TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

Protein kinase C-mediated phosphorylation of p47 phox modulates platelet-derived growth factor-induced H2O2 generation and cell proliferation in human umbilical vein endothelial cells.

Substantial evidence indicate that growth factors such as platelet-derived growth factor (PDGF) exert their effect, at least in part, through reactive oxygen species (ROS) generated via NAD(P)H oxidase. In this work, the role of p47(phox), a key component of the phagocytic NAD(P)H oxidase in cell proliferation, was addressed. The authors show that diphenylene iodonium (DPI) and apocynin, but not N(G)-nitro-L-arginine methyl esterL-NAME, reduced PDGF-induced ROS generation and proliferation in human umbilical vein endothelial cells (HUVECs). Pharmacological inhibition of protein kinase C (PKC) as well as dominant-negative mutants of p47(phox) directed to PKC-dependent phosphorylation targets inhibited PDGF-stimulated ROS production and cell proliferation. Hydrogen peroxide restored PDGF-stimulated proliferation in cells that was inhibited by apocynin, DPI, or by the dominant-negative mutants. PDGF-induced proliferation was reduced in the HUVEC-derived cell line E.A.hy926 overexpressing catalase. On the contrary, cells overexpressing superoxide dismutase 1 exhibited increased proliferation. These results demonstrate that PKC-dependent phosphorylation of p47(phox) is essential for PDGF-stimulated ROS generation and proliferation in HUVECs. More relevant, H(2)O(2) is identified as the key molecule that signals proliferation in the systems studied.

Swelling-activated ion channels: functional regulation in cell-swelling, proliferation and apoptosis.

Cell volume regulation is one of the most fundamental homeostatic mechanisms and essential for normal cellular function. At the same time, however, many physiological mechanisms are associated with regulatory changes in cell size meaning that the set point for cell volume regulation is under physiological control. Thus, cell volume is under a tight and dynamic control and abnormal cell volume regulation will ultimately lead to severe cellular dysfunction, including alterations in cell proliferation and cell death. This review describes the different swelling-activated ion channels that participate as key players in the maintenance of normal steady-state cell volume, with particular emphasis on the intracellular signalling pathways responsible for their regulation during hypotonic stress, cell proliferation and apoptosis.

Nonselective cation channels as effectors of free radical-induced rat liver cell necrosis.

Necrosis, as opposed to apoptosis, is recognized as a nonspecific cell death that induces tissue inflammation and is preceded by cell edema. In non-neuronal cells, the latter has been explained by defective outward pumping of Na(+) caused by metabolic depletion or by increased Na(+) influx via membrane transporters. Here we describe a novel mechanism of swelling and necrosis; namely the influx of Na(+) through oxidative stress-activated nonselective cation channels. Exposure of liver epithelial Clone 9 cells to the free-radical donors calphostin C or menadione induced the rapid activation of an approximately 16-pS nonselective cation channel (NSCC). Blockage of this conductance with flufenamic acid protected the cells against swelling, calcium overload, and necrosis. Protection was also achieved by Gd(3+), an inhibitor of stretch-activated cation channels, or by isosmotic replacement of extracellular Na(+) with N-methyl-D-glucamine. It is proposed that NSCCs, which are ubiquitous although largely inactive in healthy cells, become activated under severe oxidative stress. The ensuing influx of Na(+) initiates a positive feedback of metabolic and electrolytic disturbances leading cells to their necrotic demise.

Characterisation of a cell swelling-activated K+-selective conductance of ehrlich mouse ascites tumour cells.

The K+ and Cl- currents activated by hypotonic cell swelling were studied in Ehrlich ascites tumour cells using the whole-cell recording mode of the patch-clamp technique. Currents were measured in the absence of added intracellular Ca2+ and with strong buffering of Ca2+. K+ current activated by cell swelling was measured as outward current at the Cl- equilibrium potential (ECl) under quasi-physiological gradients. It could be abolished by replacing extracellular Na+ with K+, thereby cancelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4 approximately Na+ approximately Li+; Cs+ appeared to be inhibitory. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and +20 mV with a permeability coefficient of around 10(-6) cm s(-1) with both physiological and high-K+ extracellular solutions. The class III antiarrhythmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner with an IC50 of 32 microM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich cells. Cell swelling-activated K+ currents of Ehrlich cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so-called background K+ channels. Noise analysis of whole-cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by cell swelling, measured at 0 mV under a quasi-physiological K+ gradient.

Separate taurine and chloride efflux pathways activated during regulatory volume decrease.

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl- by anions that are more permeant through the volume-activated Cl- channel, as indicated by electrophysiological measurements, significantly decreased taurine efflux. In the presence of less-permeant anions, an increase in taurine efflux was observed. Simultaneous measurement of the 125I, used as a tracer for Cl-, and [3H]taurine efflux showed that the time courses for the two effluxes differed. In Cl--rich medium the increase in I- efflux was transient, whereas that for taurine was sustained. Osmosensitive Cl- conductance, assessed by measuring changes in cell volume, increased rapidly after hypotonic shock. The influx of taurine was able to counteract Cl- conductance-dependent cell shrinkage but only approximately 4 min after triggering cell swelling. This taurine-induced effect was blocked by DIDS. Differences in anion sensitivity, the time course of activation, and sensitivity to DIDS suggest that the main cell swelling-activated permeability pathways for taurine and Cl- are separate.

Modulation by extracellular and intracellular iodide of volume-activated Cl- current in HeLa cells.

The patch-clamp technique was used to study the effect of extracellular and intracellular iodide on the properties of the volume-activated anion current in HeLa cells. Upon hypotonic challenge, HeLa cells responded by activating an outwardly rectifying Cl- current. Replacement of extracellular Cl- by I-, a more permeable anion, increased the peak outward and inward current, reduced the magnitude of deactivation observed at depolarized potentials and shifted the half-maximal (V0.5) deactivation voltage towards more positive values. On the other hand, when internal Cl- was replaced by I- the volume-activated current was not observed in normal, Cl--rich hypotonic extracellular solution. However, switching to a hypotonic extracellular solution containing a mixture of Cl- and I- resulted in the activation of the volume-sensitive current. Furthermore, once the current was activated, I- could be excluded from the external solution without significantly affecting the current properties. These results suggest that the permeant anion plays a crucial role in the gating mechanism of the volume-activated Cl- current, influencing the swelling-dependent activation and the voltage-dependent deactivation processes.

Modulation by extracellular Cl- of volume-activated organic osmolyte and halide permeabilities in HeLa cells.

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.

Calcium-activated chloride currents and non-selective cation channels in a novel cystic fibrosis-derived human pancreatic duct cell line.

The modulation of ion fluxes across the plasma membrane of epithelial cells is central for fluid secretion and absorption. Their disruption can lead to pathological states. An example is cystic fibrosis (CF), a disease characterized by abnormal functioning of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-modulated chloride channel. Here we report the characterization of calcium-activated, DIDS sensitive chloride current and non-selective calcium-activated cation channels in a novel human pancreatic duct cell line (YHV-1) derived from a non-delta F508 mutation CF patient bearing a severe phenotype. Southern blot analysis of the CFTR gene indicates a distinct electrophoretic pattern for the region spanned by exons 15-24, a result presumably related to a mutation which has yet to be identified. In contrast to large calcium-activated chloride currents there were no cAMP-dependent CFTR-type chloride currents. Non-selective cation channels were blocked by intracellular ATP and activated by intracellular calcium and cAMP. We propose the cell line YHV-1 as a suitable model for studying pancreatic ion and fluid secretion alterations in CF.

A chloride channel from human placenta reconstituted into giant liposomes.

OBJECTIVE: Ion channels play important roles in epithelial transport, but they are difficult to access for conventional electrophysiologic studies in intact placenta. The purpose of this work was to explore the suitability of purified trophoblast plasma membrane as a source of ion channels for reconstitution in artificial lipid membranes. STUDY DESIGN: Human placental brush border membranes were purified by differential and gradient centrifugation and fused with small liposomes. Giant liposomes were then generated by a cycle of dehydration and rehydration. These giant liposomes are suitable for electrophysiologic studies and were probed for the presence of active ion channels by the patch-clamp method. RESULTS: The results reported here indicate the presence of a high conductance chloride channel showing some similarities with "maxi" chloride channels described in secreting and absorbing epithelia. The channel had a slight outward rectification with conductances of 232 and 300 pS at negative and positive potentials, respectively. CONCLUSIONS: For the first time successful reconstitution of a human placental ion channel is achieved in a system suited for electrophysiologic studies. The chloride channel described might play a role in transplacental transport.

Modulation of intracellular pH by secretagogues and the Na+/H+ antiporter in cultured bovine chromaffin cells.

The possible physiological role of cytosolic pH changes in adrenal medullary chromaffin cell secretion was examined by investigating the effects of catecholamine secretagogues on cytosolic pH, which was monitored using the intracellular fluorescent indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Anti-fluorescein antibodies were used to reduce background fluorescence from extracellular 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Stimulation with both cholinergic agonists (acetylcholine, nicotine) and a depolarizing agent (high K+) transiently acidified the cytosol of the chromaffin cell. This acidification was antagonized by reducing extracellular Ca2+ concentration and by Ca2+ antagonists (Co2+, verapamil), indicating that it occurred secondarily to Ca2+ influx, possibly as a result of exchange of Ca2+ ions for protons across organelle membranes. Taken together with previously published data [Kuijpers G.A. J. et al. (1989) J. biol. Chem. 264, 698-705] showing no effect of cytosolic acidification on nicotine-induced catecholamine secretion, these results indicate that secretagogue-induced cytosolic pH changes do not represent a causal step in stimulus-secretion coupling of the chromaffin cell. The cytosolic pH recovery to pre-stimulatory cytosolic pH levels was delayed by amiloride and by 5-(N,N-dimethyl)amiloride, at concentrations that otherwise substantially inhibited cytosolic pH recovery from the rebound acidification induced by a 40-fold sudden dilution of NH4Cl. This latter type of recovery results from activation of an Na+/H+ exchange mechanism. Therefore, the data suggest that Na+/H+ exchange is actively involved in the dissipation of the small acid loads generated by secretagogues.

[Physiopathology of cystic fibrosis]. / Fisiopatología de la fibrosis quística.

Cystic Fibrosis (CF) is the most common lethal genetic autosomic disease in Caucasians. The disease expresses itself in airway and other epithelial cells as a defective chloride ion absorption and secretion. At least, an abnormal cAMP-dependent regulation of an apically located chloride channel has been proposed as the underlying molecular defect. The gene responsible for CF has been identified and predicted to encode a membrane protein termed cystic fibrosis transmembrane conductance regulator (CFTR). The functional role of the predicted protein remains unclear, although strong evidence suggest that it is directly or indirectly involved in regulation of the apical chloride permeability in epithelial cells. This review discusses the fundamental issues currently being investigated in CF.

Generation and amplification of the cytosolic calcium signal during secretory responses to gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) stimulates characteristic biphasic increases in cytosolic calcium concentration ([Ca2+]i) and in luteinizing hormone (LH) release in cultured gonadotrophs, with an early peak followed by a prolonged plateau in both responses. Analysis of [Ca2+]i by dual-wavelength fluorimetric assay and of LH release at 5-sec intervals in perifused pituitary cells revealed increases in both responses within a few seconds of exposure to GnRH. The maximum elevation of [Ca2+]i occurred within 20 sec, and the peak gonadotropin release in 35 sec; the total duration of the spike phase for both [Ca2+]i and LH release was 2.5 min. Under extracellular Ca2(+)-deficient conditions, the GnRH-induced peak in [Ca2+]i was reduced by about 20% and the plateau phase was abolished. Concomitantly, the magnitude of the acute phase of LH release was reduced by 40% and that of the second phase by about 90%. Recovery of the plateau phase of LH release occurred within 25 sec after addition of 1.25 mM Ca2+ to Ca2(+)-deficient medium. In a dose-dependent manner, the non-selective Ca2+ channel blockers Co2+ and Cd2+ reduced the Ca2+ current measured by whole-cell recording in pituitary gonadotrophs and abolished the extracellular Ca2(+)-dependent component of LH release. The selective calcium channel blocker, nifedipine, decreased the magnitude of the Ca2+ current and reduced the plateau phase of LH release by 50%; conversely, the dihydropyridine agonist methyl, 1,4,dihydro-2,6-dimethyl 3-nitro-4-(2-trifluorome) (Bay K 8644) consistently enhanced the amplitudes of both Ca2+ current and GnRH-induced LH release. These data reveal a close temporal correlation between changes in [Ca2+]i and LH release during GnRH action, with Ca2+ mobilization during the spike phase and Ca2+ influx through dihydropyridine-sensitive and insensitive sets of receptor-operated calcium channels during the spike and plateau phases. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses to a wide range of GnRH concentrations in the presence and absence of extracellular Ca2+ is consistent with amplification of the [Ca2+]i signal in agonist-stimulated gonadotrops.

Effects of calcium and Bay K-8644 on calcium currents in adrenal medullary chromaffin cells.

The kinetic and steady-state characteristics of calcium currents in cultured bovine adrenal chromaffin cells were analyzed by the patch-clamp technique. Whole cell inward Ca2+ currents, recorded in the presence of either 5.2 or 2.6 mM Ca2+ exhibited a single, noninactivating component. To analyze the effects of Ca2+ and Bay K-8644 on the kinetics of the Ca2+ currents, we used a modified version of the Hodgkin-Huxley empirical model. At physiological [Ca2+] (2.5 mM) the midpoint of the steady-state Ca2(+)-channel activation curve lay at -6.9 mV. Increasing the [Ca2+] to 5.2 mM shifted the midpoint by -4.3 mV along the voltage axis. At the midpoint, changes in potential of 7.8 mV (for 5.2 mM Ca2+) and 9.2 mV (for 2.5 mM Ca2+) induced an e-fold change in the activation of the current. Increasing [Ca2+]o from 2.5 to 5.2 mM induced a marked increase in the rate constant for turning on the Ca2+ permeability. Conductances were estimated from the slope of the linear part of the current-voltage relationships as 8.7 and 4.2 nS in the presence of 5.2 and 2.5 mM Ca2+, respectively. Incubation of the cells in the presence of Bay K-8644 at increasing concentrations from 0.001 to 0.1 microM increased the slope conductance from 4.2 to 9.6 nS. Further increases in the concentration of Bay K-8644 from 1 to 100 microM induced a marked reduction in the conductance to 1.1 nS. In the presence of Bay K-8644 (0.1 microM) the midpoint of the activation curve was shifted by 6.1 mV towards more negative potentials, i.e., from -6.9 to -13 mV. At the midpoint potential of -13 mV, a change in potential of 6.9 mV caused an e-fold change in Ca2+ permeability. The kinetic analysis showed that Bay K-8644 significantly reduced the size of the rate constant for turning off the Ca2+ permeability.

Characteristics of two types of calcium channels in rat pituitary gonadotrophs.

The properties of Ca2+ channels in cultured rat pituitary gonadotrophs were analyzed by the patch-clamp technique. The inward Ca2+ currents, recorded in the presence of 5.2 mM Ca2+ or Ba2+, included a fast, transient component with activation-inactivation kinetics and a delayed component with slower activation. The midpoint of the activation curve lay at -30 mV for the transient component and at -12 mV for the delayed component. At the midpoint, changes in potential of 9.5 and 13 mV induced an e-fold change in the activation of the transient and delayed components, respectively. The rate of inactivation of the first component was strongly voltage dependent. At -43 mV, a 7.4-mV change in potential induced an e-fold change in the fraction of Ca2+ channels available to conduct Ca2+ current. During long-lasting (100-200 ms) low-frequency depolarizing voltage-clamp pulses, the size of the delayed component of the Ca2+ current remained constant. The differential effects of membrane potential on inactivation and the different time constants for activation of the two components of the Ca2+ conductance indicate the presence of two types of Ca2+ channels in the membrane of the gonadotroph: the rapidly inactivating current appears to be attributable to a T-type channel, and the noninactivating current corresponds to the L-type channel described in many other cell types.

Desensitization of pituitary gonadotropin secretion by agonist-induced inactivation of voltage-sensitive calcium channels.

Gonadotropin-releasing hormone (GnRH) stimulates calcium mobilization and influx in pituitary gonadotrophs, and agonist-induced calcium entry through voltage-sensitive channels (VSCC) is required for the maintenance of gonadotropin secretion. However, prolonged or frequent exposure to GnRH attenuates the extracellular Ca2+-dependent cytosolic Ca2+ signal and diminishes hormone secretion. Measurements of membrane Ca2+ currents revealed significant impairment of VSCC activity in gonadotrophs during desensitization by GnRH. VSSC were also inactivated in a calcium-dependent manner during exposure to high K+. Prolonged inactivation of such Ca2+ channels by high K+ reduced the calcium and secretory responses to GnRH and vice versa. The calcium-dependent inactivation of VSCC during GnRH action appears to be a primary factor in the onset of desensitization in pituitary gonadotrophs. This mechanism could also account for the development of agonist-induced refractoriness in other calcium-regulated target cells.

Synexin-mediated fusion of bovine chromaffin granule ghosts. Effect of pH.

Synexin induces chromaffin granule ghosts to fuse one to another, a process which is followed continuously and quantitatively by monitoring the mixing of the intragranular aqueous compartments. A freeze-thaw technique was used for preparing chromaffin granule ghosts loaded with a self-quenching concentration of the fluorescent, high molecular weight probe FITC-Dextran. When the loaded ghosts were mixed with empty ghosts in the presence of synexin, the two compartments fused, resulting in the dilution of the probe with the concomitant increase in fluorescence. So as to suppress possible leakage signals, anti-fluorescein antibodies which quench probe fluorescence were present in the reaction media. Synexin-mediated fusion of freeze-thaw (F/Th) ghosts and binding of 125I-synexin to these membranes were found to be dependent on Ca2+ concentration, but only in a partial manner. However, these two synexin-mediated properties were demonstrably sensitive to [H+] in the medium. A detailed pH profile of fusion revealed an apparent midpoint of activation at approx. pH 5.2, with asymptotic values at pH 4 (maximum) and pH 7.2 (minimum). In our attempt to determine whether the pH effect was on the synexin or on the membranes, we found that fusion was blocked only by treatment of the membranes with the membrane-impermeant carboxyl group modifier 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. These data suggest that membrane fusion evoked by synexin seems to be promoted by rendering the F/Th membranes relatively less negatively charged while the synexin becomes more positively charged. The fusion process was entirely dependent upon synexin concentration; the k1/2 under optimal conditions of pCa and pH was 85 nM. Similar to what has been previously found with intact granules, an anti-synexin polyclonal antibody partially (48%) blocked fusion, as did pretreatment of the chromaffin granules ghosts with trypsin (30%). We conclude that the coincident pCa and pH sensitivity of synexin-mediated binding to chromaffin granule membranes and their subsequent fusion might be associated with physiological changes in the concentration of both cations in the cytoplasm of secreting chromaffin cells.