Absence Of Extracellular Na Research Articles

Mg2+ extrusion activated by hydrogen peroxide in rat ventricular myocytes.

To investigate the effect of reactive oxygen species (ROS) on intracellular-free Mg2+ concentration ([Mg2+]i), we measured [Mg2+]i in rat ventricular myocytes using a fluorescent indicator, mag-fura-2. The treatment of hydrogen peroxide (H2O2, 500 μM) for 5 min decreased [Mg2+]i from 1.02 ± 0.08 (mean ±SD, n = 7) to 0.75 ± 0.18 mM (p = 0.018, by Wilcoxon signed-rank test) in ∼9 min (including the 5-min treatment and subsequent washout). [Mg2+]i was continuously decreased in ∼ 20 min, and then recovered to the basal level (∼1.0 mM) gradually in the Ca2+-free Tyrode's solution. The half maximal effective concentration of H2O2 on the Mg2+- decrease was estimated to be between 400 and 425 μM. The rate of decrease in [Mg2+]i by H2O2 in the Ca2+-free Tyrode's solution (−0.61± 0.42 μM/s, n = 36) was not significantly different from that obtained in the absence of extracellular Na+ or Mg2+. The addition of 1 mM Ca2+ extracellularly slowed the rate of Mg2+- decrease, on average, by ∼60% (p = 0.002, by Mann-Whitney U-test). The H2O2-induced Mg2+ decrease in Na+-free (substituted with N-methyl-d-glucamine) /Ca2+-free Tyrode's solution was inhibited by 200 μM imipramine, a known inhibitor of Na+/Mg2+ exchange. Rat hearts were perfused with the Ca2+-free Tyrode's solution containing 500 μM H2O2 for 5 min on the Langendorff apparatus. The Mg2+-concentration of the cardiac perfusate was significantly increased after H2O2 administration, suggesting that the H2O2-induced Mg2+-decrease was caused by Mg2+- extrusion. Pyocyanin, a generator of reactive oxygen species also reduced [Mg2+]i. The Mg2+-decrease by pyocyanin was inhibited by a pretreatment of n-acetyl cysteine, an anti-oxidant. In conclusion, it is suggested ROS disrupt intracellular Mg2+ homeostasis, and that the existence of a Na+-independent Mg2+- extrusion system activated by H2O2 in cardiomyocytes.

Enhancement of receptor-mediated calcium responses by phenytoin through the suppression of calcium excretion in human gingival fibroblasts.

Gingival overgrowth caused by phenytoin is proposed to be associated with Ca2+ signaling; however, the mechanisms that increase the intracellular Ca2+ concentration ([Ca2+ ]i ) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin-induced increase in [Ca2+ ]i in human gingival fibroblasts (HGFs). Effects of 100 μM phenytoin on [Ca2+ ]i in HGFs were examined at the single-cell level using fluorescence images of fura-2 captured by an imaging system consisting of an EM-CCD camera coupled to an inverted fluorescence microscope at room temperature. Exposure of HGFs to 100 μM phenytoin induced a transient increase in [Ca2+ ]i in the absence of extracellular Ca2+ , indicating that the phenytoin-induced increase in [Ca2+ ]i does not require an influx of extracellular Ca2+ . In addition, phenytoin increased [Ca2+ ]i in HGFs depleted of intracellular Ca2+ stores by thapsigargin, indicating that neither Ca2+ release from stores nor inhibition of Ca2+ uptake is involved. Furthermore, the phenytoin-induced [Ca2+ ]i elevation was reduced to 18.8% in the absence of extracellular Na+ , and [Ca2+ ]i elevation upon removal of extracellular Na+ was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca2+ ]i of HGFs by suppressing the Na+ /Ca2+ exchanger. Suppression of intracellular Ca2+ excretion is thought to enhance the Ca2+ responses induced by various stimuli. Analysis at the single-cell level showed that stimulation with 1μM ATP or 3μM histamine increased [Ca2+ ]i in 20-50% of cells, and [Ca2+ ]i increased in many unresponsive cells in the presence of phenytoin. Our findings demonstrate that phenytoin induced increase in [Ca2+ ]i by the inhibition of Ca2+ efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca2+ responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca2+ efflux. These findings suggest a possibility that phenytoin causes drug-induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.

Another Unique Feature of the Nociceptor-specific Sodium Channel NaV1.9

The voltage-gated Na+ channel NaV1.9 remains an important pain target because of its preferential expression in nociceptors, its involvement in rodent models of nociception, and the association between gain- or loss-of-function NaV1.9 mutations with the presence or absence of pain in humans. At room temperature, NaV1.9 has a low threshold for activation, carrying a persistent current that may influence the resting membrane potential, action potential threshold, and burst firing. We recently demonstrated that in the absence of extracellular Na+, NaV1.9 may also contribute to a transient outward current. Here, we used whole cell patch clamp recordings of human and rat dorsal root ganglion neurons to further characterize this outward current. We found that this current had pharmacological and biophysical properties consistent with a role for NaV1.9: insensitivity to K+, Na+, Cl-, and TRP channel blockers, sensitivity to internally-administered Na+ channel blocker QX-314, and an NaV1.9-like voltage-dependence of inactivation, steady-state inactivation, recovery from inactivation. Interestingly, in contrast to TTX- and A-803467-sensitive currents, the transient outward current persisted in the absence of intracellular Na+, suggesting that NaV1.9 is less selective than other voltage-gated Na+ channels in human and rat sensory neurons. Consistent with this suggestion, the reversal potential for persistent (NaV1.9) inward currents was left shifted relative to that of TTX- and A-803467-sensitive currents. Furthermore, the outward current persisted in the presence of even relatively large intracellular cations (Cs+ and TEA+). Our results suggest NaV1.9 is a relatively non-selective ion channel. Together, these observations have at least two important implications. First, NaV1.9-mediated currents may contaminate voltage-clamp recordings of voltage-gated K+, Ca2+, and Cl- channels in sensory neurons. Second, NaV1.9 may have a more significant impact on the action potential waveform than previously appreciated, including the attenuating the action potential overshoot. Grant support from NIH R01NS122784, NIH 4UH3TR003090.

Impact of Na+ permeation on collective migration of pulmonary arterial endothelial cells.

Collective migration of endothelial cells is important for wound healing and angiogenesis. During such migration, each constituent endothelial cell coordinates its magnitude and direction of migration with its neighbors while retaining intercellular adhesion. Ensuring coordination and cohesion involves a variety of intra- and inter-cellular signaling processes. However, the role of permeation of extracellular Na+ in collective cell migration remains unclear. Here, we examined the effect of Na+ permeation in collective migration of pulmonary artery endothelial cell (PAEC) monolayers triggered by either a scratch injury or a barrier removal over 24 hours. In the scratch assay, PAEC monolayers migrated in two approximately linear phases. In the first phase, wound closure started with fast speed which then rapidly reduced within 5 hours after scratching. In the second phase, wound closure maintained at slow and stable speed from 6 to 24 hours. In the absence of extracellular Na+, the wound closure distance was reduced by >50%. Fewer cells at the leading edge protruded prominent lamellipodia. Beside transient gaps, some sustained interendothelial gaps also formed and progressively increased in size over time, and some fused with adjacent gaps. In the absence of both Na+ and scratch injury, PAEC monolayer migrated even more slowly, and interendothelial gaps obviously increased in size towards the end. Pharmacological inhibition of the epithelial Na+ channel (ENaC) using amiloride reduced wound closure distance by 30%. Inhibition of both the ENaC and the Na+/Ca2+ exchanger (NCX) using benzamil further reduced wound closure distance in the second phase and caused accumulation of floating particles in the media. Surprisingly, pharmacological inhibition of the Ca2+ release-activated Ca2+ (CRAC) channel protein 1 (Orai1) using GSK-7975A, the transient receptor potential channel protein 1 and 4 (TRPC1/4) using Pico145, or both Orai1 and TRPC1/4 using combined GSK-7975A and Pico145 treatment did not affect wound closure distance dramatically. Nevertheless, the combined treatment appeared to cause accumulation of floating particles. Note that GSK-7975A also inhibits small inward Ca2+ currents via Orai2 and Orai3 channels, whereas Pico145 also blocks TRPC4, TRPC5, and TRPC1/5 channels. By contrast, gene silence of Orai1 by shRNAs led to a 25% reduction of wound closure in the first 6 hours but had no effect afterwards. However, in the absence of extracellular Na+ or cellular injury, Orai1 did not affect PAEC collective migration. Overall, the data reveal that Na+ permeation into cells contributes to PAEC monolayer collective migration by increasing lamellipodial formation, reducing accumulation of floating particles, and improving intercellular adhesion.

Effects of Sodium and Amino Acid Substrate Availability upon the Expression and Stability of the SNAT2 (SLC38A2) Amino Acid Transporter.

The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AAs) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis, and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that while AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (N-methylaminoisobutyric acid and glutamine), but not non-substrates. This stabilization and substrate-induced repression were critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteasome system), because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.

Role of cationic drug-sensitive transport systems at the blood-cerebrospinal fluid barrier in para-tyramine elimination from rat brain

Backgroundpara-Tyramine (p-TA) is a biogenic amine which is involved in multiple neuronal signal transductions. Since the concentration of p-TA in dog cerebrospinal fluid (CSF) has been reported to be greater than that in plasma, it is proposed that clearance of cerebral p-TA is important for normal function. The purpose of this study was to examine the role of the blood–brain barrier and blood-cerebrospinal fluid barrier (BCSFB) in p-TA clearance from the brain.MethodsIn vivo [3H]p-TA elimination from rat cerebral cortex and from CSF was examined after intracerebral and intracerebroventricular administration, respectively. To evaluate BCSFB-mediated p-TA transport, [3H]p-TA uptake by isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells, TR-CSFB3 cells, was performed.ResultsThe half-life of [3H]p-TA elimination from rat CSF was found to be 2.9 min, which is 62-fold faster than that from rat cerebral cortex. In addition, this [3H]p-TA elimination from the CSF was significantly inhibited by co-injection of excess unlabeled p-TA. Thus, carrier-mediated p-TA transport process(es) are assumed to take part in p-TA elimination from the CSF. Since it is known that transporters at the BCSFB participate in compound elimination from the CSF, [3H]p-TA transport in ex vivo and in vitro models of rat BCSFB was examined. The [3H]p-TA uptake by isolated rat choroid plexus and TR-CSFB3 cells was time-dependent and was inhibited by unlabeled p-TA, indicating carrier-mediated p-TA transport at the BCSFB. The p-TA uptake by isolated choroid plexus and TR-CSFB3 cells was not reduced in the absence of extracellular Na+ and Cl−, and in the presence of substrates of typical organic cation transporters. However, this p-TA uptake was significantly inhibited by cationic drugs such as propranolol, imipramine, amantadine, verapamil, and pyrilamine. Moreover, p-TA uptake by TR-CSFB3 cells took place in an oppositely-directed H+ gradient manner. Therefore, this suggested that p-TA transport at the BCSFB involves cationic drug-sensitive transport systems which are distinct from typical plasma membrane organic cation transporters.ConclusionOur study indicates that p-TA elimination from the CSF is greater than that from the cerebral cortex. Moreover, it is suggested that cationic drug-sensitive transport systems in the BCSFB participate in this p-TA elimination from the CSF.

207 - Mechanism Underlying 2-Deoxy-D-Ribose-Induced Oxidative Damage in a Pancreatic Beta-Cell Line

2-Deoxy- d -ribose (dRib) is known to induce oxidative damage and apoptosis by depleting GSH content in several types of cells. However, little is known about how dRib depletes intracellular GSH content. We hypothesized that dRib would deplete GSH content through inhibition of cystine uptake via system χc-. HIT-T15 cells were incubated with various concentrations of dRib, 2-mercaptoethanol (2-ME), Sulfasalazine (SASP), and homocysteic acid (HCA). We measured intracellular uptake of l -[14C]cystine, GSH content, and cell viability. Stimulation with dRib dose- and time-dependently decreases intracellular l -[14C]cystine uptake and GSH content. The addition of 2-ME, a cystine uptake enhancer, fully recovered the dRib-induced decreases in l -[14C]cystine uptake, GSH content, and cell viability. The 2-ME promoted the l -[14C]cystine uptake even in the absence of extracellular Na+, probably suggesting the involvement of the system χc-. SASP and HCA, system χc- inhibitors, significantly reduced l -[14C]cystine uptake and GSH content like dRib did, and 2-ME also restored the reductions. These findings suggest that dRib depletes intracellular GSH content through inhibition of cystine transport, and this inhibition may be mediated by system χc-. Additional experiments using a gene overexpression technique is necessary to further clarify the mechanism.

From the Cover: Selective Enhancement of Domoic Acid Toxicity in Primary Cultures of Cerebellar Granule Cells by Lowering Extracellular Na+ Concentration.

Domoic acid (DOM) is an excitatory amino acid analog of kainic acid (KA) that acts through glutamic acid (GLU) receptors, inducing a fast and potent neurotoxic response. Here, we present evidence for an enhancement of excitotoxicity following exposure of cultured cerebellar granule cells to DOM in the presence of lower than physiological Na+ concentrations. The concentration of DOM that reduced by 50% neuronal survival was approximately 3 µM in Na+-free conditions and 16 µM in presence of a physiological concentration of extracellular Na+. The enhanced neurotoxic effect of DOM was fully prevented by AMPA/KA receptor antagonist, while N-methyl-D-aspartate-receptor-mediated neurotoxicity did not seem to be involved, as the absence of extracellular Na+ failed to potentiate GLU excitotoxicity under the same experimental conditions. Lowering of extracellular Na+ concentration to 60 mM eliminated extracellular recording of spontaneous electrophysiological activity from cultured neurons grown on a multi electrode array and prevented DOM stimulation of the electrical activity. Although changes in the extracellular Na+ concentration did not alter the magnitude of the rapid increase in intracellular Ca2+ levels associated to DOM exposure, they did change significantly the contribution of voltage-sensitive calcium channels (VScaCs) and the recovery time to baseline. The prevention of Ca2+ influx via VSCaCs by nifedipine failed to prevent DOM toxicity at any extracellular Na+ concentration, while the reduction of extracellular Ca2+ concentration ameliorated DOM toxicity only in the absence of extracellular Na+, enhancing it in physiological conditions. Our data suggest a crucial role for extracellular Na+ concentration in determining excitotoxicity by DOM.

Properties of acid-induced currents in mouse dorsal root ganglia neurons.

Acid‐sensing ion channels (ASICs) are cation channels that are activated by protons (H+). They are expressed in neurons throughout the nervous system and may play important roles in several neurologic disorders including inflammation, cerebral ischemia, seizures, neurodegeneration, anxiety, depression, and migraine. ASICs generally produce transient currents that desensitize in response to a decrease in extracellular pH. Under certain conditions, the inactivation of ASICs can be incomplete and allow them to produce sustained currents. Here, we characterize the properties of both transient and sustained acid‐induced currents in cultured mouse dorsal root ganglia (DRG) neurons. At pH levels between 7.3 and 7.1 they include “window currents” through ASICs. With stronger acid signals sustained currents are maintained in the absence of extracellular Na+ or the presence of the ASIC blockers amiloride and Psalmotoxin‐1(PcTx1). These sustained responses may have several different origins in these cells, including acid‐induced stimulation of inward Cl− currents, block of outward K+ currents, and augmentation of inward H+ currents, properties that distinguish these novel sustained currents from the well‐characterized transient currents.

Broad-range TRP channel inhibitors (2-APB, flufenamic acid, SKF-96365) affect differently contraction of resistance and conduit femoral arteries of rat

Transient receptor potential (TRP) channels are proposed to contribute to membrane depolarization and Ca2+ influx into vascular smooth muscle (VSM) cells. Our aim was to study the effects of widely used broad-range TRP channel inhibitors – 2-aminoethoxydiphenyl borate (2-APB), flufenamic acid (FFA) and SKF-96365 – on the contraction of freshly isolated small and large arteries.Endothelium-denuded resistance (≈250µm) and conduit (≈1000µm) femoral arteries were isolated from adult Wistar rats and mounted in wire myograph. The effects of the above mentioned TRP channel inhibitors and voltage-dependent calcium channel inhibitor nifedipine were studied on arterial contractions induced by phenylephrine, U-46619 or K+. Phenylephrine-induced contractions were also studied in the absence of extracellular Na+. mRNA expression of particular canonical and melastatin TRP channel subunits in femoral vascular bed was determined.TRP channel inhibitors attenuated K+-induced contraction less than nifedipine. Phenylephrine-induced contraction was more influenced by 2-APB in resistance arteries, while FFA completely prevented U-46619-induced contraction in both sizes of arteries. The absence of extracellular Na+ prevented the inhibitory effects of 2-APB, but not those of FFA.The observed effects of broad-range TRP channel inhibitors, which were dependent on the size of the artery, confirmed the involvement of TRP channels in agonist-induced contractions. The inhibitory effects of 2-APB (but not those of FFA or SKF-96365) were dependent on the presence of extracellular Na+.

A Voltage Gated Dependent Na+ Channel is Activated during Apopoptosis in Xenopus Oocytes

Ion channels in the plasma membrane are important for the apoptotic process. In previous studies we and other have found that different voltage-gated ion channels are upregulated early in the apoptotic process and that block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are upregulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na+ current was upregulated. This current activated at voltages more positive than 0 mV with a midpoint of the open-probability curve around +50 mV. The current was resistant to the Na+-channel blockers tetrodotoxin (1 uM) and amiloride (10 uM), while the Ca2+-channel blocker verapamil (200 uM) in the bath solution completely blocked the current. Oocytes treated with staurosporine in the presence of verapamil, or in the absence of extracellular Na+ (replaced with Choline+) did not show an upregulated Na+ current (when measured in the absence of verapamil, or in the presence of Na+ respectively). Low extracellular Na+ concentration also prevented caspase-3 activation. Taken together, this suggests that the influx of Na+ is a crucial step in the apoptotic process in Xenopus oocytes.

A Cell Permeable NPE Caged ADP-Ribose for Studying TRPM2

Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca2+-permeable cation channel of the TRPM channel subfamily and is mainly activated by intracellular adenosine diphosphate ribose (ADPR). Here we synthesized a 1-(2-nitrophenyl)ethyl caged ADPR (NPE-ADPR) and found that uncaging of NPE-ADPR efficiently stimulated Ca2+, Mg2+, and Zn2+ influx in a concentration-dependent manner in intact human Jurkat T-lymphocytes. The cation influx was inhibited by inhibitors or knockdown of TRPM2. Likewise, uncaging of NPE-ADPR markedly induced cation entry in HEK 293 cells that overexpress TRPM2. As expected, high temperature increased the ability of the photolyzed NPE-ADPR to induce cation entry, whereas acidic pH inhibited. Moreover, the absence of extracellular Ca2+ significantly inhibited Mg2+ and Zn2+ influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na+ or Mg2+ had no effect on photolyzed NPE-ADPR induced Ca2+ entry. Taken together, our results indicated that NPE-ADPR is a cell permeable ADPR analogue that is useful for studying TRPM2-mediated cation entry in intact cells.

Mechanism of capsaicin receptor TRPV1-mediated toxicity in pain-sensing neurons focusing on the effects of Na+/Ca2 + fluxes and the Ca2 +-binding protein calretinin

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca2+ and Na+ ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na+ via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na+, the membrane blebbing was completely inhibited, but Ca2+ influx did not change under these conditions. Na+ influx was modulated by the intracellular Ca2+ concentration ([Ca2+]i). Elevation of [Ca2+]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca2+, capsaicin caused only little increase in [Ca2+]i indicating that the increase in [Ca2+]i observed after capsaicin application is derived essentially from extracellular Ca2+ and not from internal Ca2+ stores. In the absence of extracellular Ca2+ also the process of cell swelling was considerably slower. Calretinin is a Ca2+ buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca2+ signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca2+ and Na+ influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca2+-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium.

Changes in the activity of defense mechanisms against induced acidosis during meiotic maturation in mouse oocytes

Previous work has indicated that although activity of the HCO3-/Cl- exchanger (AE), which regulates intracellular alkalosis, is high in the germinal vesicle (GV) stage, the oocyte is inhibited as it progresses through meiotic maturation. In this study, we aimed to investigate the defense mechanisms against acidosis during the meiotic maturation stages. Intracellular pH (pHi) was recorded using a microspectrofluorometric technique, and Na+/H+ (NHE) and Na+-dependent HCO3-/Cl- exchanger (NDCBE) activity were determined by measuring the recovery rate from induced acidosis. Additionally, SLC9A1 (for NHE) and SLC4A8 (for NDCBE) gene transcription levels were determined by real-time PCR. The recovery rate of first meiotic prophase (GV) oocytes was high, but it decreased during the meiotic metaphase II (MII) stage in HCO3--free medium; it became high again at the pronuclear zygote (PN) stage. Recovery rate was significantly inhibited by 5-(N-ethyl-N-isopropyl) amiloride and cariporide or in the absence of extracellular Na+, implicating NHE, specifically NHE1 activity. Moreover, the level of SLC9A1 transcription correlated with the observed changes in NHE activity. The changes in NHE activity during meiotic maturation displayed a similar pattern to that of AE. The recovery rate from acidosis was significantly higher in MII stage oocytes and PN zygotes in HCO3--containing medium; however, the increase was significantly inhibited in Na+-free medium or 4,4'-diisocyanatostilbene-2,2'-disulfonic acid. Furthermore, changes in the transcription of SLC4A8 during meiotic maturation were concordant with the level of exchanger activity. These results indicate that NDCBE activity is present in mouse oocytes and zygotes, and that this activity exhibits a different pattern than that of AE and NHE during meiotic maturation.

Intracellular pH gradients in migrating cells

Cell polarization along the axis of movement is required for migration. The localization of proteins and regulators of the migratory machinery to either the cell front or its rear results in a spatial asymmetry enabling cells to simultaneously coordinate cell protrusion and retraction. Protons might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of pH(i), the Na(+)/H(+) exchanger NHE1, is essential for cell migration and accumulates at the cell front. Here, we test the hypothesis that the uneven distribution of NHE1 activity creates a pH(i) gradient in migrating cells. Using the pH-sensitive fluorescent dye BCECF, pH(i) was measured in five cell lines (MV3, B16V, NIH3T3, MDCK-F1, EA.hy926) along the axis of movement. Differences in pH(i) between the front and the rear end (ΔpH(i) front-rear) were present in all cell lines, and inhibition of NHE1 either with HOE642 or by absence of extracellular Na(+) caused the pH(i) gradient to flatten or disappear. In conclusion, pH(i) gradients established by NHE1 activity exist along the axis of movement.

The Route and Mechanism of Uncoupled Current Flow through Na/K-ATPase Pumps Lacking the Two COOH-Terminal Tyrosines

Na/K-ATPase pumps generate outward current during ATP-driven stoichiometric exchange of three intracellular Na ions for two extracellular K ions. At acidic pH, in the absence of extracellular Na and K ions, an uncoupled current flows through wild-type Na/K pumps at large negative membrane potentials, believed carried by protons. Both currents are abolished by the specific Na/K pump inhibitor ouabain. In Xenopusα1 pumps made less sensitive to ouabain by mutations Q120R/N131D or C113Y we observed a similar uncoupled current in the absence of extracellular Na and K ions even at physiological pH (7.6). After deletion of the last five (∈″KESYY), or two (∈″YY), COOH-terminal residues of those pumps, uncoupled current was recorded also in extracellular Na. We used two-microelectrode recording to measure uncoupled, and coupled Na/K transport, currents as those inhibited by 10-30 mM ouabain in Xenopus oocytes expressing C113Y or C113Y-∈″YY Na/K pumps. To investigate whether the uncoupled current traverses the same principal pathway followed by transported Na and K ions we used two methods to close that pathway. In one method, we formed stable BeFX-Na/K-pump complexes, trapped in an E2P-like state with closed cytoplasmic-side gates, by injecting oocytes with 1mM BeFX. In the other, we closed the extracellular access pathway by modifying a Cys substituted for T806, at the outer end of TM6, with 1 mM extracellular MTSET. Both methods abolished coupled Na/K pump transport current, as well as Na current flow through palytoxin-bound C113Y Na/K pump-channels. But neither method diminished uncoupled current at −180 mV, suggesting either that the responsible ions do not traverse the principal pathway shared by transported Na and K ions or, if they do follow that route, that they do not travel as hydrated cations. [NIH HL36783]

Attenuation of Canonical Transient Receptor Potential-Like Channel 6 Expression Specifically Reduces the Diacylglycerol-Mediated Increase in Intracellular Calcium in Human Myometrial Cells

An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).

About a new method to measure fractional Ca2+ currents through ligand-gated ion channels

About a new method to measure fractional Ca2+ currents through ligand-gated ion channels

Metabolic Inhibition Strongly Inhibits Na +-Dependent Mg 2+ Efflux in Rat Ventricular Myocytes

We measured intracellular Mg 2+ concentration ([Mg 2+] i) in rat ventricular myocytes using the fluorescent indicator furaptra (25°C). In normally energized cells loaded with Mg 2+, the introduction of extracellular Na + induced a rapid decrease in [Mg 2+] i: the initial rate of decrease in [Mg 2+] i (initial Δ[Mg 2+] i/Δ t) is thought to represent the rate of Na +-dependent Mg 2+ efflux (putative Na +/Mg 2+ exchange). To determine whether Mg 2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na + gradient, we estimated the initial Δ[Mg 2+] i/Δ t after metabolic inhibition. In the absence of extracellular Na + and Ca 2+, treatment of the cells with 1 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg 2+] i from ∼0.9 mM to ∼2.5 mM in a period of 5–8 min (probably because of breakdown of MgATP and release of Mg 2+) and cell shortening to ∼50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg 2+] i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for ≥90 min. The initial Δ[Mg 2+] i/Δ t was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59–85 min), a significant decrease in the initial Δ[Mg 2+] i/Δ t (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na + concentration ([Na +] i) estimated with a Na + indicator sodium-binding benzofuran isophthalate was, on average, 5.0–10.5 mM during the time required for the initial Δ[Mg 2+] i/Δ t measurements, which is well below the [Na +] i level for half inhibition of the Mg 2+ efflux (∼40 mM). Normalization of intracellular pH using 10 μM nigericin, a H + ionophore, did not reverse the inhibition of the Mg 2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly in the this study), rather than elevation of [Na +] i or intracellular acidosis, inhibits the Mg 2+ efflux, suggesting the absolute necessity of ATP for the Na +/Mg 2+ exchange.

TRPC6 knock‐down specifically attenuates diacylglycerol‐mediated elevation of intracellular calcium in human myometrial cells

Human myometrium exhibits receptor‐, store‐ and diacylglycerol (OAG)‐mediated extracellular Ca2+‐dependent increases in intracellular Ca2+ ([Ca2+]i)(SRCE) and expresses TRPC mRNAs, predominantly TRPC1, 4, and 6, that have been implicated in SRCE. To determine the role of TRPC6 in myometrial SRCE, short hairpin RNA constructs that effectively targeted a TRPC6 mRNA reporter for degradation were designed and one sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, 3, 4, 5 or 7 mRNAs in PHM1‐41 myometrial cells. Compared to uninfected cells or cells infected with empty vector, the increase in [Ca2+]i in response to OAG was specifically inhibited by TC6sh1; SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1‐41 cells were activated by OAG in the absence of extracellular Na+, the increase in [Ca2+]i was partially reduced. Furthermore, pretreatment with nifedipine, an L‐type calcium channel blocker, also reduced the OAG‐induced [Ca2+]i increase. These findings suggest that OAG targets channels containing TRPC6 in myometrial cells and that these channels act, in part, in an indirect manner via enhanced Na+ entry and activation of voltage‐dependent Ca2+ entry to promote elevation of intracellular Ca2+. Supported by March of Dimes #6‐FY05‐77 and HD38970.