Extracellular acidification elicits spatially and temporally distinct Ca2+ signals

Extracellular acidification accompanies neoplastic transformation of tissues and increases with tumor aggressiveness [1, 2]. The intracellular signaling cascade triggered by this process remains poorly understood and may be linked to recently discovered proton-activated G protein-coupled receptors such as OGR1 and G2A [3, 4]. Here, we report that OGR1 and G2A are expressed in human medulloblastoma tissue and its corresponding neuronal cell line. We show that extracellular acidification activates phospholipase C, IP(3) formation, and subsequent Ca2+ release from thapsigargin-sensitive stores in neurons. The number of responsive cells and the amount of Ca2+ released from stores correlated positively with the extent of extracellular acidification. Ca2+ release recruited the MEK/ERK pathway, providing a mechanistic explanation for how acidification stimulates cell growth. In addition, acidification activated Ca2+-permeable ion channels through a mechanism dependent on phospholipase C but independent of store depletion or a cytoplasmic Ca2+ rise. Hence, extracellular acidification, to levels seen in tumor tissue, activates temporally and spatially distinct pathways that elevate Ca2+ and may be directly relevant for tumor cell biology.     

Intracellular Ca2+ stores are essential for injury induced Ca2+ signaling and re-endothelialization

Endothelialization repairs the lining of damaged vasculature and is a key process in preventing thrombosis and restenosis. It has been demonstrated that extracellular calcium ([Ca2+](o)) influx is important for subsequent endothelialization. The role of intracellular Ca2+ stores in mechanical denudation induced intracellular calcium ([Ca2+](i)) rise and endothelialization remains to be demonstrated. Using monolayer culture of a human endothelial cell line (human umbilical vein endothelial cell, HUVEC), we investigated [Ca2+](i) wave propagation and re-endothelialization following mechanical denudation. Consistent with previous reports for other types of cells, mechanical denudation induces calcium influx, which is essential for [Ca2+](i) rise and endothelialization. Moreover, we found that intracellular Ca(2+) stores are also essential for denudation induced [Ca2+](i) wave initiation and propagation, and the subsequent endothelialization. Thapsigargin which depletes intracellular Ca2+ stores completely abolished [Ca2+](i) wave generation and endothelialization. Xestospongin C (XeC), which prevents Ca2+ release from intracellular Ca2+ stores by inhibition of inositol 1,4,5-trisphosphate (IP(3)) receptor, inhibited intercellular Ca2+ wave generation and endothelialization following denudation. Purinergic signaling through a suramin sensitive mechanism and gap junction communication also contribute to in intercellular Ca(2+) wave propagation and re-endothelialization. We conclude that intracellular Ca2+ stores, in addition to extracellular Ca2+, are essential for intracellular Ca2+ signaling and subsequent endothelialization following mechanical denudation.     

IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK

OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.     

The MEK inhibitor, U0126, alters fertilization-induced [Ca2+]i oscillation parameters and secretion: differential effects associated with in vivo and in vitro meiotic maturation

Although mitogen-activated protein kinase (MAPK) is a well-known cell cycle regulator, emerging studies have also implicated its activity in the regulation of intracellular calcium concentration ([Ca2+](i)) and secretion. Those studies raise the hypothesis that MAPK activity during oocyte maturation and early fertilization is required for normal egg Ca2+ oscillations and cortical granule (CG) secretion. We extend the findings of [Lee, B., Vermassen, E., Yoon, S.-Y., Vanderheyden, V., Ito, J., Alfandari, D., De Smedt, H., Parys, J.B., Fissore, R.A., 2006. Phosphorylation of IP(3)R1 and the regulation of [Ca2+](i) responses at fertilization: a role for the MAP kinase pathway. Development 133, 4355-4365] by demonstrating acute effects on Ca2+ oscillation frequency, amplitude, and duration in fertilized mouse eggs matured in vitro with the MAPK inhibitor, U0126. Frequency was increased, whereas amplitude and duration were greatly decreased. These effects were significantly reduced in eggs matured in vivo and fertilized in the presence of the inhibitor. Ionomycin studies indicated that intracellular Ca2+ stores were differentially affected in eggs matured in vitro with U0126. Consistent with these effects on [Ca2+](i) elevation, fertilization-induced CG exocytosis and metaphase II exit were also reduced in in vitro-matured eggs with U0126, but not in those similarly treated after in vivo maturation. These results indicate that MAPK targets Ca2+ regulatory proteins during both maturation and fertilization, as well as provide a new hypothesis for MAPK function, which is to indirectly regulate events of early development by controlling Ca2+ oscillation parameters.     

Activation of P2Y but not P2X(4) nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+](i)) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of ([Ca2+](i)) persisted on removal of extracellular Ca2+ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of ([Ca2+](i)). After store depletion in the absence of Ca, addition of Ca led to a rise of ([Ca2+](i)) consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La(3+). In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X(4) nucleotide receptors, but with no rise of ([Ca2+](i)). We conclude that nucleotide-induced elevation of [Ca(2+)](i) in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.     

Identification of signal transduction pathway in fresh and vitrified porcine oocytes

Signal transduction pathway under the influence of somatotropin have been identified basis on the analysis of Ca2+ release from intracellular stores of fresh and vitrified porcine oocytes using inhibitory analysis. Somatotropin and GTP individually stimulated Ca2+ release from intracellular stores. The joint action of somatotropin and GTP activated additional Ca2+ release from intracellular stores both in fresh and vitrified porcine oocytes. Treatment of the oocytes with inhibitor of protein kinase C caused no additional Ca2+ release from intracellular stores. Ca2+ release from intracellular stores stimulated by GTP was connected with phosphate hydrolysis. Moving between intracellular Ca2+ depots stimulated by GTP was not determined by phosphate hydrolysis. Inhibitor of protein kinase C and microtubules were involved in the interaction of various intracellular depots. The data obtained suggest that signal transduction pathway in porcine oocytes do not change after vitrification.     

Opioid elevation of intracellular free calcium: possible mechanisms and physiological relevance

Opioid receptors are seven transmembrane domain Gi/G0 protein-coupled receptors, the activation of which stimulates a variety of intracellular signalling mechanisms including activation of inwardly rectifying potassium channels, and inhibition of both voltage-operated N-type Ca2+ channels and adenylyl cyclase activity. It is now apparent that like many other Gi/G0-coupled receptors, opioid receptor activation can significantly elevate intracellular free Ca2+ ([Ca2+]i), although the mechanism underlying this phenomenon is not well understood. In some cases opioid receptor activation alone appears to elevate [Ca2+]i, but in many cases it requires concomitant activation of Gq-coupled receptors, which themselves stimulate Ca2+ release from intracellular stores via the inositol phosphate pathway. Given the number of Ca2+-sensitive processes known to occur in cells, there are therefore a myriad of situations in which opioid receptor-mediated elevations of [Ca2+](i) may be important. Here, we review the literature documenting opioid receptor-mediated elevations of [Ca2+]i, discussing both the possible mechanisms underlying this phenomenon and its potential physiological relevance.     

Effects of guanine nucleotides and protein kinase C on prolactin-stimulated release of Ca from intracellular stores of pig oocytes

The effects of guanine nucleotides and protein kinase C on prolactin-stimulated Ca2+ release from intracellular stores of pig oocytes were studied using the fluorescent dye chlorotetracycline. The effect of prolactin was related to the protein kinase C activation. Inhibition of protein kinase C stimulated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin in the presence of extracellular Ca2+ and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. In a Ca2+-free medium, prolactin did not stimulate Ca2+ release from intracellular stores of the oocytes treated with GDP in the presence of GDP. GTP inhibition of protein kinase C activated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. These data suggest the influence of guanine nucleotides and protein kinase C on calcium metabolism, stimulated by prolactin.     

The structure of SENP1-SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing

eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein-protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+]i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+]i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+]i, phosphorylation and, possibly, intracellular trafficking.     

Interferon-gamma elicits a G-protein-dependent Ca2+ signal in human neutrophils after depletion of intracellular Ca2+ stores

Interferon-gamma (IFN-gamma) has multiple effects on Ca2+ signalling in polymorphonuclear neutrophils (PMNs), including evoked cytosolic Ca2+ transients, increased capacitative calcium influx and increased sequestration of Ca2+ in intracellular stores. The present study was conducted to elucidate the mechanism behind the Ca2+ transients. As observed before, the IFN-gamma-evoked Ca2+ signals were apparent when extracellular Ca2+ was removed. A new finding was that the proportion of responding cells and the extent of calcium release increased with increasing time in EGTA buffer. As assessed by N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated Ca2+ release, the intracellular stores were depleted during this incubation period, and the extent of depletion correlated well with the appearance of IFN-gamma-induced Ca2+ signals. This store dependence of the IFN-gamma-induced Ca2+ signals was confirmed by the appearance of IFN-gamma-evoked Ca2+ signals in the presence of extracellular Ca2+ after store depletion by thapsigargin. The appearance of IFN-gamma-mediated Ca2+-signals in the presence of EGTA indicates that IFN-gamma stimulates Ca2+ release from intracellular stores. This was confirmed by the inability of the calcium transportation blocker La3+ to abolish the IFN-gamma response and the total abrogation of the response by the phospholipase C inhibitor U73122. Although these latter results imply a role for inositol 1,4,5-trisphosphate(IP3) in IFN-gamma signalling, comparison of IFN-gamma-evoked responses with fMLP responses revealed clear differences that suggest different signal-transduction pathways. However, responses to fMLP and IFN-gamma were both depressed by pertussis toxin, and the IFN-gamma responses were, in addition, inhibited by the tyrosine kinase inhibitor genistein. Further evidence of the involvement of tyrosine kinase was a slight stimulatory effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate. The PI-3K activity was of minor importance. In conclusion, we present evidence of a novel signal-transduction mechanism for IFN-gamma in PMNs, dependent on tyrosine kinase activity, a pertussis toxin-sensitive G protein and phospholipase C activity.     

Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants

Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+](i)) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal ([Ca2+](i)) Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca2+ modulators before exposure to oxidants (H2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-alpha, PKC-betaI, and PKC-zeta isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced (45)Ca2+ efflux and prevented oxidant-induced (sustained) rises in ([Ca2+](i)). PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca2+ ionophore (A-23187), exacerbated by high-Ca2+ media, and prevented by calcium removal or chelation or by Ca2+ channel antagonists. PKC activators mimicked EGF on both (45)Ca2+ efflux and ([Ca2+](i)). Membrane Ca2+-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of ([Ca2+](i)).     

Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+), and calmodulin

Ras activation induces a variety of cellular responses that depend on the specific activated effector, the intensity and amplitude of its activation, and the cellular type. Transient activation followed by a sustained but low signal of the Ras/Raf/MEK/ERK pathway is a common feature of cell proliferation in many systems. On the contrary, sustained, high activation is linked with either senescence or apoptosis in fibroblasts and to differentiation in neurones and PC12 cells. The temporal regulation of the pathway is relevant and not only depends on the specific receptor activated but also on the presence of diverse modulators of the pathway. We review here evidence showing that calcium (Ca(2+)) and calmodulin (CaM) are able to regulate the Ras/Raf/MEK/ERK pathway. CaM-binding proteins (CaMBPs) as Ras-GRF and CaM-dependent protein kinase IV (CaMKIV) positively modulate ERK1/2 activation induced by either NGF or membrane depolarisation in neurones. In fibroblasts, CaM binding to EGF receptor and K-Ras(B) may be involved in the downregulation of the pathway after its activation, allowing a proliferative signalling.     

Cytosolic free calcium spiking affected by intracellular pH change

The characteristics underlying cytosolic free calcium oscillation were evaluated by superfused dual wave-length microspectrofluorometry of fura-2-loaded single acinar cells from rat pancreas. Application of a physiological concentration of cholecystokinin octapeptide (CCK) (20 pM) induced a small basal increase in cytosolic free calcium concentration ([Ca2+]i) averaging 34 nM above the prestimulation level (69 nM) with superimposed repetitive Ca2+ spike oscillation. The oscillation amplitude averaged 121 nM above the basal increase in [Ca2+]i and occurred at a frequency of one pulse every 49 s. Although extracellular Ca2+ was required for maintenance of high frequency and amplitude of the spikes with increase in basal [Ca2+]i, the primary source utilized for oscillation was intracellular. The threshold of the peak [Ca2+]i amplitude for causing synchronized and same-sized oscillations was less than 300 nM. The [Ca2+]i oscillation was sensitive to intracellular pH (pHi) change. This is shown by the fact that the large pHi shift toward acidification (delta pHi decrease, 0.95) led to a basal increase in [Ca2+]i to the spike peak level with inhibiting Ca2+ oscillation. The pHi shift toward alkalinization (delta pHi increase, 0.33) led to a basal decrease in [Ca2+]i to the prestimulation level, possibly due to reuptake of Ca2+ into the Ca2+ stores, with inhibiting Ca2+ oscillation. Whereas extracellular pH (pHo) change had only minimal effects on Ca2+ oscillation (and/or Ca2+ release from intracellular stores), the extra-Ca2+ entry process, which was induced by higher concentrations of CCK, was totally inhibited by decreasing pHo from 7.4 to 6.5. Thus the major regulatory sites by which H+ affects Ca2+ oscillation are accessible from the intracellular space.     

Intracellular mediators of granulysin-induced cell death

Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.     

Angiotensin II activates extracellular signal regulated kinases via protein kinase C and epidermal growth factor receptor in breast cancer cells

Angiotensin II (Ang II) induces, through AT1, intracellular Ca(2+) increase in both normal and cancerous breast cells in primary culture (Greco et al., 2002 Cell Calcium 2:1-10). We here show that Ang II stimulated, in a dose-dependent manner, the 24 h-proliferation of breast cancer cells in primary culture, induced translocation of protein kinase C (PKC)-alpha, -beta1/2, and delta (but not -epsilon, -eta, -theta, -zeta, and -iota), and phosphorylated extracellular-regulated kinases 1 and 2 (ERK1/2). The proliferative effects of Ang II were blocked by the AT1 antagonist, losartan. Also epidermal growth factor (EGF) had mitogenic effects on serum-starved breast cancer cells since induced cell proliferation after 24 h and phosphorylation of ERK1/2. The Ang II-induced proliferation of breast cancer cells was reduced by (a) G?6976, an inhibitor of conventional PKC-alpha and -beta1, (b) AG1478, an inhibitor of the tyrosine kinase of the EGF receptor (EGFR), and (c) downregulation of 1,2-diacylglycerol-sensitive PKCs achieved by phorbol 12-myristate 13-acetate (PMA). A complete inhibition of the Ang II-induced cell proliferation was achieved using the inhibitor of the mitogen activated protein kinase kinase (MAPKK or MEK), PD098059, or using G?6976 together with AG1478. These results indicate that in human primary cultured breast cancer cells AT1 regulates mitogenic signaling pathways by two simultaneous mechanisms, one involving conventional PKCs and the other EGFR transactivation.     

Peroxisomes as novel players in cell calcium homeostasis

Ca2+ concentration in peroxisomal matrix ([Ca2+](perox)) has been monitored dynamically in mammalian cells expressing variants of Ca2+-sensitive aequorin specifically targeted to peroxisomes. Upon stimulation with agonists that induce Ca2+ release from intracellular stores, peroxisomes transiently take up Ca2+ reaching peak values in the lumen as high as 50-100 microm, depending on cell types. Also in resting cells, peroxisomes sustain a Ca2+ gradient, [Ca2+](perox) being approximately 20-fold higher than [Ca2+] in the cytosol ([Ca2+](cyt)). The properties of Ca2+ traffic across the peroxisomal membrane are different from those reported for other subcellular organelles. The sensitivity of peroxisomal Ca2+ uptake to agents dissipating H+ and Na+ gradients unravels the existence of a complex bioenergetic framework including V-ATPase, Ca2+/H+, and Ca2+/Na+ activities whose components are yet to be identified at a molecular level. The different [Ca2+](perox) of resting and stimulated cells suggest that Ca2+ could play an important role in the regulation of peroxisomal metabolism.     

Cholecystokinin and EGF activate a MAPK cascade by different mechanisms in rat pancreatic acinar cells

The effects of activating the G_qprotein-coupled cholecystokinin (CCK) receptor on differentproteins/signaling molecules in the mitogen-activated protein kinase(MAPK) cascade in pancreatic acinar cells were analyzed and comparedwith the effects of activating the tyrosine kinase-coupled epidermalgrowth factor (EGF) receptor. Both EGF and CCK octapeptide rapidlyincreased the activity of the MAPKs [extracellular signal-regulatedkinase (ERK) 1 and ERK2], reaching a maximum within 2.5 min when 3.9- and 8.5-fold increases, respectively, were observed. The EGF-inducedincrease of MAPK activity was transient, with only a slight elevationafter 30 min, whereas CCK-stimulated MAPK remained at a high level ofactivation to 60 min. The protein kinase C inhibitor GF-109203Xabolished the activation by phorbol ester and inhibited the effect ofCCK by 78% but had no effect on EGF-activated MAPK activity. EGF and CCK activated both forms of MAPK kinase (MEK), with CCK having a muchlarger effect, activating MEK1 by 6-fold and MEK2 by 10-fold, whereasEGF activated both MEKs by only 2-fold. Immunoblotting revealed threedifferent forms of Raf in pancreatic acinar cells. Of the total basalRaf kinase activity, 3.7% was Raf-A, 89.0% was Raf-B, and 7.3% wasc-Raf-1. All three forms of Raf were stimulated to a greater extent byCCK than by EGF, which was especially evident for Raf-A and c-Raf-1.The effect of CCK in activating Rafs was at least partially mimicked bystimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. EGF significantlyincreased GTP-bound Ras by 183 and 164% at 2.5 and 10 min,respectively; CCK and TPA had no measurable effect. Our study suggeststhat CCK and EGF activate the MAPK cascade by distinct mechanisms in pancreatic acinar cells.

     

The cytosolic calcium concentration is affected by S-nitrosocysteine in human lymphomonocytes

The homeostasis of cytosolic calcium [Ca2+](c) in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+](c), although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 microM) and at high (160 microM) concentrations by measuring the [Ca2+](c) by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+](c), whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+](c) in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+](c) homeostasis by stimulating the movement of the ion from the cytosol to other compartments.     

EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells

We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl(-) secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2). Short-circuit current (I(sc)) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2) caused a concentration-dependent change in I(sc) that was significantly reduced in Cl(-)-free buffer and by the intracellular Ca(2+) blockers thapsigargin and BAPTA-AM, but not by the Ca(2+) channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl(-) transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca(2+). Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH(2)-induced increases in I(sc). Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated I(sc). We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl(-) secretion via cAMP- and Ca(2+)-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.