NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Hypertonic environment elicits cyclooxygenase-2-driven prostaglandin E2 generation by colon cancer cells: Role of cytosolic phospholipase A2-α and kinase signaling pathways

Cyclooxygenase (COX)-2-derived prostaglandin (PG)E₂ controls many aspects of colon cancer development, modulating from apoptosis resistance and cell proliferation to angiogenesis, invasion, and metastasis. Here, we investigated the role of different phospholipases (PL)A₂ in supplying arachidonic acid (AA) for COX-2-dependent PGE₂ generation and signaling pathways involved in activation of colon cancer cells by a physiologically relevant stimulus. To emulate the hypertonic environment found physiologically in colon, the human colon cancer cell line Caco-2 was maintained in hypertonic complete DMEM medium. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked AA release, COX-2 induction and PGE₂ generation. Selective secretory (s)PLA₂ and calcium-independent (i)PLA₂ inhibitors did not modify PGE₂ generation, while either COX-2 or cytosolic (c)PLA₂ inhibitors completely inhibited PGE₂ generation. cPLA₂-α was responsible for AA supply for PGE₂ generation, but had no role in COX-2 induction. Mitogen-activated protein (MAP) kinases, ERK 1/2, p38, and JNK, participated in the signaling events that lead to PGE₂ generation by modulating AA release, but only ERK 1/2 was involved in COX-2 upregulation. Our results indicate that hypertonic stress activates PGE₂ generation by Caco-2 cells through a mechanism dependent on MAP kinase-regulated AA mobilization, increased cPLA₂-α activity, and COX-2 induction.     

Activation of Arachidonate Release and Cytosolic Phospholipase A2 via Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase in Macrophages Stimulated by Bacteria or Zymosan

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A₂ (cPLA₂). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA₂ in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA₂ activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA₂ activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA₂ activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA₂ and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.     

Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway

Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca²⁺ and Ca²⁺-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs. calcium signaling; protein kinase C; Src; G protein-coupled receptor; nonreceptor tyrosine kinase; protein G_i; protein G_q; protein tyrosine kinase 2; microvascular endothelial cell; cardiovascular remodeling     

Redistribution and abnormal activity of phospholipase A(2) isoenzymes in postinfarct congestive heart failure

Cardiacsarcolemmal (SL) cis-unsaturated fatty acid sensitivephospholipase D (cis-UFA PLD) is modulated by SLCa²⁺-independent phospholipase A₂(iPLA₂) activity via intramembrane release ofcis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca²⁺ concentration and contractileperformance of the cardiomyocyte, changes in iPLA₂ activitymay contribute to abnormal function of the failing heart. We examinedPLA₂ immunoprotein expression and activity in the SL andcytosol from noninfarcted left ventricular (LV) tissue of rats in anovert stage of congestive heart failure (CHF). Hemodynamic assessmentof CHF animals showed an increase of the LV end-diastolic pressure withloss of contractile function. In normal hearts, immunoblot analysisrevealed the presence of cytosolic PLA₂ (cPLA₂)and secretory PLA₂ (sPLA₂) in the cytosol, withcPLA₂ and iPLA₂ in the SL. IntracellularPLA₂ activity was predominantly Ca²⁺independent, with minimal sPLA₂ activity. CHF increasedcPLA₂ immunoprotein and PLA₂ activity in thecytosol and decreased SL iPLA₂ and cPLA₂immunoprotein and SL PLA₂ activity. sPLA₂activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA₂isoenzymes, suggesting that PLA₂-mediated bioprocesses arealtered in CHF.

     

Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent

Bradykinin (BK)-induced release of arachidonic acid (AA) fromMadin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol12-myristate 13-acetate (PMA) caused a synergistic increase in BK- andA-23187-induced release of AA but alone had no effect on this release.Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS)abolished the synergistic effects of PMA but did not affect AA releasecaused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMAresulted in a reduction of AA release induced by BK or A-23187addition, which corresponded to a decrease in cytoplasmic phospholipaseA₂(cPLA₂) activity as measured incell extracts. Although Western blotting revealed no differences in cPLA₂ expression as a result ofPMA treatment, phosphorylation of the enzyme, as assessed byphosphoserine content, was significantly reduced in PKC-depleted cells.These results imply that, with PKC downregulation, subsequent BKstimulation results in aCa²⁺-dependent translocation of aless phosphorylated, less active form ofcPLA₂. Any stimulation of PKC byBK addition did not appear as a significant event in onset reponsesleading to AA release. On the other hand, inhibition of themitogen-activated protein kinase (MAPK) cascade with the MAPK kinaseinhibitor, PD-98059, significantly decreased BK-induced release of AA,a finding that, with our other results, points to the existence of aPKC-independent route for stimulation of MAPK and the propagation ofonset responses.

     

Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells

Eicosanoid production is reduced when the nitric oxide (NO·) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO· and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO· and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100–500 µM) and 3-morpholinosydnonimine (SIN-1; 200 µM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A₂ (cPLA₂). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA₂ activation and arachidonic acid release. Thus studies investigating the role of the NO· pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease. reactive nitrogen species; prostaglandin H₂ synthase; extracellular signal-regulated kinase; p38; cytosolic phospholipase A₂     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

p38 MAP kinase-mediated negative inotropic effect of HIV gp120 on cardiac myocytes

Myocardial dysfunction leading to dilated cardiomyopathy has been documented with surprisingly high frequency in human immunodeficiency virus (HIV)-infected individuals. p38 MAP kinase has been implicated as a mediator of myocardial dysfunction. We previously reported p38 MAP kinase activation by the HIV coat protein gp120 in neonatal rat cardiac myocytes. We now report the direct inotropic effects of HIV gp120 on adult rat ventricular myocytes (ARVM). ARVM were continuously superfused with gp120, and percent fractional shortening (FS) was determined by automated border detection and simultaneous intracellular ionized free Ca²⁺ concentration ([Ca²⁺]_i) measured by fura 2-AM fluorescence: gp120 alone increased FS and increased [Ca²⁺]_i within 5 min and then depressed FS without a decrease in [Ca²⁺]_i by 20–60 min, which persisted for at least 2 h. Exposure of ARVM to gp120 also resulted in the phosphorylation of the upstream regulator of p38 MAP kinase MKK3/6, p38 MAP kinase itself, and its downstream effector, ATF-2, over a similar time course. ERK (p44/42) and JNK stress signaling pathways were not similarly activated. The effects of the p38 MAP kinase inhibitor were concentration dependent. SB-203580 (10 µM) blocked both p38 MAP kinase phosphorylation and the delayed negative inotropic effect of gp120. SB-203580 (5 µM) selectively blocked phosphorylation of ATF-2 without blocking the phosphorylation of MKK3/6 or p38 MAP kinase itself. SB-203580 (5 µM) administered before, with, or after gp120 blocked the negative inotropic effect of gp120 in ARVM. p38 MAP kinase activation may be a common stress-response mechanism contributing to myocardial dysfunction in HIV and other nonischemic as well as ischemic cardiomyopathies. cardiomyopathy; cell signaling     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and cPLA2 signaling pathways

Exposure of renal proximal tubule cells to oxalate may play an important role in cell proliferation, but the signaling pathways involved in this effect have not been elucidated. Thus the present study was performed to examine the effect of oxalate on ³H-labeled thymidine incorporation and its related signal pathway in primary cultured rabbit renal proximal tubule cells (PTCs). The effects of oxalate on [³H]thymidine incorporation, lactate dehydrogenase (LDH) release, Trypan blue exclusion, H₂O₂ release, activation of mitogen-activated protein kinases (MAPKs), and ³H-labeled arachidonic acid (AA) release were examined in primary cultured renal PTCs. Oxalate inhibited [³H]thymidine incorporation in a time- and dose-dependent manner. However, its analogs did not affect [³H]thymidine incorporation. Oxalate (1 mM) significantly increased H₂O₂ release, which was blocked by N-acetyl-L-cysteine (NAC) and catalase (antioxidants). Oxalate significantly increased p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun NH₂-terminal kinase (JNK) activity, not p44/42 MAPK. Oxalate stimulated [³H]AA release and translocation of cytosolic phospholipase A₂ (cPLA₂) from the cytosolic fraction to the membrane fraction. Indeed, oxalate significantly increased prostaglandin E₂ (PGE₂) production compared with control. Oxalate-induced inhibition of [³H]thymidine incorporation and increase of [³H]AA release were prevented by antioxidants (NAC), a p38 MAPK inhibitor (SB-203580), a SAPK/JNK inhibitor (SP-600125), or PLA₂ inhibitors [mepacrine and arachidonyl trifluoromethyl ketone (AACOCF₃)], but not by a p44/42 MAPK inhibitor (PD-98059). These findings suggest that oxalate inhibits renal PTC proliferation via oxidative stress, p38 MAPK/JNK, and cPLA₂ signaling pathways. kidney; mitogen-activated protein kinase; phospholipase A₂     

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

Phosphorylation of h-caldesmon has beenproposed to regulate airway smooth muscle contraction. Bothextracellular signal-regulated kinase (ERK) and p38 mitogen-activatedprotein (MAP) kinases phosphorylate h-caldesmon in vitro. To determinewhether both enzymes phosphorylate caldesmon in vivo,phosphorylation-site-selective antibodies were used to assayphosphorylation of MAP kinase consensus sites. Stimulation of culturedtracheal smooth muscle cells with ACh or platelet-derived growth factorincreased caldesmon phosphorylation at Ser789 by about twofold.Inhibiting ERK MAP kinase activation with 50 µM PD-98059 blockedagonist-induced caldesmon phosphorylation completely. Inhibiting p38MAP kinases with 25 µM SB-203580 had no effect on ACh-inducedcaldesmon phosphorylation. Carbachol stimulation increased caldesmonphosphorylation at Ser789 in intact tracheal smooth muscle, which wasblocked by the M₂ antagonist AF-DX 116 (1 µM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thusdissociating caldesmon phosphorylation from contraction. Activation ofM₂ receptors leads to activation of ERK MAP kinases andphosphorylation of caldesmon with little or no functional effect onisometric force. P38 MAP kinases are also activated by muscarinicagonists, but they do not phosphorylate caldesmon in vivo.

     

Impaired [Ca(2+)](i) and pH(i) responses to kappa-opioid receptor stimulation in the heart of chronically hypoxic rats

-Opioid receptor (-OR)stimulation with U50,488H, a selective -OR agonist, or activation ofprotein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), anactivator of PKC, decreased the electrically induced intracellularCa²⁺ ([Ca²⁺]_i) transient andincreased the intracellular pH (pH_i) in single ventricularmyocytes of rats subjected to 10% oxygen for 4 wk. The effects ofU50,488H were abolished by nor-binaltorphimine, a selective -ORantagonist, and calphostin C, a specific inhibitor of PKC, while theeffects of PMA were abolished by calphostin C andethylisopropylamiloride (EIPA), a potent Na⁺/H⁺exchange blocker. In both right hypertrophied and leftnonhypertrophied ventricles of chronically hypoxic rats, the effects ofU50,488H or PMA on [Ca²⁺]_i transient andpH_i were significantly attenuated and completely abolished,respectively. Results are first evidence that the[Ca²⁺]_i and pH_i responses to-OR stimulation are attenuated in the chronically hypoxic rat heart,which may be due to reduced responses to PKC activation. Responses toall treatments were the same for right and left ventricles, indicatingthat the functional impairment is independent of hypertrophy. -ORmRNA expression was the same in right and left ventricles of bothnormoxic and hypoxic rats, indicating no regional specificity.

     

Differential MAP kinase activation and Na(+)/H(+) exchanger phosphorylation by H(2)O(2) in rat cardiac myocytes

Bursts in reactive oxygen species productionare important mediators of contractile dysfunction duringischemia-reperfusion injury. Cellular mechanisms that mediatereactive oxygen species-induced changes in cardiac myocyte functionhave not been fully characterized. In the present study,H₂O₂ (50 µM) decreased contractility of adultrat ventricular myocytes. H₂O₂ caused aconcentration- and time-dependent activation of extracellularsignal-regulated kinases 1 and 2 (ERK1/2), p38, and c-JunNH₂-terminal kinase (JNK) mitogen-activated protein (MAP)kinases in adult rat ventricular myocytes. H₂O₂ (50 µM) caused transient activation of ERK1/2 and p38 MAP kinase thatwas detected as early as 5 min, was maximal at 20 min (9.6 ± 1.2- and 9.0 ± 1.6-fold, respectively, vs. control), and returned tobaseline at 60 min. JNK activation occurred more slowly (1.6 ± 0.2-fold vs. control at 60 min) but was sustained at 3.5 h. Theprotein kinase C inhibitor chelerythrine completely blocked JNKactivation and reduced ERK1/2 and p38 activation. The tyrosine kinaseinhibitors genistein and PP-2 blocked JNK, but not ERK1/2 and p38,activation. H₂O₂-inducedNa⁺/H⁺ exchanger phosphorylation was blocked bythe MAP kinase kinase inhibitor U-0126 (5 µM). These resultsdemonstrate that H₂O₂-induced activation of MAPkinases may contribute to cardiac myocyte dysfunction duringischemia-reperfusion.

     

Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH_i). pH_i was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH_i in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H₂O₂ decreased pH_i by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pH_i by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH_i, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pH_i recovery mediated by Na⁺/H⁺ exchange (NHE) from NH₄Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH_i. These results indicate that oxidative stress 1) decreases pH_i in some SC neurons, 2) together with hypercapnia has an additive effect on pH_i, 3) partially inhibits NHE, and 4) directly affects excitability of CO₂/H⁺-chemosensitive SC neurons independently of pH_i changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction. hyperoxic hyperventilation; O₂ toxicity; pH regulation; brain stem; reactive oxygen species     

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca²⁺ ([Ca²⁺]_i) by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca²⁺]_i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M₃) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M₁) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M₃, but not M₁, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms—pilocarpine acting via M₃ receptors and Src-dependent transactivation of EGF receptors, and carbachol via M₁/M₃ receptors and PKC—converging on the ERK pathway. muscarinic receptor; epidermal growth factor receptor; protein kinase C     

Effect of intracellular pH on depolarization-evoked calcium influx in human sperm

Human sperm are endowed with putative voltage-dependent calcium channels (VDCC) that produce measurable increases in intracellular calcium concentration ([Ca²⁺]_i) in response to membrane depolarization with potassium. These channels are blocked by nickel, inactivate in 1–2 min in calcium-deprived medium, and are remarkably stimulated by NH₄Cl, suggesting a role for intracellular pH (pH_i). In a previous work, we showed that calcium permeability through these channels increases approximately onefold during in vitro "capacitation," a calcium-dependent process that sperm require to fertilize eggs. In this work, we have determined the pH_i dependence of sperm VDCC. Simultaneous depolarization and pH_i alkalinization with NH₄Cl induced an [Ca²⁺]_i increase that depended on the amount of NH₄Cl added. VDCC stimulation as a function of pH_i showed a sigmoid curve in the 6.6–7.2 pH_i range, with a half-maximum stimulation at pH 7.00. At higher pH_i (7.3), a further stimulation occurred. Calcium release from internal stores did not contribute to the stimulating effect of pH_i because the [Ca²⁺]_i increase induced by progesterone, which opens a calcium permeability pathway that does not involve gating of VDCC, was unaffected by ammonium. The ratio of pH_i-stimulated-to-nonstimulated calcium influx was nearly constant at different test depolarization values. Likewise, depolarization-induced calcium influx in pH_i-stimulated and nonstimulated cells was equally blocked by nickel. In our capacitating conditions pH_i increased 0.11 pH units, suggesting that the calcium influx stimulation observed during sperm capacitation might be partially caused by pH_i alkalinization. Additionally, a calcium permeability pathway triggered exclusively by pH_i alkalinization was detected. mammalian sperm; capacitation; intracellular calcium     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

H(2)O(2)-mediated permeability: role of MAPK and occludin

H₂O₂-mediated elevation inendothelial solute permeability is associated with pathological eventssuch as ischemia-reperfusion and inflammation. To understand howH₂O₂ mediates increased permeability, weinvestigated the effects of H₂O₂ administrationon vascular endothelial barrier properties and tight junctionorganization and function. We report that H₂O₂exposure caused an increase in endothelial solute permeability in atime-dependent manner through extracellularly regulated kinase 1 and 2 (ERK1/ERK2) signal pathways. H₂O₂ exposurecaused the tight junctional protein occludin to be rearranged fromendothelial cell-cell junctions. Occludin rearrangement involvedredistribution of occludin on the cell surface and dissociation ofoccludin from ZO-1. Occludin also was heavily phosphorylated onserine residues upon H₂O₂ administration. H₂O₂ mediates changes in ERK1/ERK2phosphorylation, increases endothelial solute permeability, and altersoccludin localization and phosphorylation were all blocked by PD-98059,a specific mitogen-activated protein (MAP) or ERK kinase 1 inhibitor. These data strongly suggest thatH₂O₂-mediated increased endothelial solutepermeability involves the loss of endothelial tight junction integritythrough increased ERK1/ERK2 activation.