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In recent decades cancer emerged as one of the leading causes of death in the developed countries, with some types of cancer contributing to the top 10 causes of death on the list of the World Health Organization. Carcinogenesis, a malignant transformation causing formation of tumors in normal tissues, is associated with changes in the cell cycle caused by suppression of signaling pathways leading to cell death and facilitation of those enhancing proliferation. Further progression of cancer, during which benign tumors acquire more aggressive phenotypes, is characterized by metastatic dissemination through the body driven by augmented motility and invasiveness of cancer cells. All these processes are associated with alterations in calcium homeostasis in cancer cells, which promote their proliferation, motility and invasion, and dissuade cell death or cell cycle arrest. Remodeling of store-operated calcium entry (SOCE), one of the major pathways regulating intracellular Ca2+ concentration ([Ca2+]i), manifests a key event in many of these processes. This review systematizes current knowledge on the mechanisms recruiting SOCE-related proteins in carcinogenesis and cancer progression.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca

Background Purinergic P2X receptors in vascular smooth muscle cells (VSMCs) play an important role in physiological stimulatory responses to the extracellularly released ATP. The aim of this work was to identify molecular P2X receptor subunits in VSMCs isolated from rat anterior, posterior and basilar arteries using a number of contemporary laboratory techniques. Methods P2X mediated ionic currents were recorded using amphotericin B perforated patch clamp method. Gene expression analysis was performed using RT-PCR in manually collected VSMCs. The expression of proteins was confirmed by fluorescent immunocytochemistry. Results Under voltage clamp conditions VSMCs stimulated by application of 10 μmol/l selective P2X receptor agonist αβ-meATP, the biphasic currents consisting of rapidly rising rapidly desensitizing and slowly desensitizing components were observed in freshly isolated myocytes from all three arteries. Using RT-PCR, the expression of genes encoding only P2X1 and P2X4 receptor subunits was detected in preparations from all three arteries. The expression of corresponding P2X1 and P2X4 receptor subunit proteins was confirmed in isolated VSMCs. Conclusions Our work therefore identified that in major arteries of rat cerebral circulation VSMCs express only P2X1 and P2X4 receptors subunits. We can propose that these P2X receptor subunits participate in functional P2X receptor structures mediating ATP-evoked stimulatory responses in cerebral vascular myocytes in vivo .

International audience; Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8(TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca(2+)release channels.

Despite the tremendous progress in medicine, cancer remains one of the most serious global health problems awaiting new effective therapies. Here we present ferroquine (FQ), the next generation antimalarial drug, as a promising candidate for repositioning as cancer therapeutics. We report that FQ potently inhibits autophagy, perturbs lysosomal function and impairs prostate tumor growth in vivo. We demonstrate that FQ negatively regulates Akt kinase and hypoxia-inducible factor-1α (HIF-1α) and is particularly effective in starved and hypoxic conditions frequently observed in advanced solid cancers. FQ enhances the anticancer activity of several chemotherapeutics suggesting its potential application as an adjuvant to existing anticancer therapy. Alike its parent compound chloroquine (CQ), FQ accumulates within and deacidifies lysosomes. Further, FQ induces lysosomal membrane permeabilization, mitochondrial depolarization and caspase-independent cancer cell death. Overall, our work identifies ferroquine as a promising new drug with a potent anticancer activity.

Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp “golden standard”, is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.

Ionotropic P2X receptors (P2XRs) are involved in sympathetic control of the vascular tone; they mediate entry of Ca2+ in smooth muscle cells (SMCs), which results in depolarization of the latter and activation of voltage-gated L-type calcium channels. In addition, Ca2+ ions, after their entry into the cell, trigger Ca2+ release from the sarcoplasmic reticulum (SR) of SMCs via ryanodine receptors (RyRs), and this amplifies calcium signals. We found earlier that Ca2+ release mediated by inositol triphosphate (IP3) receptors (IP3Rs) also provides a considerable contribution to P2XR-mediated calcium signaling. Thus, a metabotropic signal pathway is a component of the calcium signaling system triggered by ionotropic P2XRs. Using confocal detection of changes in the intracellular Ca2+ concentration ([Ca2+] i ) and applications of the inhibitors of calcium channels (nicardipine, 5 μM), sarco-endoplasmic Ca2+ ATPase SERCA (CPA, 10 μM), IP3Rs (2-APB, 30 μM), RyRs (tetracaine, 100 μM), and phosphalipase C (PLC; U-73122, 2.5 μM), we estimated relative contributions of the above-mentioned four components to increase in the [Ca2+] i induced by the action of an agonist of P2XRs, α,β-meATP. The contributions of transmembrane Ca2+ entry via channels of P2XRs and calcium channels were comparable (11.0 ± 1.4 %, n = 14 and 8.0 ± 1.4 %, n = 14, respectively). The contribution of Ca2+ release via IP3Rs was found to be three times greater than that via RyRs (41 ± 5 %, n = 26 and 14 ± 7 %, n = 16, respectively). Blocking of calcium channels resulted in a sevenfold decrease in the contribution of IP3R-mediated Ca2+ release (from 41.0 to 5.6%); in this case, the contribution of RyR-mediated Ca2+ release underwent no significant changes. This fact allows us to suppose that there is a functional relation between activation of calcium channels and functioning of a metabotropic PLC/IP3-mediated signal cascade. The efficiency of inhibition of α,β-meATP-induced calcium responses by the blocker of PLC, on the one hand, and by the IP3R blocker and nicardipine, on the other hand, is comparable, and this fact agrees with the above hypothesis. According to our data, P2XR activation-induced increase in [Ca2+] I results not only from P2XR-mediated Ca2+ entry that triggers Ca2+ release via RyRs but also from Ca2+ release via IP3Rs. The latter process is realized due to the functioning of the PLC-mediated pathway, is in close relation with activation of calcium channels, and provides a dominant contribution in Ca2+ release from the stores after activation of the above ionotropic receptors.

ATP is one of the principal sympathetic neurotransmitters which contracts vascular smooth muscle cells (SMCs) via activation of ionotropic P2X receptors (P2XRs). We have recently demonstrated that contraction of the guinea pig small mesenteric arteries evoked by stimulation of P2XRs is sensitive to inhibitors of IP3 receptors (IP3Rs). Here we analyzed contribution of IP3Rs and ryanodine receptors (RyRs) to [Ca(2+)]i transients induced by P2XR agonist αβ-meATP (10 μM) in single SMCs from these vessels.The effects of inhibition of L-type Ca(2+) channels (VGCCs), RyRs and IP3Rs (5 μM nicardipine, 100 μM tetracaine and 30 μM 2-APB, respectively) on αβ-meATP-induced [Ca(2+)]i transients were analyzed using fast x-y confocal Ca(2+) imaging.The effect of IP3R inhibition on the [Ca(2+)]i transient was significantly stronger (67 ± 7%) than that of RyR inhibition (40 ± 5%) and was attenuated by block of VGCCs. The latter indicates that activation of VGCCs is linked to IP3R-mediated Ca(2+) release. Immunostaining of RyRs and IP3Rs revealed that RyRs are located mainly in deeper sarcoplasmic reticulum (SR) while sub-plasma membrane (PM) SR elements are enriched with type 1 IP3Rs. This structural peculiarity makes IP3Rs more accessible to Ca(2+) entering the cell via VGCCs. Thus, IP3Rs may serve as an "intermediate amplifier" between voltage-gated Ca(2+) entry and RyR-mediated Ca(2+) release.P2X receptor activation in mesenteric artery SMCs recruits IP3Rs-mediated Ca(2+) release from sub-PM SR, which is facilitated by activation of VGCCs. Sensitivity of IP3R-mediated release to VGCC antagonists in vascular SMCs makes this mechanism of special therapeutic significance.

BACKGROUND AND PURPOSE P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca2+ concentration ([Ca2+]i). Although it is well-appreciated that the myocyte Ca2+ signalling system is composed of microdomains, little is known about the structure of the [Ca2+]i responses induced by P2X receptor stimulation in vascular myocytes. EXPERIMENTAL APPROACHES Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca2+ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP). KEY RESULTS RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP3R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca2+]i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L−1αβ-meATP triggered an abrupt Ca2+ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP3Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca2+ channels (VGCCs) or IP3Rs suppressed the sub-plasmalemmal [Ca2+]i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP3R inhibition on the sub-plasmalemmal [Ca2+]i upstroke was attenuated following block of VGCCs. CONCLUSIONS AND IMPLICATIONS Depolarization of RVSMCs following P2X receptor activation induces IP3R-mediated Ca2+ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca2+ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.

Background and purpose: Ionotropic purinoreceptors (P2X) in renal vascular smooth muscle cells (RVSMCs) are involved in mediating the sympathetic control and paracrine regulation of renal blood flow (RBF). Activation of P2X receptors elevates [Ca2+]i in RVSMCs triggering their contraction, leading to renal vasoconstriction and decrease of RBF. The goal of the present work was to characterize the P2X receptor-mediated ionic current (IP2X) and to identify the types of P2X receptors expressed in myocytes isolated from interlobar and arcuate arteries of rat kidney. Experimental approach: The expression of P2X receptors in isolated RVSMCs was analysed by reverse transcription (RT)–PCR. IP2X and membrane potential were recorded using the amphotericin B-perforated patch method. Key results: RT–PCR analysis on single RVSMCs showed the presence of genes encoding P2X1 and P2X4 receptors. Under voltage clamp conditions, the selective P2X receptor agonist αβ-methylene ATP (αβ-meATP) evoked IP2X similar to that induced by ATP. Under current clamp conditions, both ATP and αβ-meATP evoked a spike-like membrane depolarization followed by a sustained depolarization, linking P2X receptors in RVSMCs to sympathetic control of renal vascular tone. A selective antagonist of P2X1 receptors, NF279, reduced IP2X amplitude by ∼65% concentration-dependently manner within the nanomolar to sub-micromolar range. The residual current was resistant to micromolar concentrations of NF279, but was inhibited by sub-millimolar to millimolar concentrations of NF279. Conclusions and implications: Two types of functional P2X receptors, monomeric P2X1 and heteromeric P2X1/4 receptors, are expressed in RVSMCs. Our study has identified important targets for possible pharmacological intervention in the sympathetic control of renal circulation. British Journal of Pharmacology (2010) 160, 987–997; 10.1111/j.1476-5381.2010.00714.x

This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle α-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmis-sion electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.

Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.

International audience; Aims: P2X receptors (P2XRs) mediate sympathetic control and autoregulation of renal circulation triggering preglomerular vasoconstriction, which protects glomeruli from elevated pressures. Although previous studies established a casual link between glomerular susceptibility to hypertensive injury and decreased preglomerular vascular reactivity to P2XR activation, the mechanisms of attenuation of the P2XR signalling in hypertension remained unknown. We aimed to analyse molecular mechanisms of the impairment of P2XR signalling in renal vascular smooth muscle cells (RVSMCs) in genetic hypertension.Methods and results: We compared the expression of pertinent genes and P2XR-linked Ca(2+) entry and Ca(2+) release mechanisms in RVSMCs of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats. We found that, in SHR RVSMCs, P2XR-linked Ca(2+) entry and Ca(2+) release from the sarcoplasmic reticulum (SR) are both significantly reduced. The former is due to down-regulation of the P2X1 subunit. The latter is caused by a decrease of the SR Ca(2+) load. The SR Ca(2+) load reduction is caused by attenuated Ca(2+) uptake via down-regulated sarco-/endoplasmic reticulum Ca(2+)-ATPase 2b and elevated Ca(2+) leak from the SR via ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors. Spontaneous activity of these Ca(2+)-release channels is augmented due to up-regulation of RyR type 2 and elevated IP3 production by up-regulated phospholipase C-β1.Conclusions: Our study unravels the cellular and molecular mechanisms of attenuation of P2XR-mediated preglomerular vasoconstriction that elevates glomerular susceptibility to harmful hypertensive pressures. This provides an important impetus towards understanding of the pathology of hypertensive renal injury.

Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non‐contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c‐kit. These vascular cells have been termed “interstitial cells” by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca(2+)‐release events from endo/sarcoplasmic reticulum including fast localised Ca(2+) transients (Ca(2+) sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca(2+) oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight‐seal patch‐clamp recording, and fluorescence confocal imaging techniques.

The dynamics of carbachol (CCh)-induced [Ca(2+)](i) changes was related to the kinetics of muscarinic cationic current (mI(cat)) and the effect of Ca(2+) release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) on mI(cat) was evaluated by fast x-y or line-scan confocal imaging of [Ca(2+)](i) combined with simultaneous recording of mI(cat) under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca(2+)-sensitive indicator fluo-3, x-y confocal imaging revealed CCh (10 microM)-induced Ca(2+) waves, which propagated from the cell ends towards the myocyte centre at 45.9 +/- 8.8 microms(-1) (n = 13). Initiation of the Ca(2+) wave preceded the appearance of any measurable mI(cat) by 229 +/- 55 ms (n = 7). Furthermore, CCh-induced [Ca(2+)](i) transients peaked 1.22 +/- 0.11s (n = 17) before mI(cat) reached peak amplitude. At -50 mV, spontaneous release of Ca(2+) through RyRs, resulting in Ca(2+) sparks, had no effect on CCh-induced mI(cat) but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca(2+) release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI(cat) (n = 14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI(cat) when [Ca(2+)](i) was strongly buffered with Ca(2+)/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca(2+) sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca(2+) upon cell dialysis with 5 mM NP-EGTA/3.8 mM Ca(2+) potentiated mI(cat) in the same way as in control). In contrast, IP(3)R-mediated Ca(2+) release upon flash photolysis of "caged" IP(3) (30 microM in the pipette solution) augmented mI(cat) (n = 15), even though [Ca(2+)](i) did not reach the level required for potentiation of mI(cat) during photorelease of Ca(2+) (n = 10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca(2+) indicator fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP(3)R type 1 and to RyRs revealed that IP(3)Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP(3)R-mediated Ca(2+) release plays a central role in the modulation of mI(cat) in the guinea-pig ileum and that IP(3) may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca(2+).

A rise in the intracellular concentration of ionized calcium ([Ca2+]i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca2+ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca2+ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca2+]i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP3Rs) is an important determinant of the [Ca2+]i dynamics and recruits neighboring Ca2+-release sites to generate [Ca2+]i waves; (v) [Ca2+]i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca2+-dependent K+ and Cl- channels sense the local changes in [Ca2+]i during a Ca2+ spark and thereby may couple changes in [Ca2+]i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI cat) does not “mirror” changes in [Ca2+]i, thus reflecting the complexity of [Ca2+]i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca2+ release, either spontaneous or caffeine-induced, does not augment mI cat; (ix) intracellular flash release of Ca2+ is less effective in augmentation of mI cat than flash release of IP3, suggesting that IP3 may sensitize muscarinic cationic channels to Ca2+; (x) intracellular flash release of IP3 fails to augment mI cat in SMCs, in which [Ca2+]i was strongly buffered, suggesting that IP3 exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca2+]i rather than depletion of the IP3-dependent Ca2+ store; and (xi) predominant expression of IP3R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP3R-mediated Ca2+ release and muscarinic cationic channel opening.

1. In visceral smooth muscles, both M(2) and M(3) muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) and shifted their activation curves positively. 4. U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5. InsP(3) (1 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](i). Our present results explain the previously established 'permissive' role of the M(3) receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.

Calcium is essential for contraction of smooth muscle cells (SMC). The contractile proteins are activated by calcium released from the stores within the cell in response to calcium entry through voltage-dependent channels and/or activation of receptors, which often increase D-myoinositol 1,4,5-trisphosphate (IP3) concentration in the cell through stimulation of phospholipase C (PLC). A global rise in the concentration of ionized calcium, [Ca2+] i , which gives rise to contraction or shortening, is initiated at preferred locations in the cell, termed frequent discharge sites (FDS). In many SMC these sites often spontaneously discharge calcium packets; this is caused by bursts of openings of calcium channels (commonly ryanodine receptors, RyR, or IP3 receptors) in the sarcoplasmic reticulum (SR). The rise in [Ca2+] i may be detected by introducing calcium indicator dyes into the cell; the release of a calcium packet then gives rise to a rapid increase in fluorescence, or “spark.” A spark may activate a burst of openings of calcium-activated potassium or chloride channels in the cell membrane, so giving rise to spontaneous transient outward currents (STOC) or spontaneous transient inward currents (STIC), respectively. The term “spark” should probably be reserved for a calcium event resulting from the discharge of a single cluster, or domain, of RyR channels; when IP3; concentrations are raised, adjacent domains may discharge closely in time, giving rise to larger calcium events, activation of more distant domains by a fire-diffuse-fire mechanism, and saltatory propagation of a calcium wave leading to a global rise in [Ca2+] i and contraction of the cell. In many smooth muscle tissues, including some blood vessels, SMC are associated with interstitial cells (IC); well-known examples are the IC of Cajal in the gut muscles. In the media of small mesenteric arteries and portal vein, the IC share many properties with the SMC but, unlike the latter, have many thin processes and do not contract to agents, which contract the SMC. The role of these IC in blood vessels is unknown.

Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40-70 microm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 microm, and spindle-shaped cells from 40 to 300 microm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90-150 microm). The cell membrane capacitance was 46.5+/-2.2 pF in SMCs, 39.7+/-2.4 pF in spindle-shaped ICs and 27.8+/-0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca(2+) sparks and Ca(2+) waves. Ca(2+) waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca(2+)](i) transients (lasting about 2s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1-10 microM), caffeine (1-10 mM) or high-K(+) solution (60mM) led to a rise of [Ca(2+)](i) in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current-clamp conditions was -46.5+/-2.0 mV in spindle-shaped ICs and -45.6+/-2.7 mV in SMCs. Under voltage-clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca(2+) current, a set of K(+) currents, including spontaneous transient outward currents (STOCs) but no Na(+) current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.

In smooth muscle the spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) occurs at preferred locations called frequent discharge sites (FDSs) giving rise to localized intracellular Ca2+ transients (Ca2+ sparks). Laser scanning confocal microscopy of fluo-3-loaded single myocytes freshly isolated from small mesenteric arteries of guinea-pig was used to investigate the action of nitric oxide (NO) donors and noradrenaline on the position and activity of FDSs and on global intracellular Ca2+ concentration ([Ca2+]i). In 8 % of cells 'microsparks', Ca2+ release events smaller in duration, spread and amplitude than Ca2+ sparks were observed. The location of the initiation point of Ca2+ sparks observed during line-scan imaging was found to 'jitter' by +/- 0.41 microm. However, the general position of an FDS within the cell did not change; most FDSs were close (within 1.2 +/- 0.1 microm) to the cell membrane and often multiple FDSs occurred in one confocal plane of the cell. In the resting state, NO donors S-nitroso-N-acetylpenicillamine (SNAP; 50 microM) and sodium nitroprusside (SNP; 100 microM) did not change the general position of FDSs and slightly depressed their activity, but did not affect the global [Ca2+]i significantly. Application of noradrenaline (1-10 microM) increased Ca2+ spark frequency at existing FDS(s) leading to a Ca2+ wave. The increase in FDS activity and in global [Ca2+]i produced by noradrenaline were inhibited by the presence of SNAP or SNP but not by 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP; 100 microM). In the presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), inhibitor of soluble guanylate cyclase, SNAP and SNP still exerted their effects on the noradrenaline response. These results suggest that SNAP and SNP inhibit the noradrenaline-evoked rise in global [Ca2+]i by a cGMP-independent mechanism and that part of this effect is due to inhibition of the activity of FDSs; moreover, only the activity, but not the position, of FDSs is changed by either stimulant or inhibitory substances.

The present study investigated the effect of redox agents on Ca2+-activated Cl- currents ( ICl(Ca)) recorded in smooth muscle cells isolated from rabbit portal vein. In perforated-patch experiments on portal vein cells the amplitude of ICl(Ca) evoked by either spontaneous release of Ca2+ from internal stores or Ca2+ influx through voltage-dependent Ca2+ channels was markedly and irreversibly enhanced by the non-specific oxidant, diamide (10-200 microM). Diamide also prolonged the decay of both currents. The reductant dithiothreitol had no effect on control ICl(Ca) but reversed the increase of current amplitude produced by diamide. Diamide also increased global intracellular Ca2+ at rest but had no effect on the time-course of Ca "sparks" determined by confocal microscopy. Diamide and the endogenous oxidant hydrogen peroxide increased the amplitude and prolonged the kinetics of ICl(Ca)evoked by pipette solutions containing free Ca2+ clamped at 500 nM. Similar effects were observed with the hydrophilic thiol-reactants thimerosal and p-chloromercuriphenylsulphonic acid (PCMPS). Therefore, the gating and activation of Ca2+-activated Cl- conductance is sensitive to redox modification.

Localized Ca(2+)-release events, Ca(2+)sparks, have been suggested to be the 'elementary building blocks' of the calcium signalling system in all types of muscles. In striated muscles these occur at regular intervals along the fibre corresponding to the sarcomeric structures which do not exist in smooth muscle. We showed previously that in visceral and vascular myocytes Ca(2+)sparks occurred much more frequently at certain sites (frequent discharge sites [FDSs]). In this paper, we have related the position of FDSs to the distribution of the sarcoplasmic reticulum in the same living myocyte. The three-dimensional distribution of the SR in freshly isolated rabbit portal vein myocytes was visualized by means of high-resolution confocal imaging after staining with DiOC(6)and/or BODIPY TR-X ryanodine. Both fluorochromes revealed a similar staining pattern indicating a helical arrangement of well-developed superficial SR which occupied about 6% of the cell volume. Computing the frequency of spontaneous Ca(2+)sparks detected by means of fluo-4 fluorescence revealed that in about 70% of myocytes there was only one major FDS located on a prominent portion of superficial SR network usually within 1-2 microm of the nuclear envelope, although a few sparks occurred at other sites scattered generally in superficial locations throughout the cell. Polarized mitochondria were readily identified by accumulation of tetramethylrhodamine ethyl ester (TMRE). These were closely associated with the SR network in extra-nuclear regions. TMRE staining, however, failed to reveal any mitochondria near the FDS-related SR element. When observed, propagating [Ca(2+)](i)waves and associated myocyte contractions were initiated at FDSs. This study provide first insight into the three-dimensional arrangement of the SR in living smooth muscle cells and relates the peculiarity of the structural organization of the myocyte to the features of Ca(2+)signalling at subcellular level.

Ionized calcium plays a central role as a second messenger in a number of physiologically important processes determining smooth muscle function. To regulate a wide range of cellular activities the mechanisms of subcellular calcium signalling should be very diverse. Recent progress in development of visible light-excitable fluorescent dyes with high affinity for Ca2+ (such as oregon green 488 BAPTA indicators, fluo-3 and fura red) and confocal laser scanning microscopy provides an opportunity for direct visualization of subcellular Ca2+ signalling and reveals that many cell function are regulated by the microenvironment within small regions of the cytoplasm ('local control' concept). Here confocal imaging is used to measure and locate changes in [Ca2+]i on a subcellular level in response to receptor stimulation in visceral myocytes. We show that stimulation of muscarinic receptors in ileal myocytes with carbachol leading to activation of inositol 1,4,5-trisphosphate receptors (IP3Rs) accelerates the frequency of spontaneous calcium sparks (discharged via ryanodine receptors, RyRs) and gives rise to periodic propagating Ca2+ waves oscillating with a frequency similar to that of carbachol-activated cationic current oscillations. Furthermore, by combining the whole-cell patch clamp technique with simultaneous confocal imaging of [Ca2+]i in voltage-clamped vascular myocytes we demonstrate that calcium sparks may lead to the opening of either Ca2+-activated Cl- channels or Ca2+-activated K+ channels, and the discharge of a spontaneous transient inward current (STIC) or a spontaneous transient outward current (STOC), respectively.

▪ Abstract The main contributors to increases in [Ca2+]i and tension are the entry of Ca2+ through voltage-dependent channels opened by depolarization or during action potential (AP) or slow-wave discharge, and Ca2+ release from store sites in the cell by the action of IP3 or by Ca2+-induced Ca2+-release (CICR). The entry of Ca2+ during an AP triggers CICR from up to 20 or more subplasmalemmal store sites (seen as hot spots, using fluorescent indicators); Ca2+ waves then spread from these hot spots, which results in a rise in [Ca2+]i throughout the cell. Spontaneous transient releases of store Ca2+, previously detected as spontaneous transient outward currents (STOCs), are seen as sparks when fluorescent indicators are used. Sparks occur at certain preferred locations—frequent discharge sites (FDSs)—and these and hot spots may represent aggregations of sarcoplasmic reticulum scattered throughout the cytoplasm. Activation of receptors for excitatory signal molecules generally depolarizes the cell while it increases the production of IP3 (causing calcium store release) and diacylglycerols (which activate protein kinases). Activation of receptors for inhibitory signal molecules increases the activity of protein kinases through increases in cAMP or cGMP and often hyperpolarizes the cell. Other receptors link to tyrosine kinases, which trigger signal cascades interacting with trimeric G-protein systems.

Localized [Ca2+]i transients ('sparks') first directly detected in cardiac myocytes were considered to represent 'elementary' Ca(2+)-release events playing a key role during excitation-contraction coupling (Cheng et al. 1993). In this study we employed confocal [Ca2+]i imaging to characterize subcellular calcium signalling in fluo-3 loaded visceral and vascular smooth muscle cells. In some experiments membrane potential of the myocyte was controlled using whole-cell patch clamp technique and changes in membrane current were recorded simultaneously with [Ca2+]i imaging. Some local [Ca2+]i transients were very similar to 'Ca2+ sparks' observed in heart, i.e. lasting approximately 200 ms with a peak fluorescence ratio of 1.75 +/- 0.23 (mean +/- SD, n = 33). Ca2+ sparks were found to occur in certain preferred locations in the cell, termed frequent discharge sites. Other events were faster and smaller, lasting only approximately 40 ms with a peak normalized fluorescence of 1.36 +/- 0.09 (mean +/- SD, n = 28). A high correlation between spontaneous transient outward currents and spark occurrence was observed. Proliferating waves of elevated [Ca2+]i initiated during membrane depolarization seem to arise from spatio-temporal recruitment of local Ca(2+)-release events. The spatial non-uniformity of sarcoplasmic reticulum and ryanodine receptor distribution within the cell may account for the existence of 'frequent discharge sites' and the wide variation in the Ca2+ wave propagation velocities observed.

International audience; To achieve and maintain skin architecture and homeostasis, keratinocytes must intricately balance growth, differentiation, and polarized motility known to be governed by calcium. Orai1 is a pore subunit of a store-operated Ca(2+) channel that is a major molecular counterpart for Ca(2+) influx in nonexcitable cells. To elucidate the physiological significance of Orai1 in skin, we studied its functions in epidermis of mice, with targeted disruption of the orai1 gene, human skin sections, and primary keratinocytes. We demonstrate that Orai1 protein is mainly confined to the basal layer of epidermis where it plays a critical role to control keratinocyte proliferation and polarized motility. Orai1 loss of function alters keratinocyte differentiation both in vitro and in vivo. Exploring underlying mechanisms, we show that the activation of Orai1-mediated calcium entry leads to enhancing focal adhesion turnover via a PKCβ-Calpain-focal adhesion kinase pathway. Our findings provide insight into the functions of the Orai1 channel in the maintenance of skin homeostasis.

SummaryStimulation of μ-opioid receptors (OPRMs) brings powerful pain relief, but it also leads to the development of tolerance and addiction. Ensuing withdrawal in abstinent patients manifests itself with severe symptoms, including cold hyperalgesia, often preventing addicted patients from successfully completing the rehabilitation. Unsurprisingly, OPRMs have been a central point of many studies. Nonetheless, a satisfactory understanding of the pathways leading to distorted sensory responses during opiate administration and abstinence is far from complete. Here, we present a mechanism that leads to modulation by OPRMs of one of the sensory responses, thermosensation. Activation of OPRM1 leads to internalization of a cold-sensor TRPM8, which can be reversed by a follow-up treatment with the inverse OPRM agonist naloxone. Knockout of TRPM8 protein leads to a decrease in morphine-induced cold analgesia. The proposed pathway represents a universal mechanism that is probably shared by regulatory pathways modulating general pain sensation in response to opioid treatment.

Background There is growing evidence suggesting involvement of L-type voltage-gated Ca 2+ channels (VGCCs) in purinergic signaling mechanisms. However, detailed interplay between VGCCs and P2X receptors in intracellular Ca 2+ mobilization is not well understood. This study examined relative contribution of the Ca 2+ entry mechanisms and induced by this entry Ca 2+ release from the intracellular stores engaged by activation of P2X receptors in smooth muscle cells (SMCs) from the guinea-pig small mesenteric arteries. Methods P2X receptors were stimulated by the brief local application of αβ-meATP and changes in [Ca 2+ ] i were monitored in fluo-3 loaded SMCs using fast x-y confocal Ca 2+ imaging. The effects of the block of L-type VGCCs and/or depletion of the intracellular Ca 2+ stores on ab -meATP-induced [Ca 2+ ] i transients were analyzed. Results Our analysis revealed that Ca 2+ entry via L-type VGCCs is augmented by the Ca 2+ -induced Ca 2+ release significantly more than Ca 2+ entry via P2X receptors, even though net Ca 2+ influxes provided by the two mechanisms are not significantly different. Conclusions Thus, arterial SMCs upon P2X receptor activation employ an effective mechanism of the Ca 2+ signal amplification, the major component of which is the Ca 2+ release from the SR activated by Ca 2+ influx via L-type VGCCs. This signaling pathway is engaged by depolarization of the myocyte membrane resulting from activation of P2X receptors, which, being Ca 2+ permeable, per se form less effective Ca 2+ signaling pathway. This study, therefore, rescales potential targets for therapeutic intervention in purinergic control of vascular tone.

One important mechanism of the regulation of membrane ion channels involves their nonfunctional isoforms generated by alternative splicing. However, knowledge of such isoforms for the members of the transient receptor potential (TRP) superfamily of ion channels remains quite limited. This study focuses on the TRPM8, which functions as a cold receptor in sensory neurons but is also expressed in tissues not exposed to ambient temperatures, as well as in cancer tissues. We report the cloning from prostate cancer cells of new short splice variants of TRPM8, termed short TRPM8α and short TRPM8β. Our results show that both variants are in a closed configuration with the C-terminal tail of the full-length TRPM8 channel, resulting in stabilization of its closed state and thus reducing both its cold sensitivity and activity. Our findings therefore uncover a new mode of regulation of the TRPM8 channel by its splice variants., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The repertoire of ionic channels expressed in myocytes freshly isolated from microdissected interlobar and arcuate arteries of rat kidney and their integrative behavior in response to endothelin-1 (ET-1) were studied by identification and characterization of major whole cell current components using patch-clamp technique. In renal microvascular smooth muscle cells (RMSMC) dialyzed with K(+)-containing solution, rapidly inactivating (Ito) and sustained outward K+ currents were identified. Voltage-dependent Ito was categorized as "A" current based on its kinetics, sensitivity to 4-aminopyridine (4-AP), and refractoriness to tetraethylammonium (TEA+). Ca(2+)-activated component of K+ current was completely blocked by 10 mM TEA+, whereas 5 mM 4-AP did not affect this current. Maximal Ca2+ current (ICa) recorded in Cs(+)-loaded RMSMC reached 250 pA when cells were bathed in a solution with 2.5 mM Ca2+. Two patterns of ICa differing in kinetics, voltage range of activation and inactivation, and sensitivity to nifedipine were identified as T and L currents. Ca(2+)-dependent current component showing reversal potential near Cl- current (ECl) and sensitivity to blocking action of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was identified as Ca(2+)-activated ECl. Activation of RMSMC with ET-1 (1-10 nM) induced elevation of [Ca2+]i and subsequent activation of Ca(2+)-activated ICl, which led to membrane depolarization sufficient to activate voltage-gated Ca2+ channels. ET-1-evoked transient reduction of ICa carried through voltage-gated Ca2+ channels was followed by augmentation of L-type ICa. ET-1-induced mobilization of intracellular Ca2+, accompanied by membrane depolarization, resulted in activation of Ca(2+)-dependent K+ channels, which can play the role of a feedback element terminating ET-1-induced membrane depolarization.

Valproic acid (VPA) is the most highly prescribed epilepsy treatment worldwide and is also used to prevent bipolar disorder and migraine. Surprisingly, very little is known about its mechanisms of cellular uptake. Here, we employ a range of cellular, molecular and genetic approaches to characterize VPA uptake using a simple biomedical model, Dictyostelium discoideum. We show that VPA is taken up against an electrochemical gradient in a dose-dependent manner. Transport is protein-mediated, dependent on pH and the proton gradient and shows strong substrate structure specificity. Using a genetic screen, we identified a protein homologous to a mammalian solute carrier family 4 (SLC4) bicarbonate transporter that we show is involved in VPA uptake. Pharmacological and genetic ablation of this protein reduces the uptake of VPA and partially protects against VPA-dependent developmental effects, and extracellular bicarbonate competes for VPA uptake in Dictyostelium. We further show that this uptake mechanism is likely to be conserved in both zebrafish (Danio rerio) and Xenopus laevis model systems. These results implicate, for the first time, an uptake mechanism for VPA through SLC4-catalysed activity.

International audience; Despite remarkable advances in the therapy and prevention of prostate cancer it is still the second cause of death from cancer in industrialized countries. Many therapies initially shown to be beneficial for the patients were abandoned due to the high drug resistance and the evolution rate of the tumors. One of the prospective therapeutical agents even used in the first stage clinical trials, 1,25-dihydroxyvitamin D3, was shown to be either unpredictable or inefficient in many cases. We have already shown that TRPV6 calcium channel, which is the direct target of 1,25-dihydroxyvitamin D3 receptor, positively controls prostate cancer proliferation and apoptosis resistance (Lehen'kyi et al., Oncogene, 2007). However, how the known 1,25-dihydroxyvitamin D3 antiproliferative effects may be compatible with the upregulation of pro-oncogenic TRPV6 channel remains a mystery. Here we demonstrate that in low steroid conditions 1,25-dihydroxyvitamin D3 upregulates the expression of TRPV6, enhances the proliferation by increasing the number of cells entering into S-phase. We show that these pro-proliferative effects of 1,25-dihydroxyvitamin D3 are directly mediated via the overexpression of TRPV6 channel which increases calcium uptake into LNCaP cells. The apoptosis resistance of androgen-dependent LNCaP cells conferred by TRPV6 channel is drastically inversed when 1,25-dihydroxyvitamin D3 effects were combined with the successful TRPV6 knockdown. In addition, the use of androgen-deficient DU-145 and androgen-insensitive LNCaP C4-2 cell lines allowed to suggest that the ability of 1,25-dihydroxyvitamin D3 to induce the expression of TRPV6 channel is a crucial determinant of the success or failure of 1,25-dihydroxyvitamin D3-based therapies.

Direct measurement of nitric oxide (NO) release is pivotal for understanding its role in the regulation of vascular tone. However, data on the direct measurement of NO have been scarce. Recent description of NO-selective electrode has prompted us to examine NO release from endothelial cells using this approach. In the present study, we continuously monitored [NO] in the incubation medium conditioned by cultured human umbilical vein endothelial cells (HUVEC) with an amperometric NO-sensor. The HUVEC released NO on stimulation with several agonists such as α-thrombin, bradykinin, L-arginine and ionomycin; the responses were characterized by an initial rise and a subsequent sustained increase. Activation of Ca/calmodulin system resulted in a robust elevation in [NO], occasionally displaying an oscillatory component. Calmidazolium pretreatment attenuated the ionomycin-induced response. Pretreatment with phorbol ester suppressed the ionomycin-induced NO release from HUVEC. Forskolin pretreatment did not modify NO release elicited by ionomycin. These findings indicate that the synthesis/release of NO in endothelial cells is a Ca/calmodulin dependent step. Activation of protein kinase C interferes with the Ca/calmodulin-induced activation of NOS in endothelial cells. Thus, the present study shows that NO synthase is a substrate for phosphorylation by different kinases which modulate the activity of the enzyme as determined by continuous monitoring of NO release from endothelial cells using a specific NO-sensor.

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca(2+) concentration ([Ca(2+) ](i) ). Although it is well-appreciated that the myocyte Ca(2+) signalling system is composed of microdomains, little is known about the structure of the [Ca(2+) ](i) responses induced by P2X receptor stimulation in vascular myocytes.Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca(2+) signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP(3) R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca(2+) ](i) transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L(-1) αβ-meATP triggered an abrupt Ca(2+) release from sub-plasmalemmal ('junctional') sarcoplasmic reticulum enriched with IP(3) Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca(2+) channels (VGCCs) or IP(3) Rs suppressed the sub-plasmalemmal [Ca(2+) ](i) upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP(3) R inhibition on the sub-plasmalemmal [Ca(2+) ](i) upstroke was attenuated following block of VGCCs.Depolarization of RVSMCs following P2X receptor activation induces IP(3) R-mediated Ca(2+) release from sub-plasmalemmal ('junctional') sarcoplasmic reticulum, which is activated mainly by Ca(2+) influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.

Ionotropic purinoreceptors (P2X) in renal vascular smooth muscle cells (RVSMCs) are involved in mediating the sympathetic control and paracrine regulation of renal blood flow (RBF). Activation of P2X receptors elevates [Ca(2+)](i) in RVSMCs triggering their contraction, leading to renal vasoconstriction and decrease of RBF. The goal of the present work was to characterize the P2X receptor-mediated ionic current (I(P2X)) and to identify the types of P2X receptors expressed in myocytes isolated from interlobar and arcuate arteries of rat kidney.The expression of P2X receptors in isolated RVSMCs was analysed by reverse transcription (RT)-PCR. I(P2X) and membrane potential were recorded using the amphotericin B-perforated patch method.RT-PCR analysis on single RVSMCs showed the presence of genes encoding P2X1 and P2X4 receptors. Under voltage clamp conditions, the selective P2X receptor agonist alphabeta-methylene ATP (alphabeta-meATP) evoked I(P2X) similar to that induced by ATP. Under current clamp conditions, both ATP and alphabeta-meATP evoked a spike-like membrane depolarization followed by a sustained depolarization, linking P2X receptors in RVSMCs to sympathetic control of renal vascular tone. A selective antagonist of P2X1 receptors, NF279, reduced I(P2X) amplitude by approximately 65% concentration-dependently manner within the nanomolar to sub-micromolar range. The residual current was resistant to micromolar concentrations of NF279, but was inhibited by sub-millimolar to millimolar concentrations of NF279.Two types of functional P2X receptors, monomeric P2X1 and heteromeric P2X1/4 receptors, are expressed in RVSMCs. Our study has identified important targets for possible pharmacological intervention in the sympathetic control of renal circulation.

Mitochondria are proposed to be a major oxygen sensor in hypoxic pulmonary vasoconstriction (HPV), a unique response of the pulmonary circulation to low oxygen tension. Mitochondrial factors including reactive oxygen species, cytochrome c, ATP, and magnesium are potent modulators of voltage-gated K+ (Kv) channels in the plasmalemmal membrane of pulmonary arterial (PA) smooth muscle cells (PASMCs). Mitochondria have also been found close to the plasmalemmal membrane in rabbit main PA smooth muscle sections. Therefore, we hypothesized that differences in mitochondria localization in rat PASMCs and systemic mesenteric arterial smooth muscle cells (MASMCs) may contribute to the divergent oxygen sensitivity in the two different circulations. Cellular localization of mitochondria was compared with immunofluorescent labeling, and differences in functional coupling between mitochondria and Kv channels was evaluated with the patch-clamp technique and specific mitochondrial inhibitors antimycin A (acting at complex III of the mitochondrial electron transport chain) and oligomycin A (which inhibits the ATP synthase). It was found that mitochondria were located significantly closer to the plasmalemmal membrane in PASMCs compared with MASMCs. Consistent with these findings, the effects of the mitochondrial inhibitors on Kv current ( IKv) were significantly more potent in PASMCs than in MASMCs. The cytoskeletal disruptor cytochalasin B (10 μM) also altered mitochondrial distribution in PASMCs and significantly attenuated the effect of antimycin A on the voltage-dependent parameters of IKv. These findings suggest a greater structural and functional coupling between mitochondria and Kv channels specifically in PASMCs, which could contribute to the regulation of PA excitability in HPV.

Although smooth muscle cells are not organized in sarcomeres, as are striated muscles, nevertheless Ca2+ for contraction is released from the sarcoplasmic reticulum (SR) at certain preferred sites. These sites commonly discharge packets of Ca2+ spontaneously and have been called frequent discharge sites (FDSs). Each spontaneous release of a Ca2+ packet usually leads to a burst of openings of Ca2+-activated K+ channels in the cell membrane which produces a spontaneous transient outward current (STOC) in smooth muscle cells under voltage clamp. When fluorescent Ca2+ indicators such as Fluo-3 became available, the spontaneous transient increases in [Ca2+]i produced by Ca2+ packets released from the SR were also detected in cardiac muscle as flashes of fluorescence or 'sparks'. Sparks in smooth muscle consist of smaller Ca2+ packets that can give rise to 'microsparks'. In some smooth muscles which have Ca2+-activated Cl- channels, STICs (spontaneous transient inward currents) are also found to be associated with sparks. FDSs have been found to be important initiating sites for a Ca2+ wave in response to an action potential or in response to receptor activation and possibly other stimuli, such as stretch. In both cases Ca2+-induced Ca2+ release seems to be crucially involved.

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

Interstitial cells of Cajal (ICCs) freshly isolated from rabbit portal vein and loaded with the Ca(2+)-sensitive indicator fluo-3 revealed rhythmical [Ca(2+)](i) changes occurring at 0.02-0.1 Hz. Each increase in [Ca(2+)](i) originated from a discrete central region of the ICC and propagated as a [Ca(2+)](i) wave towards the cell periphery, but usually became attenuated before reaching the ends of the cell. In about 40% of ICCs each rhythmical change in [Ca(2+)](i) consisted of an initial [Ca(2+)](i) increase (phase 1) followed by a faster rise in [Ca(2+)](i) (phase 2) and then a decrease in [Ca(2+)](i) (phase 3); the frequency correlated with the rate of rise of [Ca(2+)](i) during phase 1, but not with the peak amplitude. Rhythmical [Ca(2+)](i) changes persisted in nicardipine, but were abolished in Ca(2+)-free solution as well as by SK&F96365, cyclopiazonic acid, thapsigargin, 2-APB, xestospongin C or ryanodine. Intracellular Ca(2+) stores visualised with the low-affinity Ca(2+) indicator fluo-3FF were found to be enriched with ryanodine receptors (RyRs) detected with BODIPY TR-X ryanodine. Rhythmical [Ca(2+)](i) changes originated from a perinuclear S/ER element showing the highest RyR density. Immunostaining with anti-TRPC3,6,7 antibodies revealed the expression of these channel proteins in the ICC plasmalemma. This suggests that these rhythmical [Ca(2+)](i) changes, a key element of ICC pacemaking activity, result from S/ER Ca(2+) release which is mediated via RyRs and IP(3) receptors and is modulated by the activity of S/ER-Ca(2+)-ATPase and TRP channels but not by L-type Ca(2+) channels.

Interstitial cells of Cajal (ICCs) were identified in the intact fixed media of the rabbit portal vein (RPV) using c-kit staining. The following experiments were performed using single cell preparations of the enzyme-dispersed vessel. Surviving contacts between the processes of single ICCs and the bodies of smooth muscle cells (SMCs) were observed in electron micrographs and by confocal microscopy. Spontaneous rhythmical [Ca 2+ ] i oscillations were observed in ICCs after loading with the calcium indicator fluo-3 and were associated with depolarizations of the ICCs recorded by tight-seal patch pipette. To investigate signal transmission from ICCs to SMCs in dispersed cell pairs, or within small surviving fragments of the ICC network, an ICC was stimulated under voltage-clamp, while changes in [Ca 2+ ] i in the stimulated cell as well as in a closely adjacent SMC or ICCs were monitored using fast x - y confocal imaging of fluo-3 fluorescence. After stimulation of single voltage-clamped ICC by a depolarizing step similar in duration to depolarizations associated with spontaneous [Ca 2+ ] i oscillations, a depolarization and transient elevation of [Ca 2+ ] i was observed in a closely adjacent SMCs after a delay of up to 4 seconds. In contrast, signal transmission from ICC to ICC was much faster, the delay being less than 200 ms. These results suggest that the an ICC may, in addition to generating an electrical signal (such as a slow wave) and thereby acting as a pacemaker for vascular SMCs of RPV, also release some unknown diffusible substance, which depolarizes the SMCs.

A rise in intracellular ionised calcium concentration ([Ca(2+)](i)) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca(2+) sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca(2+)](i) upon myocyte excitation; (2) spontaneous Ca(2+) sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca(2+)-dependent K(+) and Cl(-) channels sense the local changes in [Ca(2+)](i) during a calcium spark and thereby couple changes in [Ca(2+)](i) within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP(3)Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.

Voltage-dependent Na+ current (INa) was identified in cultured endothelial cells derived from rat interlobar artery (RIAE cells) and human umbilical vein (HUVE cells). Tetrodotoxin (TTX) reduced INa in a dose-dependent manner with the apparent dissociation constant (Kd) of 1.4 microM. Low sensitivity of INa to TTX as well as its kinetics and voltage-dependent properties indicates that voltage-gated Na channels expressed in vascular endothelial cells belong to the so-called TTX-resistant type.

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca(2+)-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca(2+) indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca(2+)-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca(2+) sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F(0)) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 microm) for the 230 localised Ca(2+)-release events analysed. The existence of two populations of Ca(2+)-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca(2+)-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 microM nicardipine, but were abolished after 10-15 min exposure to 50-100 microM ryanodine, 0.1 microM thapsigargin or 10 microM cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, 10 microM xestospongin C or 30 microM 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 microM U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca(2+)-release domains giving rise to spontaneous [Ca(2+)](i) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP(3) receptors to Ca(2+), which can give rise to 'loose coupling' between release sites. These results suggest that activation of IP(3) receptors (due to the tonic activity of PLC and ongoing production of IP(3)) recruits neighbouring domains of ryanodine receptors, leading to larger Ca(2+) releases and saltatory propagation of [Ca(2+)](i) waves in portal vein myocytes.

International audience; The growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the 'two-in-one' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy.

1. Spontaneous, localized transient increases in [Ca2+]i ('Ca2+ sparks') were observed in about 40 % of fluo-3-loaded myocytes examined using laser scanning confocal microscopy. Ca2+ sparks persisted after application of Cd2+ (200 microM), but were abolished by ryanodine (30 microM) or thapsigargin (0.1 microM), suggesting that they arise from the spontaneous activation of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). 2. Ca2+ sparks occurred much more frequently at certain sites (or 'frequent discharge sites', FDSs) within any confocal plane of the cell and line-scan imaging revealed a wide variation in their spatial size, amplitude and time course. Some spontaneous local transients were very similar to 'Ca2+ sparks' observed in heart, i.e. lasting approximately 200 ms with a peak fluorescence ratio of 1.75 +/- 0.23 (mean +/- s.d., n = 33). Other events were faster and smaller, lasting only approximately 40 ms with a peak normalized fluorescence of 1.36 +/- 0.09 (mean +/- s.d., n = 28). 3. Spontaneous Ca2+ waves with a wide range of propagation velocities (between 30 and 260 micron s-1) were also observed. In about 60 % of records (n = 33), Ca2+ sparks could be detected at the sites of wave initiation. Waves of elevated [Ca2+]i propagated with non-constant velocity and in some cases terminated. These observations could be explained by heterogeneity in the distribution of subcellular release sites as well as variability in the contribution of each release site to the wave. 4. Spontaneous [Ca2+]i transients in single dispersed visceral smooth muscle cells have a wide spectrum of behaviour that is likely to be the result of spatio-temporal recruitment of smaller local events, probably via a calcium-induced calcium release (CICR) mechanism. The spatial non-uniformity of SR and RyR distribution within the cell may account for the existence of 'frequent discharge sites' firing the majority of the smooth muscle Ca2+ sparks and the wide variation in the Ca2+ wave propagation velocities observed.

Membrane currents were studied in single human blood eosinophils using the whole cell voltage clamp technique. The whole cell current-voltage relationship exhibited rectification about the membrane potential which followed the potassium equilibrium potential when [K+]o was raised. Elevation of [K+]o considerably potentiated inward current amplitude, and in some cells channel activity was discernible in the whole cell membrane current recordings. The single channel conductance was 24 ± 1 pS ([K+]o, 100 mm [K+]i, 140 mm), and eosinophils were found to have as few as three, and on average twenty, inward rectifier channels each. The inward current was inhibited in a voltage-dependent manner by extracellular cations in order of potency Ba2+ > Cs+ > Na+. Intracellular acidification inhibited while alkalization augmented the inward current. Mg2+ contributed to rectification as dialysis with nominally Mg2+-free pipette solution was associated with an increase in the outward current during membrane polarization. By reverse transcription-polymerase chain reaction (RT-PCR) using suitable primers on human eosinophil mRNA, an inward rectifier channel, Kir2.1, was identified, which is known from expression studies to have very similar properties to those found in this study. Superoxide anion production or its stimulation by phorbol 12-myristate 13-acetate (PMA) was not significantly affected by depolarization with 140 mm [K+]o, or by 1 mm BaCl2. It is concluded that the single channel currents and the whole cell current rectification observed in human blood eosinophils resulted from the presence of an inwardly rectifying potassium channel, probably Kir2.1. The potential role of ion channels in regulating the behaviour of eosinophils was essentially unexplored until recently. Two independent studies have reported the existence and characterization of proton currents in freshly isolated human eosinophils (Gordienko, Tare, Parveen, Fenech, Robinson & Bolton, 1996; Schrenzel, Lew & Krause, 1996) and additionally, Ca2+-activated K+ channels have been identified in EoL-1, a cell line with properties claimed to resemble those of human eosinophils (Saito, Sato, Hisatome & Narahashi, 1996). Alterations in the resting membrane potential produced as a result of channel activity may play a role in modulating cellular function in eosinophils. In a variety of cell types (see Hille, 1992) including cells of myeloid origin (for review see Gallin, 1991), anomalously or inwardly rectifying potassium channels have been identified and are presumed to have a role in the regulation of cell resting membrane potential. Inwardly rectifying potassium channels conduct ions more readily in the inward direction at membrane potentials negative to the equilibrium potential for potassium ions (EK). Activation of the inward rectifier is dependent on the electrochemical potential for potassium (Hagiwara & Yoshi, 1979) and is susceptible to inhibition by various extracellular cations such as Cs+, Ba2+ and Na+ (Gay & Stanfield, 1977; Standen & Stanfield, 1978, 1979) and intracellular cations including protons (Moody & Hagiwara, 1982). Cytoplasmic Mg2+ and/or polyamines (Matsuda, Saigusa & Irisawa, 1987; Lopatin, Makhina & Nichols, 1994) are responsible for rectification of the current by inhibiting the outward flow of K+ through inward rectifier channels during membrane polarization. This study reveals that the behaviour of the whole cell inward current upon membrane hyperpolarization of human blood eosinophils can be attributed to the presence of inward rectifier potassium channels. Some of the characteristics of this current are similar to those described in several other cells of myeloid origin (Gallin, 1991). It may serve to set the membrane potential of eosinophils close to the equilibrium potential for K+. Preliminary accounts of this work have previously been reported in abstract form (Tare, Gordienko, Parveen, Fenech, Robinson & Bolton, 1996; Tare, Gordienko, Parveen, Robinson & Bolton, 1997).