Regulation of phosphatidylserine synthesis in Jurkat T cell clones: caffeine bypasses CD3/TCR-induced protein tyrosine kinases and calcium signals

Phosphatidylserine synthesis as measured by the incorporation of [(3)H]serine into phosphatidylserine (PtdSer) through the serine-base exchange enzyme system (serine-BEES) is markedly inhibited in Jurkat cells treated with caffeine. The caffeine-induced inhibition was compared to that observed in cells treated with either CD3 mAb or thapsigargin. While CD3- and thapsigargin-induced inhibition was related to the release of Ca(2+) from the endoplasmic reticulum (ER), a process that deprives the serine-BEES of its major cofactor, caffeine modified PtdSer synthesis in the absence of decreased Ca(2+) content of ER. Using Jurkat clones differing by the expression of cell surface markers or protein tyrosine kinases implicated in the CD3/TCR signal transmission pathway, we have shown that CD3 mAb-induced inhibition of PtdSer synthesis necessitates the expression of both the CD3/TCR and the protein tyrosine phosphatase CD45 at the cell surface as well as the presence of p56(lck) and ZAP-70 protein tyrosine kinases. By contrast, thapsigargin, a blocker of the Ca(2+)-ATPase of the ER, known for its Ca(2+) releasing properties, inhibited PtdSer synthesis in all the Jurkat clones tested, indicating that this compound bypasses the CD3/TCR-induced signals. Despite its lack of effect on Ca(2+) release from ER and on protein tyrosine phosphorylations, caffeine inhibited PtdSer synthesis in all the Jurkat clones. The use of several cAMP-inducing drugs and of others xanthine derivatives indicated that caffeine modify PtdSer synthesis either by a direct action on the serine-BEES or by a modification of the structure of the phospholipids used as substrate by the enzyme.     

Inhibition of phosphatidylserine synthesis during Jurkat T cell activation. The phosphatase inhibitor, sodium ortho-vanadate bypasses the CD3/T cell receptor-induced second messenger signaling pathway.

Sodium ortho-vanadate (Na3VO4), an inhibitor of protein tyrosine phosphatase, induces a rapid (15 min) and strong inhibition of phosphatidylserine synthesis with an IC50 = 100 microM. The mode of action of Na3VO4 was compared to that of CD3 mAbs. It was found that Na3VO4 bypasses the major CD3-induced T cell activation signals including protein tyrosine phosphorylation, p56lck activation and the generation of second messengers including inositol phosphates and its subsequent Ca2+ mobilization as well as diacylglycerol production. These facts were confirmed by using a panel of Jurkat clones that differs by the expression of either tyrosine kinases involved in the CD3-induced T cell activation pathway such as p56lck, p72syk and ZAP-70 or some cell surface receptors such as the CD3/TCR complex or the CD45 phosphatase.     

Modulation of CCK-8-evoked intracellular Ca2+ waves by hydrogen peroxide in mouse pancreatic acinar cells.

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.     

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.     

XOD-catalyzed ROS generation mobilizes calcium from intracellular stores in mouse pancreatic acinar cells.

In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactive oxygen species (ROS) generation caused an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) by release of Ca(2+) from intracellular stores. The ROS-induced Ca(2+) signals showed large variability in shape and time-course and resembled in part Ca(2+) signals in response to physiological secretagogues. ROS-induced Ca(2+) mobilization started at the luminal cell pole and spread towards the basolateral side in a wave manner. ROS-evoked Ca(2+) responses were not inhibited by the phospholipase C (PLC) inhibitor U73122 (10 microM). Neither 2-aminoethoxy-diphenylborate (2-APB) (70 microM) nor ryanodine (50 microM) suppressed ROS-evoked Ca(2+) release. ROS still released Ca(2+) when the endoplasmic reticulum Ca(2+)-ATPase was blocked with thapsigargin (1 microM), or when rotenone (10 microM) was added to release Ca(2+) from mitochondria. Our results suggest that pancreatic acinar cells ROS do not unspecifically affect Ca(2+) homeostasis. ROS primarily affect Ca(2+) stores located in the luminal cell pole, which is also the trigger zone for agonist-induced Ca(2+) signals. Release of Ca(2+) induces Ca(2+) waves carried by Ca(2+)-induced Ca(2+) release and produces thereby global Ca(2+) signals. Under oxidative stress conditions, the increase in [Ca(2+)](i) could be one mechanism contributing to an overstimulation of the cell which could result in cell dysfunction and cell damage.     

Galectin-1 signaling in leukocytes requires expression of complex-type N-glycans

Dimeric galectin-1 (dGal-1) is a homodimeric lectin with multiple proposed functions. Although dGal-1 binds to diverse glycans, it is unclear whether dGal-1 preferentially binds to specific subsets of glycans on cell surfaces to transmit signals. To explore this question, we selectively inhibited major glycan biosynthetic pathways in human HL60, Molt-4, and Jurkat cells. Inhibition of N-glycan processing blocked surface binding of dGal-1 and prevented dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. By contrast, inhibition of O-glycan or glycosphingolipid biosynthesis did not affect dGal-1 binding or dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. These results demonstrate that dGal-1 preferentially binds to and signals through glycoproteins containing complex-type N-glycans in at least some leukocyte subsets.     

Inhibition of store-operated calcium entry in human lymphocytes by radiation: protection by glutathione

The influence of gamma radiation on basal compared to activation-dependent Ca(2+) influx in human lymphocytes was investigated. A new quantitative fluorescence technique termed differential ratiometric fluorescence spectroscopy (DRFS) was employed. DRFS facilitated the real-time detection of changes in fluorescence in experimental and control cell samples simultaneously, enabling the resolution of acute moderate changes ( congruent with10-30%) in Ca(2+) (manganese) influx after exposure to ionizing radiation and other oxidant interventions. Exposure to radiation inhibited thapsigargin-stimulated store-operated Ca(2+) influx but not basal Ca(2+) influx in Jurkat T cells and human peripheral blood lymphocytes. The response of store-operated Ca(2+) influx to gamma radiation was dependent on dose between 5 and 40 Gy and was inhibited by preincubation with the Ca(2+) channel blocker Ni(2+), as determined with Jurkat T cells. Elevation of the intracellular concentration of glutathione significantly reduced the inhibition of Ca(2+) influx by gamma radiation. Similar to radiation, both the superoxide anion-generating xanthine/xanthine oxidase system and hydrogen peroxide inhibited thapsigargin-stimulated Ca(2+) influx in Jurkat T cells, and this inhibition was reversed in the presence of the antioxidant N-acetyl-l-cysteine. In conclusion, (1) ionizing radiation inhibited store-operated Ca(2+) entry in human lymphocytes, (2) the sensitivity of Ca(2+) influx to radiation was strictly dependent on depletion of Ca(2+) stores, and (3) glutathione protected against the inhibition of store-operated Ca(2+) entry by gamma radiation.     

Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3 microM Ca2+, and up to 80% inhibition occurring at 3-320 microM Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca(2+)-stimulated binding, but showed reduced Ca(2+)-independent binding. Ca(2+)-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca(2+)-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca(2+)-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.     

MAPK and PKC activity are not required for H(2)O(2)-induced arterial muscle contraction

H(2)O(2)-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca(2+) and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H(2)O(2)-induced contraction. Porcine PASM strips were stimulated with 1 mM H(2)O(2), 120 mM KCl, or 10 microM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 microM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing (32)P and CaCl(2) to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H(2)O(2) do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H(2)O(2), high K(+), or phorbol esters.     

Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer

The role of H(2)O(2) and protein thiol oxidation in oxidative stress-induced epithelial paracellular permeability was investigated in Caco-2 cell monolayers. Treatment with a H(2)O(2) generating system (xanthine oxidase + xanthine) or H(2)O(2) (20 microM) increased the paracellular permeability. Xanthine oxidase-induced permeability was potentiated by superoxide dismutase and prevented by catalase. H(2)O(2)-induced permeability was prevented by ferrous sulfate and potentiated by deferoxamine and 1,10-phenanthroline. GSH, N-acetyl-L-cysteine, dithiothreitol, mercaptosuccinate, and diethylmaleate inhibited H(2)O(2)-induced permeability, but it was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. H(2)O(2) reduced cellular GSH and protein thiols and increased GSSG. H(2)O(2)-mediated reduction of GSH-to-GSSG ratio was prevented by ferrous sulfate, GSH, N-acetyl-L-cysteine, diethylmaleate, and mercaptosuccinate and potentiated by 1,10-phenanthroline and 1, 3-bis(2-chloroethyl)-1-nitrosourea. Incubation of soluble fraction of cells with GSSG reduced protein tyrosine phosphatase (PTPase) activity, which was prevented by coincubation with GSH. PTPase activity was also lower in H(2)O(2)-treated cells. This study indicates that H(2)O(2), but not O(2)(-). or.OH, increases paracellular permeability of Caco-2 cell monolayer by a mechanism that involves oxidation of GSH and inhibition of PTPases.     

Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

We examined the effect of angiotensin II (ANG II) on epithelial Na(+) channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NP(o) (a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca(2+)-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H(2)O(2) diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca(2+)-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.     

Inhibition of PTPs by H(2)O(2) regulates the activation of distinct MAPK pathways

It has been shown that endogenous production of reactive oxygen species (ROS) during T cell activation regulates signaling events including MAPK activation. Protein tyrosine phosphatases (PTPs) have been regarded as targets of ROS which modify the catalytic cysteine residues of the enzymes. We have analyzed the interplay between the inhibition of PTPs and the activation of MAPK by H(2)O(2). Stimulation of Jurkat T cells with H(2)O(2) induces the phosphorylation of ERK, p38, and JNK members of MAPK family. H(2)O(2) stimulation of T cells was found to inhibit the PTP activity of CD45, SHP-1, and HePTP. Transfection of cells with wtSHP-1 decreased H(2)O(2)-induced ERK and JNK phosphorylation without affecting p38 phosphorylation. Transfection with wtHePTP inhibited H(2)O(2)-induced ERK and p38 phosphorylation without inhibiting JNK phosphorylation. The Src-family kinase inhibitor, PP2, inhibited the H(2)O(2)-induced phosphorylation of ERK, p38, and JNK. The phospholipase C (PLC) inhibitor, U73122, or the protein kinase C (PKC) inhibitor, Ro-31-8425, blocked H(2)O(2)-induced ERK phosphorylation, whereas the same treatment did not inhibit p38 or JNK phosphorylation. Taken together, these results suggest that inhibition of PTPs by H(2)O(2) contributes to the induction of distinct MAPK activation profiles via differential signaling pathways.     

Polynitroxyl alphaalpha-hemoglobin (PNH) inhibits peroxide and superoxide-mediated neutrophil adherence to human endothelial cells.

Experimental hemoglobin-based O2 carriers e.g. cross-linked alphaalpha-hemoglobin (alphaalpha-Hb), are under investigation as potential blood substitutes. However, some Hb-based products form strong oxidant species in vivo that may cause adverse clinical effects. We report the prototype of a new class of modified Hb-based O2 carrier, polynitroxylated alphaalpha-Hb (PNH), which has antioxidant activities that may reduce inflammatory effects mediated by oxidant formation. We compared the effects of alphaalpha-Hb and PNH on xanthine oxidase and H2O2-induced neutrophil-endothelial adhesion in vitro. Both peroxide (>0.1 mM), and superoxide/peroxide generated by xanthine oxidase (XO) (> 10 mU/ml) + 0.1 mM xanthine (X), increased endothelial-neutrophil adhesion. At 30 microM, alphaalpha-Hb significantly increased X/XO-mediated adhesion, while PNH inhibited peroxide or X/XO induced adhesion, with maximal inhibition at 10 microM PNH. These data indicate that PNH has antioxidant-anti-inflammatory properties that suggest its use as a potentially safer blood substitute in reperfusion injury, stroke, myocardial infarction and other forms of inflammation.     

TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca(2+)-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H(2)O(2)) evokes Ca(2+) influx through TRPM2 to activate Ca(2+)-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-kappaB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H(2)O(2)-induced Ca(2+) influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca(2+) influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.     

Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Inhibition of protein synthesis in cortical neurons during exposure to hydrogen peroxide

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H(2)O(2) formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate-induced increase in cytosolic Ca(2+) is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn(2+) preferentially inhibits the initiation step. In this study, we provide evidence indicating that H(2)O(2) and thapsigargin mobilized a common intracellular Ca(2+) pool. H(2)O(2) treatment stimulated a slow increase in intracellular Ca(2+), and precluded the effect of thapsigargin on Ca(2+) mobilization. H(2)O(2) stimulated the phosphorylation of both eIF-2alpha and eEF-2, in a time- and dose-dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H(2)O(2)-induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H(2)O(2) treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF-2. In conclusion, H(2)O(2) inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF-2alpha and eEF-2 and the relative contribution of these two events depends on the duration of H(2)O(2) treatment.     

Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H(2)O(2) are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 microM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca(2+)-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca(2+) sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca(2+)-independent PKC.     

Common mechanisms for declines in oxidative stress tolerance and proliferation with aging

Aging is often characterized by reduced stress tolerance and decreased proliferative capacity, but little is known about the effects of aging on signaling pathways important in regulating these responses. Recent studies from our laboratory have implicated impairments in epidermal growth factor receptor (EGFR) signaling and extracellular signal-regulated kinase (ERK) activation to both effects in rat hepatocytes. Here we investigated the responsiveness of hepatocytes derived from young (4-5 months) and aged (24-29 months) mice to proliferative signals (low concentrations of H2O2 and epidermal growth factor [EGF] stimulation), and oxidant injury (high H2O2 concentrations). Old hepatocytes displayed lower levels of DNA synthesis in response to low H(2)O(2) concentrations (5-10 microM) and EGF stimulation, and reduced survival following treatment with high H2O2 concentrations (20-50 microM). Both effects were associated with reduced activation of ERK and diminished phosphorylation of EGFR tyrosine residue 1173. p38 was also activated by H2O2, but to a greater extent in old cells. Pharmacologic inhibition of ERK increased the sensitivity of young cells to H2O2-induced cell death, while inhibition of p38 decreased the sensitivity of old cells. Our findings suggest that impairments in common signaling events underlie age-related declines in proliferative capacity and oxidative stress tolerance in mouse hepatocytes, and that an imbalance in ERK and p38 activities contributes to the greater sensitivity of aged cells to H2O2.     

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.