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Increased expression of the TRPM4 channel has been reported to be associated with the progression of prostate cancer. However, the molecular mechanism underlying its effect remains unknown. This work found that decreasing TRPM4 levels leads to the reduced proliferation of PC3 cells. This effect was associated with a decrease in total β-catenin protein levels and its nuclear localization, and a significant reduction in Tcf/Lef transcriptional activity. Moreover, TRPM4 silencing increases the Ser33/Ser37/Thr41 β-catenin phosphorylated population and reduces the phosphorylation of GSK-3β at Ser9, suggesting an increase in β-catenin degradation as the underlying mechanism. Conversely, TRPM4 overexpression in LNCaP cells increases the Ser9 inhibitory phosphorylation of GSK-3β and the total levels of β-catenin and its nonphosphorylated form. Finally, PC3 cells with reduced levels of TRPM4 showed a decrease in basal and stimulated phosphoactivation of Akt1, which is likely responsible for the decrease in GSK-3β activity in these cells. Our results also suggest that the effect of TRPM4 on Akt1 is probably mediated by an alteration in the calcium/calmodulin-EGFR axis, linking TRPM4 activity with the observed effects in β-catenin-related signaling pathways. These results suggest a role for TRPM4 channels in β-catenin oncogene signaling and underlying mechanisms, highlighting this ion channel as a new potential target for future therapies in prostate cancer., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Cerebral ischemia-reperfusion injury triggers a deleterious process ending in neuronal death. This process has two components, a glutamate-dependent and a glutamate-independent mechanism. In the glutamate-independent mechanism, neurons undergo a slow depolarization eventually leading to neuronal death. However, little is known about the molecules that take part in this process. Here we show by using mice cortical neurons in culture and ischemia-reperfusion protocols that TRPM4 is fundamental for the glutamate-independent neuronal damage. Thus, by blocking excitotoxicity, we reveal a slow activating, glibenclamide- and 9-phenanthrol-sensitive current, which is activated within 5 min upon ischemia-reperfusion onset. TRPM4 shRNA-based silenced neurons show a reduced ischemia-reperfusion induced current and depolarization. Neurons were protected from neuronal death up to 3 hours after the ischemia-reperfusion challenge. The activation of TRPM4 during ischemia-reperfusion injury involves the increase in both, intracellular calcium and H 2 O 2 , which may act together to produce a sustained activation of the channel.

Angiotensin II regulation of L-type calcium currents in cardiac muscle is controversial and the underlying signaling events are not completely understood. Moreover, the possible role of auxiliary subunit composition of the channels in Angiotensin II modulation of L-type calcium channels has not yet been explored. In this work we study the role of Ca(v)β subunits and the intracellular signaling responsible for L-type calcium current modulation by Angiotensin II. In cardiomyocytes, Angiotensin II exposure induces rapid inhibition of L-type current with a magnitude that is correlated with the rate of current inactivation. Semi-quantitative PCR of cardiomyocytes at different days of culture reveals changes in the Ca(v)β subunits expression pattern that are correlated with the rate of current inactivation and with Angiotensin II effect. Over-expression of individual b subunits in heterologous systems reveals that the magnitude of Angiotensin II inhibition is dependent on the Ca(v)β subunit isoform, with Ca(v)β(1b) containing channels being more strongly regulated. Ca(v)β(2a) containing channels were insensitive to modulation and this effect was partially due to the N-terminal palmitoylation sites of this subunit. Moreover, PLC or diacylglycerol lipase inhibition prevents the Angiotensin II effect on L-type calcium channels, while PKC inhibition with chelerythrine does not, suggesting a role of arachidonic acid in this process. Finally, we show that in intact cardiomyocytes the magnitude of calcium transients on spontaneous beating cells is modulated by Angiotensin II in a Ca(v)β subunit-dependent manner. These data demonstrate that Ca(v)β subunits alter the magnitude of inhibition of L-type current by Angiotensin II.

Chloride permeability pathways and progesterone (P4) secretion elicited by human chorionic gonadotropin (hCG) in human granulosa cells were studied by electrophysiological techniques and single-cell volume, membrane potential and Ca2+i measurements. Reduction in extracellular Cl(-) and equimolar substitution by the membrane-impermeant anions glutamate or gluconate significantly increased hCG-stimulated P4 accumulation. A similar result was achieved by exposing the cells to hCG in the presence of a hypotonic extracellular solution. Conversely, P4 accumulation was drastically reduced in cells challenged with hCG exposed to a hypertonic solution. Furthermore, conventional Cl(-) channel inhibitors abolished hCG-mediated P4 secretion. In contrast, 25-hydroxycholesterol-mediated P4 accumulation was unaffected by Cl(-) channel blockers. In human granulosa cells, hCG triggered the activation of a tamoxifen-sensitive outwardly rectifying Cl(-) current comparable to the volume-sensitive outwardly rectifying Cl(-) current. Exposure of human granulosa cells to hCG induced a rapid 4,4'-diisothiocyanatostilbene-2,2-disulphonic acid-sensitive cell membrane depolarization that was paralleled with an approximately 20% decrease in cell volume. Treatment with hCG evoked oscillatory and nonoscillatory intracellular Ca2+ signals in human granulosa cells. Extracellular Ca2+ removal and 4,4'-diisothiocyanatostilbene-2,2-disulphonic acid abolished the nonoscillatory component while leaving the Ca2+ oscillations unaffected. It is concluded that human granulosa cells express functional the volume-sensitive outwardly rectifying Cl(-) channels that are activated by hCG, which are critical for plasma membrane potential changes, Ca2+ influx, and P4 production.

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.

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.

The role of Ca2+ in the signaling transduction pathway involved in osmosensitive taurine efflux in HeLa cells was studied using radiotracer efflux techniques. Taurine efflux induced by extracellular hypotonicity was decreased by 85% by removal of extracellular Ca2+ and simultaneous depletion of intracellular Ca2+ stores with thapsigargin. Extracellular Ca2+ removal, thapsigargin treatment, or addition of Gd3+ all decreased taurine efflux by approximately 50%. To explore the putative signal transduction pathways involved in swelling-induced taurine efflux, HeLa cells were exposed to PP1, an inhibitor of the Src family of tyrosine kinases, the phospholipase C inhibitor U73122, the IP3 receptor antagonist 2-APB, and the generic protein kinase C inhibitor chelerythrine. All of these treatments caused approximately 50% inhibition of taurine release in Ca2+-rich extracellular medium and approximately 85%-90% in Ca2+-free conditions. The inhibitors of the conventional protein kinase C isoforms BIM-1 and Gö6976 reduced taurine efflux to a lesser extent. Acute (10-min) exposure to the phorbol ester tetradecanoyl phorbol acetate (TPA) increased taurine efflux in 25%, whilst overnight exposure had an inhibitory effect decreasing efflux by 22%. A working model for activation of osmosensitive taurine efflux in HeLa cells involving different Ca2+ signaling pathways is presented.

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.

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.

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.

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.

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.

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.