Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) through controlling voltage-gated K(+) channel expression and membrane potential (E(m)) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca(2+)](cyt) activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca(2+)](cyt), by either polyamine depletion or exposure to the Ca(2+)-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca(2+)](cyt) also inhibited cell migration after wounding. Elevation of [Ca(2+)](cyt) induced by the Ca(2+) ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca(2+)](cyt) can interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.     

Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes

Activity of voltage-gated K+ (KV) channels regulates membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). A rise in ([Ca2+](cyt))in pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (PO(2) 30-35 mmHg for 60-72 h) decreased mRNA expression of KV channel alpha-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced KV current [I(KV)]; caused E(m) depolarization; and increased ([Ca2+](cyt)) in PASMCs but negligibly affected KV channel expression, increased I(KV), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KV channel expression, reduces I(KV), and induces E(m) depolarization in PASMCs. The subsequent rise in ([Ca2+](cyt)) plays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on KV channel alpha-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KV channel gene expression.     

Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines

Maintenance of intestinal mucosal epithelial integrity requires polyamines that are involved in the multiple signaling pathways controlling gene expression and different epithelial cell functions. Integrity of the intestinal epithelial barrier depends on a complex of proteins composing different intercellular junctions, including tight junctions, adherens junctions, and desmosomes. E-cadherin is primarily found at the adherens junctions and plays a critical role in cell-cell adhesions that are fundamental to formation of the intestinal epithelial barrier. The current study determined whether polyamines regulate intestinal epithelial barrier function by altering E-cadherin expression. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced intracellular free Ca2+ concentration ([Ca2+]cyt), decreased E-cadherin expression, and increased paracellular permeability in normal intestinal epithelial cells (IEC-6 line). Polyamine depletion did not alter expression of tight junction proteins such as zona occludens (ZO)-1, ZO-2, and junctional adhesion molecule (JAM)-1. Addition of exogenous polyamine spermidine reversed the effects of DFMO on [Ca2+]cyt and E-cadherin expression and restored paracellular permeability to near normal. Elevation of [Ca2+]cyt by the Ca2+ ionophore ionomycin increased E-cadherin expression in polyamine-deficient cells. In contrast, reduction of [Ca2+]cyt by polyamine depletion or removal of extracellular Ca2+ not only inhibited expression of E-cadherin mRNA but also decreased the half-life of E-cadherin protein. These results indicate that polyamines regulate intestinal epithelial paracellular barrier function by altering E-cadherin expression and that polyamines are essential for E-cadherin expression at least partially through [Ca2+]cyt.     

Rapid spatiotemporal patterning of cytosolic Ca2+ underlies flagellar excision in Chlamydomonas reinhardtii.

Ca(2+)-dependent signalling processes are implicated in many aspects of flagella function in the green alga, Chlamydomonas. In this study, we examine the spatiotemporal dynamics of cytosolic Ca2+ ([Ca2+](cyt)) in single Chlamydomonas cells during the process of flagellar excision, using biolistically loaded calcium-responsive dyes. Acid-induced deflagellation occurred in parallel with a single transient elevation in whole-cell [Ca2+](cyt), which was absent in the acid deflagellation-deficient adf1 mutant. Deflagellation could also be induced by elevated external Ca2+ ([Ca2+](ext)), which promoted very rapid spiking of [Ca2+](cyt) across the whole cell and in the flagella. We also detected very rapid apically localised Ca2+ signalling events with an approximate duration of 500 msec. Ninety-seven per cent of deflagellation events coincided with a rapid elevation in [Ca2+](cyt) in the apical region of the cell, either in the form of a whole cell or an apically localised increase, indicating that [Ca2+](cyt) elevations in the apical region play an underlying role in deflagellation. Our data indicate that elevated [Ca2+](ext) acts to disrupt Ca2+ homeostasis which induces deflagellation by both Adf1-dependent and Adf1-independent mechanisms. Elevated [Ca2+](ext) also results in further [Ca2+](cyt) elevations after the main period of whole cell spiking which are very strongly associated with deflagellation, exhibit a high degree of apical localisation and are largely absent in the adf1 mutant. We propose that these later elevations may act as specific signals for deflagellation.     

Polyamines are required for phospholipase C-gamma1 expression promoting intestinal epithelial restitution after wounding

Intestinal mucosal restitution occurs by epithelial cell migration, rather than by proliferation, to reseal superficial wounds after injury. Polyamines are essential for the stimulation of intestinal epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca2+ homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca2+ concentration ([Ca2+]cyt) remains unclear. Phospholipase C (PLC)-gamma1 catalyzes the formation of inositol (1,4,5)-trisphosphate (IP3), which is implicated in the regulation of [Ca2+]cyt by modulating Ca2+ store mobilization and Ca2+ influx. The present study tested the hypothesis that polyamines are involved in PLC-gamma1 activity, regulating [Ca2+]cyt and cell migration after wounding. Depletion of cellular polyamines by alpha-difluoromethylornithine inhibited PLC-gamma1 expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells), as indicated by substantial decreases in levels of PLC-gamma1 mRNA and protein and its enzyme product IP3. Polyamine-deficient cells also displayed decreased [Ca2+]cyt and inhibited cell migration. Decreased levels of PLC-gamma1 by treatment with U-73122 or transfection with short interfering RNA specifically targeting PLC-gamma1 also decreased IP3, reduced resting [Ca2+]cyt and Ca2+ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-gamma1 by 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide induced IP3, increased [Ca2+]cyt, and promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are absolutely required for PLC-gamma1 expression in IECs and that polyamine-mediated PLC-gamma1 signaling stimulates cell migration during restitution as a result of increased [Ca2+]cyt.     

Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells

Occludin is an integral membrane protein that forms the sealing element of tight junctions and is critical for epithelial barrier function. Polyamines are implicated in multiple signaling pathways driving different biological functions of intestinal epithelial cells (IEC). The present study determined whether polyamines are involved in expression of occludin and play a role in intestinal epithelial barrier function. Studies were conducted in stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) associated with a highly differentiated phenotype. Polyamine depletion by alpha-difluoromethylornithine (DFMO) decreased levels of occludin protein but failed to affect expression of its mRNA. Other tight junction proteins, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3, were also decreased in polyamine-deficient cells. Decreased levels of tight junction proteins in DFMO-treated cells were associated with dysfunction of the epithelial barrier, which was overcome by exogenous polyamine spermidine. Decreased levels of occludin in polyamine-deficient cells was not due to the reduction of intracellular-free Ca(2+) concentration ([Ca(2+)](cyt)), because either increased or decreased [Ca(2+)](cyt) did not alter levels of occludin in the presence or absence of polyamines. The level of newly synthesized occludin protein was decreased by approximately 70% following polyamine depletion, whereas its protein half-life was reduced from approximately 120 min in control cells to approximately 75 min in polyamine-deficient cells. These findings indicate that polyamines are necessary for the synthesis and stability of occludin protein and that polyamine depletion disrupts the epithelial barrier function, at least partially, by decreasing occludin.     

Putrescine does not support the migration and growth of IEC-6 cells

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.     

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration

Polyamines are essential for cell migrationduring early mucosal restitution after wounding in the gastrointestinaltract. Activity of voltage-gated K⁺ channels (Kv) controlsmembrane potential (E_m) that regulates cytoplasmicfree Ca²⁺ concentration([Ca²⁺]_cyt) by governing thedriving force for Ca²⁺ influx. This study determinedwhether polyamines are required for the stimulation of cell migrationby altering K⁺ channel gene expression,E_m, and[Ca²⁺]_cyt in intestinal epithelialcells (IEC-6). The specific inhibitor of polyamine synthesis,-difluoromethylornithine (DFMO, 5 mM), depleted cellularpolyamines (putrescine, spermidine, and spermine), selectivelyinhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression,and resulted in membrane depolarization. Because IEC-6 cells did notexpress voltage-gated Ca²⁺ channels, the depolarizedE_m in DFMO-treated cells decreased [Ca²⁺]_cyt as a result of reduceddriving force for Ca²⁺ influx through capacitativeCa²⁺ entry. Migration was reduced by 80% in thepolyamine-deficient cells. Exogenous spermidine not only reversed theeffects of DFMO on Kv1.1 channel expression, E_m,and [Ca²⁺]_cyt but also restoredcell migration to normal. Removal of extracellular Ca²⁺ orblockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cellmigration by exogenous spermidine in polyamine-deficient cells. Theseresults suggest that polyamine-dependent intestinal epithelial cellmigration may be due partially to an increase of Kv1.1 channelexpression. The subsequent membrane hyperpolarization raises[Ca²⁺]_cyt by increasing the drivingforce (the electrochemical gradient) for Ca²⁺ influx andthus stimulates cell migration.

     

Pulmonary vascular response to normoxia and K(Ca) channel activity is developmentally regulated

To address developmental regulation of pulmonary vascular O(2) sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O(2), 2) Ca2+-sensitive K(+) (K(Ca)) channel activity decreases with maturation, and 3) PASMC K(Ca) channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O(2) tension. Acute normoxia induced a 22 +/- 2% decrease in cytosolic Ca2+ concentration ([Ca2+](i)) in fetal PASMCs and no change in ([Ca2+](i)) in adult PASMCs (P < 0.01). The effects of K(+) channel antagonists were studied on fetal and adult PASMC ([Ca2+](i)). Iberiotoxin (10(-9) M) caused PASMC ([Ca2+](i)) to increase by 694 +/- 22% in the fetus and caused no change in adult PASMCs. K(Ca) channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both K(Ca) channel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O(2) sensing render the fetal PASMCs uniquely sensitive to an acute increase in O(2) tension at a biologically critical time point.     

Arachidonic acid induces both Na+ and Ca2+ entry resulting in apoptosis

Marked accumulation of arachidonic acid (AA) and intracellular Ca2+ and Na+ overloads are seen during brain ischemia. In this study, we show that, in neurons, AA induces cytosolic Na+ ([Na+](cyt)) and Ca2+ ([Ca2+](cyt)) overload via a non-selective cation conductance (NSCC), resulting in mitochondrial [Na+](m) and [Ca2+](m) overload. Another two types of free fatty acids, including oleic acid and eicosapentaenoic acid, induced a smaller increase in the [Ca2+](i) and [Na+](i). RU360, a selective inhibitor of the mitochondrial Ca2+ uniporter, inhibited the AA-induced [Ca2+](m) and [Na+](m) overload, but not the [Ca2+](cyt) and [Na+](cyt) overload. The [Na+](m) overload was also markedly inhibited by either Ca2+-free medium or CGP3715, a selective inhibitor of the mitochondrial Na+(cyt)-Ca2+(m) exchanger. Moreover, RU360, Ca2+-free medium, Na+-free medium, or cyclosporin A (CsA) largely prevented AA-induced opening of the mitochondrial permeability transition pore, cytochrome c release, and caspase 3-dependent neuronal apoptosis. Importantly, Na+-ionophore/Ca2+-free medium, which induced [Na+](m) overload, but not [Ca2+](m) overload, also caused cyclosporin A-sensitive mitochondrial permeability transition pore opening, resulting in caspase 3-dependent apoptosis, indicating that [Na+](m) overload per se induced apoptosis. Our results therefore suggest that AA-induced [Na+](m) overload, acting via activation of the NSCC, is an important upstream signal in the mitochondrial-mediated apoptotic pathway. The NSCC may therefore act as a potential neuronal death pore which is activated by AA accumulation under pathological conditions.     

Heterogeneity of hypoxia-mediated decrease in I(K(V)) and increase in [Ca2+](cyt) in pulmonary artery smooth muscle cells

Hypoxic pulmonary vasoconstriction is caused by a rise in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) via multiple mechanisms. PASMC consist of heterogeneous phenotypes defined by contractility, proliferation, and apoptosis as well as by differences in expression and function of various genes. In rat PASMC, hypoxia-mediated decrease in voltage-gated K(+) (Kv) currents (I(K(V))) and increase in [Ca(2+)](cyt) were not uniformly distributed in all PASMC tested. Acute hypoxia decreased I(K(V)) and increased [Ca(2+)](cyt) in approximately 46% and approximately 53% of PASMC, respectively. Using combined techniques of single-cell RT-PCR and patch clamp, we show here that mRNA expression level of Kv1.5 in hypoxia-sensitive PASMC (in which hypoxia reduced I(K(V))) was much greater than in hypoxia-insensitive cells (in which hypoxia negligibly affected I(K(V))). These results demonstrate that 1) different PASMC express different Kv channel alpha- and beta-subunits, and 2) the sensitivity of a PASMC to acute hypoxia partially depends on the expression level of Kv1.5 channels; hypoxia reduces whole-cell I(K(V)) only in PASMC that express high level of Kv1.5. In addition, the acute hypoxia-mediated changes in [Ca(2+)](cyt) also vary in different PASMC. Hypoxia increases [Ca(2+)](cyt) only in 34% of cells tested, and the different sensitivity of [Ca(2+)](cyt) to hypoxia was not related to the resting [Ca(2+)](cyt). An intrinsic mechanism within each individual cell may be involved in the heterogeneity of hypoxia-mediated effect on [Ca(2+)](cyt) in PASMC. These data suggest that the heterogeneity of PASMC may partially be related to different expression levels and functional sensitivity of Kv channels to hypoxia and to differences in intrinsic mechanisms involved in regulating [Ca(2+)](cyt).     

The phosphorylation state of MRLC is polyamine dependent in intestinal epithelial cells

Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

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.     

Calcium: Just Another Regulator in the Machinery of Life?

*BACKGROUND: Current hypotheses imply that stimulus-response systems in plants are networks of signal transduction pathways. It is usually assumed that these pathways connect receptors with effectors via chains of molecular events. Diverse intermediate signalling components (transducers) participate in these processes. However, many cellular transducers respond to several stimuli. Hence, there are no discrete chains but rather pathways that interconnect network-modules of different command structure. In particular, the cytosolic free Ca2+ concentration ([Ca2+](cyt)) is thought to perform many different tasks in a wide range of cellular events. However, this range of putative functions is so wide that it is often questioned how Ca2+ can comply with the definition of a second messenger. *THE Ca2+ SIGNATURE HYPOTHESIS: Some authors have suggested the concept of a specific signature of the ([Ca2+](cyt)) response. This implies that characteristics of the time course of changes in ([Ca2+](cyt)) and their localized sites of appearance in cells are used by the plant to identify the type and intensity of the stimulus. This hypothesis has triggered many investigations, which have yielded contradictory results. * THE CURRENT PICTURE: Much evidence suggests that the functions of calcium can be grouped into three classes: Ca2+ as a protective agent, Ca2+ as a chemical switch and Ca2+ as a 'digital' information carrier. Examples of the first two classes are presented here. The third is more controversial; while some investigations seem to support this idea, others call the Ca2+ signature hypothesis into question. Further investigations are needed to shed more light on Ca(2+)-driven signalling cascades.     

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)).     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

K(Ca) channel blockers reveal hyperpolarization and relaxation to K+ in rat isolated mesenteric artery

Raising extracellular K+ concentration ([K+](o)) around mesenteric resistance arteries reverses depolarization and contraction to phenylephrine. As smooth muscle depolarizes and intracellular Ca(2+) and tension increase, this effect of K+ is suppressed, whereas efflux of cellular K+ through Ca(2+)-activated K+ (K(Ca)) channels is increased. We investigated whether K+ efflux through K(Ca) suppresses the action of exogenous K+ and whether it prestimulates smooth muscle Na(+)-K(+)-ATPase. Under isometric conditions, 10.8 mM [K+](o) had no effect on arteries contracted >10 mN, unless 100 nM iberiotoxin (IbTX), 100 nM charybdotoxin (ChTX), and/or 50 nM apamin were present. Simultaneous measurements of membrane potential and tension showed that phenylephrine depolarized and contracted arteries to -32.2 +/- 2.3 mV and 13.8 +/- 1.6 mN (n = 5) after blockade of K(Ca), but 10.8 mM K+ reversed fully the responses (107.6 +/- 8.6 and 98.8 +/- 0.6%, respectively). Under isobaric conditions and preconstriction with phenylephrine, 10.7 mM [K+](o) reversed contraction at both 50 mmHg (77.0 +/- 8.5%, n = 9) and 80 mmHg (83.7 +/- 5.5%, n = 5). However, in four additional vessels at 80 mmHg, raising K+ failed to reverse contraction unless ChTX was present. Increases in isometric and decreases in isobaric tension with phenylephrine were augmented by either ChTX or ouabain (100 microM), whereas neither inhibitor altered tension under resting conditions. Inhibition of cellular K+ efflux facilitates hyperpolarization and relaxation to exogenous K+, possibly by indirectly reducing the background activation of Na(+)-K(+)-ATPase.     

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.     

Localized activation of m-calpain in migrating human umbilical vein endothelial cells stimulated by shear stress

Using a parallel-plate flow-chamber and confocal laser scanning microscopy (CLSM), we studied the mode of cytoskeletal reorganization in migrating HUVECs stimulated by shear stress. Activation of m-calpain associated with a change in the spatial distribution of cytoplasmic ionized Ca2+ concentration ([Ca2+](i)) was studied. Shear stress (10 dyne/cm(2)) caused migration and decrease in the F-actin content of HUVECs. Migrating individual HUVECs showed the lamellipodium formed in the direction of cell migration, in which [Ca2+](i) elevated to 148 +/- 12 nM in a localized fashion. We found the appearance of activated m-calpain in the local area of the migrating HUVECs, which was associated with a decrease in the amounts of pp125FAK and ezrin. The localized rise in [Ca2+](i) might be closely related to morphological change to regulate the direction of cell migration induced by shear stress through localized activation of m-calpain.