellular calcium or when the cells are treated with the L-type Ca(2+) channel blocker, nifedipine, but is stimulated upon treatment with the L-type Ca(2+) channel agonist, Bay K 8644, or under high K(+) conditions. The rise in [Ca(2+)](i) is severely attenuated after treatment of the cells with thapsigargin, a selective endoplasmic reticulum Ca(2+) pump inhibitor. TGF-beta1 enhancement of HOB adhesion to tissue culture polystyrene is also inhibited in cells treated with nifedipine. These data suggest that intracellular Ca(2+) signaling is an important second messenger of the TGF-beta1 signal transduction pathway in osteoblast function.     

Characteristic features of kynurenine aminotransferase allosterically regulated by (alpha)-ketoglutarate in cooperation with kynurenine

Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5'-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.     

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.     

The mechanism of excitation-contraction coupling in phenylephrine-stimulated human saphenous vein

The human saphenous vein (HSV) is the most widely used graft in coronary artery revascularization procedures and is susceptible to spasm perioperatively. The aim of this study is to elucidate the mechanism(s) of agonist-induced excitation-contraction coupling in this vessel. Isometric contraction experiments were combined with in situ smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) imaging by confocal microscopy of intact undistended HSV segments during activation with phenylephrine (PE; 50 microM). Stimulation with PE produced a sustained contraction. Preincubation with 5 microM nifedipine, a blocker of the L-type voltage-operated Ca(2+) channel, or 50 microM SKF-96365, a blocker of both the voltage- and receptor-operated channels, reduced force generation by 25-30%. Ca(2+) imaging revealed that PE elicited only a transient rise in [Ca(2+)](i), suggesting that Ca(2+) plays only a minor role. However, a requirement for basal Ca(2+) levels was demonstrated when PE contractions could not be maintained in Ca(2+)-free medium. In light of the transient Ca(2+) response, it appears that signals other than Ca(2+) must maintain the tonic contraction elicited by PE, such as those that sensitize the myofilaments to Ca(2+). Application of HA-1077 (a Rho kinase inhibitor) at the peak of the contraction completely abolished the plateau phase of the response, whereas application of genistein (a tyrosine kinase inhibitor) reduced this phase by approximately 50%. The foregoing results suggest that, whereas the transient Ca(2+) signal can contribute to the development of force, maintenance of the plateau phase of the PE contraction in the HSV is the result of myofilament Ca(2+) sensitization by Rho kinase and tyrosine phosphorylation. The elucidation of the mechanisms of excitation-contraction coupling in the HSV may be useful for the development of therapeutic strategies for the alleviation of vein graft spasm.     

The grateful dead: calcium and cell death in plant innate immunity

Plant cells sensing pathogenic microorganisms evoke defence systems that can confer resistance to infection. This innate immune reaction can include triggering of basal defence responses as well as programmed cell death, or hypersensitive response (HR). In both cases (basal defence and HR), pathogen perception is translated into elevated cytosolic Ca(2+) (mediated by plasma membrane and intracellular channels) as an early step in a signalling cascade. Cyclic nucleotide-gated channels contribute to this influx of Ca(2+) into the cell. The molecular nature of other transport proteins contributing to the Ca(2+) elevation is unclear. Pathogen recognition occurs at two levels: the perception of pathogen-associated molecular pattern (PAMP) molecules widely present in microorganisms, and an interaction between pathogen avirulence gene products (if present) and corresponding plant R (resistance) gene products. The Ca(2+) elevation occurring in response to PAMP perception or R gene interactions could occur due to phosphorylation events, G-protein signalling and/or an increase in cyclic nucleotides. Downstream from the initial Ca(2+) rise, the signalling cascade includes: activation of calmodulin and protein kinases, and nitric oxide and reactive oxygen species generation. Some of these downstream events amplify the Ca(2+) signal by further activation of Ca(2+) transporters.     

Norepinephrine induces slow calcium signalling in murine brown preadipocytes through the beta-adrenoceptor/cAMP/protein kinase A pathway

The mechanism of adrenergically activated calcium signalling in isolated murine brown preadipocytes (stromal-vascular fraction) was studied with Fura-2. Norepinephrine (NE) generated in preadipocytes a slow Ca(2+)-response ( approximately 10 nM/min) without a burst and a maximum, whereas in mature brown adipocytes, the quick burst reached 1.5 microM [Ca(2+)](i). Thapsigargin, which is known to discharge Ca(2+) ions from the IP(3)-sensitive stores, initiated a huge capacitative calcium entry in mature brown adipocytes but failed to stimulate a response in preadipocytes. The beta-selective antagonist nadolol almost completely prevented the effect of NE on [Ca(2+)](i), while the antagonist of alpha-adrenoceptors phentolamine caused only a approximately 25% reduction of the cellular response. Forskolin or the cell-permeable Br-cAMP caused [Ca(2+)](i) rise, which were even higher than with NE. The protein kinase A (PKA) inhibitor N-[2-(p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) reduced and the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX), N-cyclohexyl-N-(2-hydroxyethyl)-4-(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide (OPC-3911), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidone (Ro 20-1724) or the protein phosphatase inhibitor okadaic acid enhanced the NE-, isoproterenol- or forskolin-initiated cellular calcium responses. It was concluded that (i) brown preadipocytes lacked a trigger mechanism of initiation of [Ca(2+)](i) rises and (ii) the cAMP- and protein kinase A-mediated phosphorylation played an important role in the beta-adrenoceptor-initiated calcium signalling in these cells. All these features distinguish brown adipocyte precursors from differentiated brown adipocytes, where calcium signalling is initiated exclusively via alpha(1)-adrenoceptors and the trigger mechanism.     

Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum

We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca(2+) release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca(2+) uptake and (ii) in maintaining a sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)). For this purpose, we expressed in HeLa cells aequorin-based Ca(2+)-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H(1) receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca(2+)](c) rise and drop in ER luminal [Ca(2+)] ([Ca(2+)](er)), and then the ER refilled with [Ca(2+)](c) at resting values. With histamine, [Ca(2+)](c) after the initial peak stabilized at a sustained plateau, and [Ca(2+)](er) decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca(2+) response than glutamate ( approximately 15 versus approximately 65 microm). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca(2+)](c) plateau and the sustained ER Ca(2+) release and markedly increased the mitochondrial Ca(2+) response. Conversely, mitochondrial Ca(2+) uptake evoked by histamine was drastically reduced by very transient ( approximately 2-s) agonist applications. These data indicate that efficient mitochondrial Ca(2+) uptake depends on the preservation of high Ca(2+) microdomains at the mouth of ER Ca(2+) release sites close to mitochondria. This in turn depends on continuous Ca(2+) release balanced by Ca(2+) reuptake into the ER and maintained by Ca(2+) influx from the extracellular space.     

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)

The rapid generation of H(2)O(2) by Cd(2+)-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H(2)O(2) has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H(2)O(2) induced by Cd(2+). These data suggest the involvement of an NADPH oxidase-like enzyme leading to H(2)O(2) production through O(2)(*-) dismutation by superoxide dismutase enzymes. To investigate the implication of Ca(2+) channels in a Cd(2+)-induced oxidative burst, different inhibitors of Ca(2+) channels were used. Only La(3+) totally inhibited the generation of H(2)O(2) induced by Cd(2+). However, verapamil and nifedipine, inhibitors of Ca(2+) channels, were not effective. Calmodulin or a Ca(2+)-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H(2)O(2) induced by Cd(2+), suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.     

Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi

Our goal was to evaluate early signaling events that occur as epithelial cells make initial contact with a substrate and to correlate them with phosphorylation. The corneal epithelium was chosen to study signaling events that occur with adhesion because it represents a simple system in which the tissue adheres to a basal lamina, is avascular, and is bathed by a tear film in which changes in the local environment are hypothesized to alter signaling. To perform these experiments we developed a novel adhesion assay to capture the changes in intracellular Ca(2+) and pH that occur as a cell makes its initial contact with a substrate. The first transient cytosolic Ca(2+) peak was detected only as the cell made contact with the substrate and was demonstrated using fluorimetric assays combined with live cell imaging. We demonstrated that this transient Ca(2+) peak always preceded a cytoplasmic alkalization. When the intracellular environment was modified, the initial response was altered. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N, N'N'-tetraacetic acid (BAPTA), an intracellular chelator, inhibited Ca(2+) mobilization, whereas benzamil altered the duration of the oscillations. Thapsigargin caused an initial Ca(2+) release followed by a long attenuated response. An inositol triphosphate analog induced a large initial response, whereas heparin inhibited Ca(2+) oscillations. Inhibitors of tyrosine phosphorylation did not alter the initial mobilization of cytosolic Ca(2) but clearance of cytosolic Ca(2+) was inhibited. Exposing corneal epithelial cells to BAPTA, benzamil, or thapsigargin also attenuated the phosphorylation of the focal adhesion protein paxillin. However, although heparin inhibited Ca(2+) oscillations, it did not alter phosphorylation of paxillin. These studies demonstrate that the initial contact that a cell makes with a substrate modulates the intracellular environment, and that changes in Ca(2+) mobilization can alter later signaling events such as the phosphorylation of specific adhesion proteins. These findings may have implications for wound repair and development.