TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence.

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 Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.

Mechanisms of the sarcoplasmic reticulum Ca2+ release induced by P2X receptor activation in mesenteric artery myocytes.

BACKGROUND: 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 αß-meATP (10 µM) in single SMCs from these vessels. METHODS: 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 αß-meATP-induced [Ca(2+)]i transients were analyzed using fast x-y confocal Ca(2+) imaging. RESULTS: 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. CONCLUSIONS: 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.

Mechanisms of [Ca2+]i elevation following P2X receptor activation in the guinea-pig small mesenteric artery myocytes.

BACKGROUND: There is growing evidence suggesting involvement of L-type voltage-gated Ca2+ channels (VGCCs) in purinergic signaling mechanisms. However, detailed interplay between VGCCs and P2X receptors in intracellular Ca2+ mobilization is not well understood. This study examined relative contribution of the Ca2+ entry mechanisms and induced by this entry Ca2+ 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 αß-meATP and changes in [Ca2+]i were monitored in fluo-3 loaded SMCs using fast x-y confocal Ca2+ imaging. The effects of the block of L-type VGCCs and/or depletion of the intracellular Ca2+ stores on αß-meATP-induced [Ca2+]i transients were analyzed. RESULTS: Our analysis revealed that Ca2+ entry via L-type VGCCs is augmented by the Ca2+-induced Ca2+ release significantly more than Ca2+ entry via P2X receptors, even though net Ca2+ influxes provided by the two mechanisms are not significantly different. CONCLUSIONS: Thus, arterial SMCs upon P2X receptor activation employ an effective mechanism of the Ca2+ signal amplification, the major component of which is the Ca2+ release from the SR activated by Ca2+ influx via L-type VGCCs. This signaling pathway is engaged by depolarization of the myocyte membrane resulting from activation of P2X receptors, which, being Ca2+ permeable, per se form less effective Ca2+ signaling pathway. This study, therefore, rescales potential targets for therapeutic intervention in purinergic control of vascular tone.

TRPV6 determines the effect of vitamin D3 on prostate cancer cell growth.

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.

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries.

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 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. 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 Ca(2+) signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αß-methylene ATP (αß-meATP). KEY RESULTS: 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 αß-meATP concentration. Depolarization induced by 10 µmol·L(-1) αß-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. CONCLUSIONS AND IMPLICATIONS: 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.

Purinoreceptor-mediated current in myocytes from renal resistance arteries.

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 [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. EXPERIMENTAL APPROACH: 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. 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 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. 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.

Interstitial cells from rat middle cerebral artery belong to smooth muscle cell type.

It is now established that non-contractile cells with thin filopodia, also called vascular interstitial cells (VICs), are constitutively present in the media of many, if not all, blood vessels. The aim of this study was to determine the type of cell lineage to which arterial VICs belong using immunocytochemical, and real-time and reverse transcription PCR (RT-PCR). Using RT-PCR, we compared gene expression profiles of single VICs and smooth muscle cells (SMCs) freshly dispersed from rat middle cerebral artery. Both VICs and SMCs expressed the SMC marker, smooth muscle myosin heavy chain (SM-MHC), but did not express fibroblast, pericyte, neuronal, mast cell, endothelial or stem cell markers. Freshly isolated VICs also did not express c-kit, which is the marker for interstitial cells of Cajal in the gastrointestinal tract. Immunocytochemical labelling of contractile proteins showed that VICs and SMCs expressed SM-MHC similarly to the same degree, but VICs in contrast to SMCs had decreased expression of alpha-SM-actin and very low or no expression of calponin. Real-time RT-PCR was consistent with immunocytochemical experiments and showed that VICs had four times lower gene expression of calponin comparing to SMCs, which may explain VICs' inability to contract. VICs had greater expression than SMCs of structural proteins such as non-muscular beta-actin and desmin. The results obtained suggest that VICs represent a subtype of SMCs and may originate from the same precursor as SMCs, but later develop filopodia and a non-contractile cell phenotype.

Cellular localization of mitochondria contributes to Kv channel-mediated regulation of cellular excitability in pulmonary but not mesenteric 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(+) (K(v)) 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 K(v) 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 K(v) current (I(Kv)) were significantly more potent in PASMCs than in MASMCs. The cytoskeletal disruptor cytochalasin B (10 microM) also altered mitochondrial distribution in PASMCs and significantly attenuated the effect of antimycin A on the voltage-dependent parameters of I(Kv). These findings suggest a greater structural and functional coupling between mitochondria and K(v) channels specifically in PASMCs, which could contribute to the regulation of PA excitability in HPV.

Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell.

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 alpha-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 transmission electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.

Function of interstitial cells of Cajal in the rabbit portal vein.

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 [Ca2+]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 [Ca2+]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 [Ca2+]i oscillations, a depolarization and transient elevation of [Ca2+]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.

Phospholipase C, but not InsP3 or DAG, -dependent activation of the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells.

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.

Effect of nitric oxide donors and noradrenaline on Ca2+ release sites and global intracellular Ca2+ in myocytes from guinea-pig small mesenteric arteries.

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.

Modulation of ICl(Ca) in vascular smooth muscle cells by oxidizing and cysteine-reactive reagents.

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.