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.     

Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I-encoded p40tax protein in the human T-cell line, Jurkat.

We examined the ability of the trans-acting factor p40tax of human T-cell leukemia virus type I (HTLV-I), which is thought to be a crucial molecule in T-cell transformation by HTLV-I, to activate expression of a set of endogenous cellular genes related to T-cell proliferation. For this purpose we established a subclone (JPX-9) of Jurkat cells that was stably transfected with an expression plasmid containing the p40tax gene, whose expression is definitely dependent on heavy-metal ions. Expression of the interleukin-2 receptor alpha chain in JPX-9 cells was induced in response to the induction of p40tax expression, as has been demonstrated by others in transient transfection experiments with Jurkat cells. In addition, we found that significant enhancement of expression of the nuclear proto-oncogene c-fos was closely associated with expression of p40tax. Continous enhancement in the level of c-fos mRNA was observed in the presence of p40tax. In contrast, mRNA levels of other nuclear proto-oncogenes (c-myc, c-myb, and c-jun) were not appreciably effected by the expression of p40tax. These results suggest that (i) in addition to the interleukin-2-interleukin-2 receptor system, cellular genes such as c-fos, which regulate normal T-cell growth, are also activated directly or indirectly by p40tax and (ii) p40tax-induced modulation of gene expression plays a crucial role in T-cell transformation by HTLV-I.     

Cytoplasmic Ca2+ is necessary for thrombin-induced platelet activation

alpha-Thrombin induces a dose-dependent rapid transient increase in platelet cytosolic Ca2+ levels, coming solely from intracellular stores, since EGTA has no effect. In contrast, the post-stimulation equilibrium [Ca2+]in depends upon an influx from the extracellular milieu, and is lower in the presence of EGTA. We measured the Ca2+ transient (with Indo-1, 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2'-amino-5'-methylp henoxy)- ethane-N,N,N',N'-tetraacetic acid), cytosolic alkalinization (with BCECF, 2',7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein), membrane depolarization (with diS-C3-(5), 3,3'-dipropylthiodi-carbocyanide iodide), and degranulation (by beta-glucuronidase release) induced in washed human platelets by 9 nM thrombin in the absence or presence of extracellular or intracellular Ca2+ chelating agents (EGTA and BAPTA, 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, respectively). Platelets loaded simultaneously with 2 microM Indo-1 and 15 microM BAPTA (each as the acetoxymethyl ester) before addition of thrombin exhibited no cytoplasmic Ca2+ transient or alkalinization, no depolarization or degranulation. Replenishment of such cells with extracellular CaCl2 restored resting [Ca2+]in. Upon stimulation with 9 nM thrombin these replenished platelets exhibited no Ca2+ transient, and a slow gradual increase in [Ca2+]in from extracellular stores, a slow alkalinization and depolarization, and partial degranulation, all abolished by extracellular EGTA. Thus thrombin-induced platelet activation exhibits a biphasic Ca2+ requirement: the initial transient increase in [Ca2+]in comes from intracellular stores only, while the later steps of depolarization, alkalinization, and degranulation can proceed, albeit more slowly, if only extracellular Ca2+ is available.     

The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes.

The low-Ca2+ response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37 degrees C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response. An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of lcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2+ response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2+ response virulence operon, and virulence in mice. The mutant had lost its Ca2+ and nucleotide requirement for growth, showed diminished expression of Ca2(+)-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2+ response virulence genes in addition to its putative role as a secreted virulence protein.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Store-operated Ca2+ influx causes Ca2+ release from the intracellular Ca2+ channels that is required for T cell activation

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.     

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen.

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.     

Inhibitors of IL-2 production and IL-2 receptor expression in human leukemic T-cell line, Jurkat

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, suppressed interleukin 2 (IL-2) production and IL-2 receptor (IL-2R) expression of the human leukemic T-cell line, Jurkat, induced by 12-O-tetradecanoyl-phorbol-13-acetate and phytohemagglutinin-P. This effect was significant at 5 microM H-7 without loss of cell viability. Such activity was not observed with N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), a potent inhibitor of cGMP- and cAMP-dependent kinases, and a weak inhibitor of Ca2+-phospholipid-dependent protein kinase (protein kinase C). These findings suggest that protein kinase C is more closely associated with IL-2 receptor expression and IL-2 production of T cells than cGMP- or cAMP-dependent kinases. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, suppressed both IL-2 production and IL-2R expression. Cycrosporin A (Cy A), a potent immunosuppressive drug, markedly inhibited IL-2 production of Jurkat cells whereas it did not affect the IL-2R expression. Thus, the mechanism of action of Cy A appears to differ from that of the protein kinase inhibitor, H-7, and the calmodulin inhibitor, W-7.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells

The ryanodinereceptor mediates intracellularCa²⁺ mobilization in muscle andnerve, but its physiological role in nonexcitable cells is less welldefined. Like adenosine 3',5'-cyclic monophosphate andinositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 µM) andADP (1-25 µM) produced a concentration-dependent rise incytosolic Ca²⁺ in permeabilizedrat parotid acinar cells. Adenosine and AMP were less effective.Ryanodine markedly depressed theCa²⁺-mobilizing action of theadenine nucleotides and forskolin in permeabilized cells and waslikewise effective in depressing the action of forskolin in intactcells. Cyclic ADP-ribose-evoked Ca²⁺ release was enhanced bycalmodulin and depressed by W-7, a calmodulin inhibitor. Afluorescently labeled ligand,4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression anddistribution of ryanodine receptors. In addition, ryanodine receptorexpression was detected in rat parotid cells with a sequence highlyhomologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of aryanodine-sensitive intracellularCa²⁺ store in rat parotid cellsthat shares many of the characteristics of stores in muscle and nerveand may mediate Ca²⁺-inducedCa²⁺ release or a modified form ofthis process.

     

Increased intracellular Ca2+ and SR Ca2+ load contribute to arrhythmias after acidosis in rat heart. Role of Ca2+/calmodulin-dependent protein kinase II

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.     

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.     

Interaction between injected Ca2+ and intracellular Ca2+ stores in Xenopus oocytes.

Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200-300 microns under the membrane), the amplitude of the transient Cl- current induced by the second injection is several-fold higher than that of the first one. This 'potentiation' persists even at 60-90 min intervals between injections. However, in oocytes permeabilized to Ca2+ by the ionophore A23187 in a Ca2(+)-free solution, the potentiation completely disappears after 30 min. It is proposed that the injected Ca2+ is largely taken up by the stores, whereas following the second injection, a higher proportion of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore-treated oocytes, the stores lose the accumulated Ca2+ over several minutes and are then ready to take up Ca2+ again, hindering its arrival at the membrane.     

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation-contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.