TRPV5 is internalized via clathrin-dependent endocytosis to enter a Ca2+-controlled recycling pathway

The epithelial Ca(2+) channel TRPV5 plays an essential role in transcellular Ca(2+) transport and is one of the most Ca(2+)-selective members of the transient receptor potential superfamily. Regulation of the abundance of TRPV5 at the cell surface is critical in body Ca(2+) homeostasis. However, little is known about the mechanisms underlying TRPV5 endo- and exocytosis. Here, we show that TRPV5 is constitutively internalized in a dynamin- and clathrin-dependent manner. Internalized TRPV5 first appears in small vesicular structures and then localizes to perinuclear structures positive for Rab11a. TRPV5 has a half-life of more than 8 h and is stable even after internalization from the cell surface for more than 3 h. Disruption of cell surface delivery of newly synthesized TRPV5 by brefeldin A does not reduce TRPV5-mediated Ca(2+) influx in cells, suggesting the presence of a stable intracellular pool of the channel capable of recycling back to the surface. Furthermore, the endocytic recycling kinetics is decreased upon treatment with Ca(2+) chelator BAPTA-AM, indicating that the channel's trafficking pathways are dynamically controlled by Ca(2+).     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

     

Sphingosine 1-phosphate formation and intracellular Ca2+ mobilization in human platelets: evaluation with sphingosine kinase inhibitors.

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [3H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [3H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [3H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca2+ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca2+ mobilization, in platelets.     

Intracellular sphingosine 1-phosphate production: a novel pathway for Ca2+ release

Sphingolipids such as sphingosine 1-phosphate (SPP) and sphingosylphosphorylcholine have long been recognized to possess Ca2+ mobilizing activity, yet to date little is known about their mechanism of action, or indeed their significance as Ca2+ mobilizing intracellular messengers. The recent discovery of extracellular receptors for the sphingolipids has further complicated the interpretation of many experiments in this field. This paper reviews the current literature in which molecular and pharmacological approaches have begun to uncover the signalling components associated with intracellular SPP production and Ca2+ mobilization. The functional significance of this novel Ca2+ release pathway is also discussed.     

Prostaglandin D2 induces interleukin-6 synthesis via Ca2+ mobilization in osteoblasts: regulation by protein kinase C

We previously showed that prostaglandin (PG) D2 stimulates Ca2+ influx from extracellular space and activates phosphoinositidic (PI)-hydrolyzing phospholipase C and phosphatidylcholine (PC)-hydrolyzing phospholipase D independently from PGE2 or PGF2alpha in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of PGD2 on the synthesis of interleukin-6 (IL-6) and its regulatory mechanism in MC3T3-E1 cells. PGD2 significantly stimulated IL-6 synthesis dose-dependently in the range between 10 nM and 10 microM. The depletion of extracellular Ca2+ by EGTA reduced the PGD2-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, significantly inhibited the PGD2-induced IL-6 synthesis. On the other hand, calphostin C, a specific inhibitor of protein kinase C (PKC), enhanced the synthesis of IL-6 induced by PGD2. In addition, U-73122, an inhibitor of phospholipase C, and propranolol, a phosphatidic acid phosphohydrolase inhibitor, enhanced the PGD2-induced IL-6 synthesis. These results strongly suggest that PGD2 stimulates IL-6 synthesis through intracellular Ca2+ mobilization in osteoblasts, and that the PKC activation by PGD2 itself regulates the over-synthesis of IL-6.     

Lysophosphatidic acid induces Ca2+ mobilization and c-Myc expression in mouse embryonic stem cells via the phospholipase C pathway

Embryonic stem cells (ESC) are pluripotent and could be maintained in vitro in a self-renewing state indefinitely, at the same time preserving their potential to differentiate towards more specific lineages. Despite the progress in the field, the complex network of signalling cascades involved in the maintenance of the self-renewing and pluripotent state remains not fully understood. In the present study, we have investigated the role of lysophosphatidic acid (LPA), a potent mitogen present in serum, in Ca²⁺ signalling and early gene activation in mouse ESC (mESC). In these cells, we detected the expression of the G-protein coupled LPA receptor subtypes LPA₁, LPA₂ and LPA₃. Using fluorescence Ca²⁺ imaging techniques, we showed that LPA induced an increase in intracellular Ca²⁺ concentration. This increase was also observed in the absence of extracellular Ca²⁺, suggesting the involvement of internal stores. Pre-treatment with BAPTA-AM, thapsigargin or U-73122 efficiently blocked this Ca²⁺ release, indicating that LPA was evoking Ca²⁺ mobilization from the endoplasmic reticulum via the phospholipase C (PLC) pathway. Interestingly, this signalling cascade initiated by LPA was involved in inducing the expression of the Ca²⁺-dependent early response gene c-myc, a key gene implicated in ESC self-renewal and pluripotency. Additionally, LPA increased the proliferation rate of mESC. Our findings therefore outline the physiological role of LPA in mESC.     

Targeting the sphingosine kinase/sphingosine 1-phosphate pathway in disease: Review of sphingosine kinase inhibitors

Sphingosine 1-phosphate (S1P) is an important bioactive sphingolipid metabolite that has been implicated in numerous physiological and cellular processes. Not only does S1P play a structural role in cells by defining the components of the plasma membrane, but in the last 20 years it has been implicated in various significant cell signaling pathways and physiological processes: for example, cell migration, survival and proliferation, cellular architecture, cell–cell contacts and adhesions, vascular development, atherosclerosis, acute pulmonary injury and respiratory distress, inflammation and immunity, and tumorogenesis and metastasis [ and ]. Given the wide variety of cellular and physiological processes in which S1P is involved, it is immediately obvious why the mechanisms governing S1P synthesis and degradation, and the manner in which these processes are regulated, are necessary to understand. In gaining more knowledge about regulation of the sphingosine kinase (SK)/S1P pathway, many potential therapeutic targets may be revealed. This review explores the roles of the SK/S1P pathway in disease, summarizes available SK enzyme inhibitors and examines their potential as therapeutic agents. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Cutting edge: Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase

One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca(2+) that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca(2+)-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.     

Voltage-independent inhibition of P/Q-type Ca2+ channels in adrenal chromaffin cells via a neuronal Ca2+ sensor-1-dependent pathway involves Src family tyrosine kinase

In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-independent pathways for Ca(2+) channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca(2+) currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca(2+)-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca(2+) currents revealed that it did not involve protein kinase C-dependent signaling pathways but did require the activity of a Src family tyrosine kinase. Two structurally distinct Src kinase inhibitors, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and a Src inhibitory peptide, increased the Ca(2+) currents, and no further increase in Ca(2+) currents was elicited by addition of naloxone and suramin. In addition, the Src-like kinase appeared to act in the same pathway as NCS-1. In contrast, addition of PP1 did not prevent a voltage-dependent facilitation elicited by a strong pre-pulse depolarization indicating that this pathway was independent of Src kinase activity. PPI no longer increased Ca(2+) currents after addition of the P/Q-type channel blocker omega-agatoxin TK. The alpha(1A) subunit of P/Q-type Ca(2+) channels was immunoprecipitated from chromaffin cell extracts and found to be phosphorylated in a PP1-sensitive manner by endogenous kinases in the immunoprecipitate. A high molecular mass (around 220 kDa) form of the alpha(1A) subunit was detected by anti-phosphotyrosine, suggesting a possible target for Src family kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca(2+) channel currents in chromaffin cells that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca(2+) channel regulation.     

Regulation of Ca2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca2+-ATPase type 2

Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk.     

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.     

Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKbeta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKbeta was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKKbeta or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca2+ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca2+/CaMKKbeta dependent and one AMP/LKB1 dependent.     

Protein kinase C enhances Ca2+ mobilization in human platelets by activating Na+/H+ exchange

Control of cytoplasmic pH (pHi) by a Na+/H+ antiport appears a general property of most eukaryotic cells. In human platelets activation of the Na+/H+ exchanger enhances Ca2+ mobilization and aggregation induced by low concentrations of thrombin (Siffert, W., and Akkerman, J. W. N. (1987) Nature 325, 456-458). Several observations indicate that the exchanger is regulated by protein kinase C. (i) Inhibitors of protein kinase C (trifluoperazine, sphingosine) inhibit the increase in pHi seen during thrombin stimulation as well as Ca2+ mobilization; artificially increasing pHi by monensin or NH4Cl then restores Ca2+ mobilization. (ii) Direct activation of protein kinase C by 1-oleoyl-2-acetylglycerol initiates an increase in pHi that depends on the presence of extracellular Na+ and is sensitive to inhibition by ethylisopropylamiloride. The pHi sensitivity of thrombin-induced Ca2+ mobilization is particularly evident in the range between pH 6.8 and 7.4 and at low thrombin concentrations, whereas thrombin concentrations of more than 0.2 unit/ml bypass the pH sensitivity. In the absence of thrombin an increase in pHi, either induced artificially (by addition of the ionophores nigericin or monensin) or via activation of protein kinase C (by addition of 1-oleoyl-2-acetylglycerol), does not induce Ca2+ mobilization. We conclude that activation of protein kinase C is essential for Ca2+ mobilization in platelets stimulated by low concentrations of thrombin and that protein kinase C exerts this effect via activation of the Na+/H+ exchanger.     

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca(2+) in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca(2+) concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca(2+) transients induced by photolysis of caged S1P were caused by Ca(2+) mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P.     

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248 000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.     

Adenosine inhibits the renal plasma-membrane (Ca2+ + Mg2+)-ATPase through a pathway sensitive to cholera toxin and sphingosine.

Adenosine, a potent autacoid produced and released in kidneys, affects nearly all aspects of renal function, and an increase in cytosolic calcium has been implicated in adenosine effects. The aim of this work was to investigate whether adenosine modifies the calcium pump present in basolateral membranes of kidney proximal tubule cells. Adenosine exerts a biphasic influence on (Ca2+ + Mg2+)-ATPase activity. Inhibition occurs up to 0.1 microM and then gradually disappears as the adenosine concentration increases to 100 microM, an effect mimicked by the adenosine analog N6-cyclohexyladenosine, which preferentially binds to A1-type receptors. In contrast, the A2 receptor agonist 5', N-ethylcarboxamideadenosine is ineffective. The A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine blocks the inhibitory effect of 0.1 microM adenosine and stimulates (Ca2+ + Mg2+)-ATPase activity in the presence of 1 mM adenosine, a concentration high enough to occupy the low-affinity A2 receptors. Inhibition by adenosine increases as medium ATP is lowered to micromolar concentrations, is maintained in the presence of pertussis toxin, and is completely abolished with 0.1 microM cholera toxin or 1 microM sphingosine. The inhibitory effect of adenosine can be reproduced by guanosine 5'-[gamma-thio]triphosphate, inositol 1,4, 5-trisphosphate or the diacylglycerol analog 12-O-tetradecanoylphorbol 13-acetate. In conjunction with the selectivity for its analogs and for its receptor agonist, the concentration profile of adenosine effects indicates that both inhibitory (A1) and stimulatory (A2) receptors are involved. The results obtained with the toxins indicate that a pathway that is modulated by G-proteins, involves a phospholipase C and a protein kinase C, and is affected by local variations in adenosine concentrations participates in the regulation of the (Ca2+ + Mg2+)-ATPase resident in basolateral membranes of kidney proximal tubules.     

Sphingosine kinase 2 inhibitor SG-12 induces apoptosis via phosphorylation by sphingosine kinase 2

Sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosine to generate sphingosine 1-phosphate, has two mammalian isotypes, SPHK1 and SPHK2. Both isozymes are promising anti-cancer therapeutic targets. In this report, we found that SG-12, a synthetic analogue of sphingosine that acts as a SPHK2 inhibitor, induces apoptosis via phosphorylation by SPHK2. The present results revealed the novel anti-cancer potential of a sphingosine analogue in the pathological setting where SPHK2 is upregulated.