Sodium block and depolarization diminish P2Z-dependent Caentry in human B lymphocytes

Despite a high Ca²⁺-permeability of the P2Z receptor in human B lymphocytes, extracellular ATP⁴has only a minor effect on global [Ca²⁺]_i. The aim of this study was to reveal the mechanisms responsible for this discrepancy. We investigated the relationship between ATP⁴⁻-application, Ca_i²-response, membrane current and membrane potential in two human B cell lines and in human tonsillar B cells. This was achieved by a combination of FACS- and voltage clamp measurements and the usage of appropriate voltage- and Ca²-sensitive fluorescent dyes. ATP⁴-induced changes in whole-cell current and [Ca²]_iwere blocked by extracellular as well as intracellular Na⁺. Under current clamp conditions, ATP⁴⁻-induced Na⁺-entry diminished the Ca²⁺entry via reduction of the driving force. A substantial increase in [Ca²⁺]_iinduced by ATP⁴⁻was only observed in Na⁺-free solutions.The pathway of signal transduction activated by ATP⁴via P2Z receptor of human B lymphocytes under physiological conditions seems not to operate by an increase in the global intracellular Ca₂-concentration, but rather by the depolarization of the cell membrane as a result of the Na⁺-influx.     

Ethanol inhibits ligand-activated Ca2+ channels in human B lymphocytes.

Ethanol reportedly is immunosuppressive, interfering with lymphocyte proliferation. To investigate the basis for this immunosuppression, the effects of acute treatment with ethanol were studied on Ca2+ mobilization in tonsillar B lymphocytes and the human lymphoblastoid B-cell line, Ramos. The level of intracellular Ca2+ was monitored in cells loaded with the fluorescent dye indo-1 following stimulation with either anti-IgM antibody or platelet activating factor. The effect of ethanol was also examined on the induction of the early proto-oncogene c-fos in these cells. Ethanol inhibited ligand-activated Ca2+ mobilization due to transmembrane influx but not intracellular store release, in a dose- and time-dependent manner. This inhibition was not due to the inability of anti-IgM to bind to its surface receptor nor to membrane depolarization induced by ethanol. Ethanol also inhibited the Ca2(+)-dependent induction by anti-IgM of c-fos in these cells. The inhibitory effects of ethanol on ligand-activated Ca2+ channels and subsequent induction of c-fos may provide the basis for its immunosuppressive action.     

Detection of ligand-activated conductive Ca2+ channels in human B lymphocytes

It has been assumed that uptake of extracellular Ca2+ occurs through ligand-activated Ca2+ channels in anti-IgM stimulated human B cells. If so, then uptake should be associated with a depolarizing inward current. Instead, a hyperpolarization due to Ca2+-sensitive K+ conductance is observed. To demonstrate conductive Ca2+ channels in human B lymphocytes, we loaded the cells with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA), an intracellular Ca2+ chelating agent. This increased the magnitude of the Ca2+ current and delayed the Ca2+-dependent K+ conductance. In BAPTA-loaded B cells suspended in Ca2+-free medium and activated with anti-IgM, reintroduction of Ca2+ resulted in a depolarization that was inhibited by high (microM) concentrations of verapamil and was observed when Ca2+ was replaced by Ba2+ but not Mg2+. These data demonstrate the opening of selective, Ca2+ conductive channels in human B cells following cross-linking of surface immunoglobulins.     

Progesterone inhibits capacitative Ca2+ entry in Jurkat T lymphocytes by a membrane delimited mechanism, independently of plasma membrane depolarization

The non-genomic inhibitory effect of progesterone on capacitative calcium entry was studied in Jurkat T lymphocytes. Capacitative calcium entry was induced by depleting intracellular calcium stores with thapsigargin and evaluated by a calcium free/calcium readmission protocol, in Fura-2 loaded cells. Progesterone (10-40 microg/ml) inhibited calcium entry and concomitantly depolarized cells, as revealed by measuring the plasma membrane potential with the fluorescent probe bis-oxonol. However, experiments run under depolarizing conditions (i.e. by substituting for Na+ with K+ ions in the medium) revealed that progesterone (10-40 microg/ml) could inhibit capacitative calcium entry independently of plasma membrane depolarization. The direct inhibition of calcium entry by progesterone was: (i) reverted by a treatment suitable to remove progesterone bound to cell surface, (ii) apparently related to the extent of membrane bound progesterone (measured radioisotopically), and (iii) specific, in that other related steroid compounds did not inhibit calcium entry.     

Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.     

Ca2+ uptake and IP3-induced Ca2+ release in permeabilized human lymphocytes

The 45Ca2+ uptake and 45Ca2+ release in saponin-permeabilized human lymphocytes were studied. An ATP-dependent Ca2+ uptake into a nonmitochondrial, intracellular Ca2+ store is observed which is approx. 2 orders of magnitude greater than the ATP-independent Ca2+ uptake. The Ca2+ uptake is inhibited by vanadate, but it is insensitive to oligomycin and ruthenium red. IP3 induces dose-dependent 45Ca2+ release. For half-maximum Ca2+ release 0.25-0.5 microM IP3 is required. The results of our studies suggest that 45Ca2+ is predominantly stored within the endoplasmic reticulum of the lymphocytes.     

Multiple effects of caffeine on Ca2+ release and influx in human B lymphocytes

Caffeine has been used as a pharmacological tool to study the ryanodine receptor (RYR)-mediated Ca2+ release from caffeine-sensitive, inositol 1,4,5,-trisphosphate (IP3)-insensitive pools. In the present study, we demonstrate multiple effects of caffeine on Ca2+ homeostasis in human B lymphocytes. Although B cells express a functional RYR, which can be activated by 4-chloro-m-cresol following depletion of IP(3)-sensitive pools, caffeine does not activate RYR-mediated Ca2+ release. Instead, caffeine dose-dependently inhibited IP3 receptor (IP3R)-mediated Ca2+ release, RYR-mediated Ca2+ release and B cell receptor-initiated Ca2+ influx, while high concentrations of caffeine (> or = 25 mM) induced a Ca2+ influx. In contrast with its ability to suppress receptor-stimulated Ca2+ influx, caffeine had no significant effect on the store-operated Ca2+ (SOC) channel-dependent Ca2+ influx induced by thapsigargin. Thus, caffeine may act as an inhibitor on a single or multiple site(s) responsible for regulating the IP3R channel, RYR channel and presumably the receptor-mediated SOC channel. The present report may be the first demonstration of multiple effects of caffeine on Ca2+ mobilization in single cell type. Our results suggest the need for caution regarding use of caffeine simply as a RYR-activator to study Ca2+ homeostasis in eucaryotic cells.     

Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin

Human neutrophils, preloaded with the fluorescent probe, Fura-2, were exposed to Ca2+-releasing agents. The monitored traces of fluorescence were transformed by computer to cytosolic Ca2+ concentration ([ Ca2+]i). Due to quenching of Fura-2, the addition of Mn2+ enabled us to compute the cytosolic concentration of total manganese ([Mn]i). The agents used were the novel Ca2+-mobilizing agent, thapsigargin (Tg), the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), and the divalent cation ionophore, A23187. The agents caused transient rises of [Ca2+]i and monotonous rises of [Mn]i, suggesting influx but no efflux of Mn2+. The rise time of [Ca2+]i and the time constants and magnitude of the apparent Mn2+ influx were strongly dependent on the sequence of addition of the agonist and Ca2+. Contrary to FMLP, Tg needed several minutes to exert its full effect on the rise of [Ca2+]i and on the influx of Mn2+, the latter being dependent on two phases, activation and partial inactivation. Pretreatment with phorbol 12-myristate 13-acetate (PMA) inhibited the responses of Tg, FMLP and A23187. For comparison, human red blood cells were tested. Contrary to A23187, Tg did not induce Ca2+ uptake in ATP-depleted red cells but increased the Ca2+ pump flux in intact red cells by 10%. The experimental data and computer simulations of the granulocyte data suggest that time-dependent changes of both passive Ca2+ flux into the cytosol and Ca2+ flux of the plasma membrane pump are involved in the transient [Ca2+]i response.     

Arachidonic acid inhibits capacitative Ca2+ entry and activates non-capacitative Ca2+ entry in cultured astrocytes

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca²⁺ entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La³⁺; (ii) AA (10 μM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and γ-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca²⁺ influx, which is not inhibited by 2-APB or La³⁺, indicating that AA activates a second Ca²⁺ entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca]_i overload is to cause delayed cell death in astrocytes.     

Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake

Store-operated Ca(2+) channels, which are activated by the emptying of intracellular Ca(2+) stores, provide one major route for Ca(2+) influx. Under physiological conditions of weak intracellular Ca(2+) buffering, the ubiquitous Ca(2+) releasing messenger InsP(3) usually fails to activate any store-operated Ca(2+) entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca(2+) that has been released by InsP(3), enabling stores to empty sufficiently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses store-operated Ca(2+) influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP(3)-sensitive store depletion, can be distinguished from Ca(2+)-dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca(2+) uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated influx and raise the possibility of bidirectional Ca(2+)-dependent crosstalk between mitochondria and store-operated Ca(2+) channels.     

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction

The correct spatial and temporal control of Ca²⁺ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca²⁺ concentration. In this review we provide an updated view of how caveolae may regulate both Ca²⁺ entry into cells and Ca²⁺-dependent signal transduction     

Activity-dependent development of P2X7 current and Ca2+ entry in rabbit osteoclasts

Bone remodeling is regulated by local factors and modulated by mechanical stimuli. Mechanical stimulation can cause release of ATP, an agent that stimulates osteoclastic resorption at low concentrations and inhibits at high concentrations. We examined whether osteoclasts express P2X(7) receptors, which are activated by high concentrations of ATP and can behave as ion channels or cause the formation of membrane pores. Rabbit osteoclasts were studied using patch clamp techniques. Successive or prolonged applications of 2'- & 3'-O-(4-benzoylbenzoyl)-ATP (BzATP, a relatively potent P2X(7) agonist) or high concentrations of ATP caused the development of a slowly deactivating inward current. The underlying channel was permeable only to small cations, ruling out pore formation. Divalent cations reduced current magnitude, consistent with the presence of P2X(7) receptors, a finding confirmed in rat osteoclasts by immunocytochemistry. Successive applications of BzATP also elicited [Ca(2+)](i) elevations that required extracellular Ca(2+). The BzATP-induced current and the rise of [Ca(2+)](i) were temporally associated, and both were inhibited by PPADS, a P2X(7) antagonist. This study demonstrates that high concentrations of ATP activate P2X(7) receptors and provides the first functional evidence for an extracellular ligand-gated Ca(2+) influx pathway in osteoclasts. ATP released in response to mechanical stimuli may act through P2X(7) receptors to inhibit osteoclastic resorption.     

Sphingomyelinase D inhibits store-operated Ca2+ entry in T lymphocytes by suppressing ORAI current

Infections caused by certain bacteria including Mycobacterium tuberculosis and Corynebacterium pseudotuberculosis provoke inflammatory responses characterized by the formation of granulomas with necrotic foci—so-called caseous necrosis. The granulomas of infected animals show prominent infiltration by T lymphocytes, and T cell depletion increases host mortality. Notorious zoonotic C. pseudotuberculosis secretes sphingomyelinase (SMase) D, a phospholipase that cleaves off the choline moiety of sphingomyelin, a phospholipid found primarily in the outer leaflet of host cell plasma membranes. Experimental C. pseudotuberculosis strains that lack SMase D are markedly less infectious and unable to spread in hosts, indicating that this enzyme is a crucial virulence factor for sustaining the caseous lymphadenitis infections caused by this microbe. However, the molecular mechanism by which SMase D helps bacteria evade the host’s immune response remains unknown. Here, we find that SMase D inhibits store-operated Ca²⁺ entry (SOCE) in human T cells and lowers the production of the SOCE-dependent cytokines interleukin-2, which is critical for T cell growth, proliferation, and differentiation, and tumor necrosis factor α, which is crucial for the formation and maintenance of granulomas in microbial infections. SMase D inhibits SOCE through a previously unknown mechanism, namely, suppression of Orai1 current, rather than through altering gating of voltage-gated K⁺ channels. This finding suggests that, whereas certain genetic mutations abolish Orai1 activity causing severe combined immunodeficiency (SCID), bacteria have the ability to suppress Orai1 activity with SMase D to create an acquired, chronic SCID-like condition that allows persistent infection. Thus, in an example of how virulence factors can disrupt key membrane protein function by targeting phospholipids in host cell membranes, our study has uncovered a novel molecular mechanism that bacteria can use to thwart host immunity.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

Spatial microenvironment defines Ca2+ entry and Ca2+ release in salivary gland cells

The difference of Ca(2+) mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic-cholinergic agonist, induced intracellular Ca(2+) release and extracellular Ca(2+) entry through store-operated Ca(2+) entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca(2+) entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca(2+) stores, the machinery for intracellular Ca(2+) release was intact in the duct cells. By immunocytochemical experiments, IP(3)R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP(3)R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP(3)R2. These results suggest that the expression of the IP(3)Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca(2+) stores. Therefore, the microenvironment probably affects intracellular Ca(2+) release and Ca(2+) entry.     

TRPV6 is a Ca2+ entry channel essential for Ca2+-induced differentiation of human keratinocytes

Ca(2+) is an essential factor inducing keratinocyte differentiation due to the natural Ca(2+) gradient in the skin. However, the membrane mechanisms that mediate calcium entry and trigger keratinocyte differentiation had not previously been elucidated. In this study we demonstrate that Ca(2+)-induced differentiation up-regulates both mRNA and protein expression of a transient receptor potential highly Ca(2+)-selective channel, TRPV6. The latter mediates Ca(2+) uptake and accounts for the basal [Ca(2+)](i) in human keratinocytes. Our results show that TRPV6 is a prerequisite for keratinocyte entry into differentiation, because the silencing of TRPV6 in human primary keratinocytes led to the development of impaired differentiated phenotype triggered by Ca(2+). The expression of such differentiation markers as involucrin, transglutaminase-1, and cytokeratin-10 was significantly inhibited by small interfering RNA-TRPV6 as compared with differentiated control cells. TRPV6 silencing affected cell morphology and the development of intercellular contacts, as well as the ability of cells to stratify. 1,25-Dihydroxyvitamin D3, a cofactor of differentiation, dose-dependently increased TRPV6 mRNA and protein expression in human keratinocytes. This TRPV6 up-regulation led to a significant increase in Ca(2+) uptake in both undifferentiated and differentiated keratinocytes. We conclude that TRPV6 mediates, at least in part, the pro-differentiating effects of 1,25-dihydroxyvitamin D3 by increasing Ca(2+) entry, thereby promoting differentiation. Taken together, these data suggest that the TRPV6 channel is a key element in Ca(2+)/1,25-dihydroxyvitamin D3-induced differentiation of human keratinocytes.