Calcium transport and calcium-ATPase activity in human lymphocyte plasma membrane vesicles

We have studied Ca transport and the Ca-activated Mg-ATPase in plasma membrane vesicles prepared from normal human lymphocytes. Membrane vesicles that were exposed to oxalate as a Ca-trapping agent accumulated Ca in the presence of Mg2+ and ATP. ADP, AMP, GTP, UTP, ITP, TTP, or CTP did not substitute for ATP in energizing uptake. The Vmax for Ca uptake was 2.4 pmol of Ca/micrograms of protein/min, and the Km values for Ca and ATP were 1.0 and 80 microM, respectively. One microM A23187, added initially, completely inhibited net Ca uptake and, if added later, caused the release of Ca accumulated previously. Cyanide, oligomycin, ouabain, or varying Na+ or K+ concentrations had no effect on Ca uptake. A Ca-activated ATPase was present in the same membrane vesicles, which had a Vmax of 25 pmol of Pi/micrograms of protein/min at a free Ca concentration of 4-5 microM. This Ca-ATPase had Km values for Ca and ATP of 0.6 and 90 microM, respectively. These kinetic parameters were similar to those observed for uptake of Ca by the vesicles. The Ca-ATPase activity was insensitive to azide, oligomycin, ouabain, or varying Na+ or K+ concentrations. No Ca-activated hydrolysis of GTP or UTP was observed. Both Ca transport and the Ca-ATPase activity of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-treated lymphocyte plasma membranes were stimulated 2-fold by a cytoplasmic component (calmodulin) that was purified 500-fold from lymphocyte cytoplasm. Thus, human lymphocyte plasma membranes have both a Ca transport activity and a Ca-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivities to calmodulin.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

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.     

Calcium mobilization and Na+/H+ antiport activation by endothelin in human skin fibroblasts

Endothelin (ET-1) has been shown to exert vasoconstrictor activity in vivo and mobilize Ca2+ in vascular smooth muscle cells in culture. In this paper we show that the human skin fibroblast exhibits specific receptors to ET-1 and that activation of these receptors results in increased intracellular Ca2+ (Ca2+i) and accelerated Na+/H+ antiport activity. ET-1 raised Ca2+i in a dose-response manner; the peak Ca2+i rise was from basal levels of 112.2 +/- 21.9 to 299.2 +/- 49.7 nM at 300 nM ET-1. This rise was attenuated by removal of extracellular Ca2+i0. Although ET-1 did not alter basal intracellular pH, it enhanced Na+/H+ antiport activity of acidified cells. Fibroblasts demonstrated 156 +/- 18 (mean +/- SE) ET-1 receptors per unit cell and an equilibrium dissociation constant of 203.4 +/- 35.6 pM. Inasmuch as ET-1 plays a role in the metabolism of cells such as the undifferentiated fibroblast, an important action of this peptide may be to act as a growth factor.     

Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry.

We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.     

Thrombopoietin acts synergistically on Ca(2+) mobilization in platelets caused by ADP or thrombin receptor agonist peptide.

Thrombopoietin (TPO) is the main regulator of megakaryopoiesis and influences also the function of mature platelets. TPO has been shown to synergize in multiple platelet activation processes induced by various agonists. Our aim was to elucidate whether TPO affects calcium signaling during platelet activation processes. TPO demonstrated a synergistic effect on the exocytosis induced by suboptimal doses of adenosine diphosphate (ADP) and the thrombin receptor agonist peptide (TRAP). We detected synergistic effects of TPO on the ADP or TRAP induced Ca(2+) mobilization in a small range of very low agonist concentrations. The TPO synergism on Ca(2+) mobilization and CD62P expression was measurable in different, nonoverlapping ranges of ADP or TRAP concentrations. Sustaining the agonist-induced calcium signal with thapsigargin led to a detectable TPO synergism in CD62P expression even in agonist concentrations in which the synergism only occurs in Ca(2+) signaling without thapsigargin.     

Calcium mobilization by muscarinic receptors in human astrocytoma cells: measurements with quin 2

Activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells leads to Ca2+ mobilization as measured by quin 2 fluorescence. Acetylcholine and methacholine were full and potent agonists, while carbachol and muscarine, were fully efficacious but 6- and 10-fold less potent than acetylcholine. The carbachol-induced Ca2+ response was also observed in absence of extracellular Ca2+ and was blocked by muscarinic receptor antagonists but not by organic Ca2+ channel blockers, tetrodotoxin (TTX), tetraethylammonium (TEA) or metal cations, suggesting that Ca2+ is mobilized from intracellular storage sites rather than through plasma membrane ion channels. Muscarinic receptor-mediated Ca2+ release was also detected in kidney epithelial cells but not in rat fibroblasts, glial cells or differentiated neuroblastoma x glioma hybrid cells.     

Effects of sodium removal on calcium mobilization and dense granule secretion induced by thrombin in human platelets

Removal of extracellular sodium decreased calcium mobilization from intracellular stores induced by thrombin in aspirin-treated human platelets. ATP and serotonin secretion were also significantly reduced. Secretion was positively correlated with calcium mobilization, but the presence or absence of sodium did not modify the slope of the regression line. Half-maximal secretion was reached when [Ca2+]i was increased by about 0.1 microM. Calcium mobilization induced by the divalent cation ionophore ionomycin was not modified by sodium removal. Secretion induced by ionomycin was much smaller than the thrombin-induced one for the same increases of [Ca2+]i. These results suggest that the presence of external sodium is required for normal thrombin-induced calcium release from the intracellular stores and hence for dense granule secretion. However, secretion cannot be only attributed to the increase of cell [Ca2+]i but also to other process(es) which are not affected by external sodium.     

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

Calcium fluxes in human trophoblast (BeWo) cells: calcium channels,calcium-ATPase,and sodium-calcium exchanger expression

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are poorly understood. In the current study, we have investigated the characteristics of Ca(2+) fluxes in relation with cell Ca(2+) homeostasis in the human placental trophoblast cell line BeWo. Time-courses of Ca(2+) uptake by BeWo cells displayed rapid initial entry (initial velocity (V(i)) of 3.42 +/- 0.35 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid declined of cell-associated (45)Ca(2+) with a V(i) of efflux (Ve(i)) of 3.30 +/- 0.08 nmol/mg protein/min. Further identification of membrane gates for Ca(2+) entry in BeWo cells was carried out. Expression of Ca(2+) transporter/channel CaT1 and L-type alpha(1S) subunit was showed by RT-PCR. However, mRNA for CaT2 channel and L-type alpha(1C) and alpha(1D) subunits were not revealed. Membrane systems responsible for intracellular Ca(2+) extrusion from BeWo cells were also investigated. Plasma membrane Ca(2+)-ATPases (PMCA) and Na/Ca exchangers (NCX) were detected by Western blot in BeWo cells. Expression of specific isoforms of PMCA and NCX was further investigated by RT-PCR. Messenger RNAs of four isoforms of PMCA (PMCA 1-4) were detected. The presence of messenger RNAs of two NCX isoforms (NCX1 and NCX3) was observed. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. Inorganic ions such as cadmium and manganese did not modify the Ca(2+) fluxes, however, barium increased cell-associated (45)Ca(2+) by, in part, by reducing radiolabel exit.     

Calcium sequestration in human platelets: is it stimulated by protein kinase C?

Sequestration of calcium into an intracellular storage site is an important mechanism in helping to maintain a low cytoplasmic Ca2+ level in many cells. In platelets, increasing cytoplasmic cAMP lowers the free calcium level in correlation with the phosphorylation of a 22 kD protein. This protein has been thought to enhance uptake of calcium into a platelet membrane bound storage site by activating a calcium-ATPase activity by analogy with phospholamban in cardiac muscle. The evidence for an analogue of phospholamban in platelets is unclear. A pathway involving cAMP dependent kinase also seems unlikely to account for the transience of the calcium signal following agonists in platelets, some of which inhibit the cAMP dependent kinase. Here we discuss the issue of whether activation of protein kinase C, which follows agonist action, leads to enhanced calcium sequestration in platelets and if so, what indications there are for a mechanism. The evidence from our experiments with phorbol myristate acetate treated platelets shows that such an enhancement can be produced by activating protein kinase C. Phosphorylation studies suggest the involvement of a polypeptide or polypeptides distinct from the 22 kD polypeptide. Further work to test this idea is necessary. A brief overview of research on the role of phosphoproteins in calcium regulation in platelets and comparison with their role in cardiac muscle is also presented.     

Interaction between GPIbalpha and FcgammaIIA receptor in human platelets

Glycoprotein (GP) Ib (alpha and beta) in platelets forms a noncovalent hetero-oligomeric complex with GPIX and GPV and serves as a receptor for von Willebrand factor (vWF), which mediates the initial adhesion of platelets to the subendothelium after vascular damage and also plays a role in thrombin-induced platelet activation. We investigated the interaction between GPIbalpha and FcgammaIIA receptor using a yeast two-hybrid system and mutagenesis, and we identified residues R542G543R544 in GPIbalpha and D298D299D300 in FcgammaIIA receptor as the primary interaction sites. These results further confirmed the interaction between GPIbalpha and FcgammaIIA receptor and support the hypothesis that the signal transduction of GPIb-IX-V that leads to platelet activation may be partially mediated through FcgammaIIA receptor.     

Paradoxical effects of amiloride analogs on protein phosphorylation and serotonin release induced by agonists in human platelets

We examined the effects of newly exploited amiloride analogs on protein phosphorylation and serotonin secretion induced by various agonists in human platelets. 3', 4'-dichlorobenzamil (DCB) and to a lesser extent, 2', 4'-dimethylbenzamil (DMB), which in many cells highly specific inhibitors of Na+/Ca2+-pump, inhibited the phosphorylation of 47K- and 20K-dalton proteins and serotonin secretion in human platelets independently of the action on the pump. DCB also induced dephosphorylation of 47K and 20K after the phosphorylation of these proteins by thrombin and released serotonin by itself.     

GLP-1 receptor agonists and DPP-4 inhibitors in the treatment of type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone with antidiabetic action through its ability to stimulate insulin secretion, increase beta cell neogenesis, inhibit beta cell apoptosis, inhibit glucagon secretion, delay gastric emptying and induce satiety. It has therefore been explored as a novel treatment of type 2 diabetes. A problem is, however, that GLP-1 is rapidly inactivated by the dipeptidyl peptidase-4 (DPP-4) enzyme, which results in a short circulating half-life of the active form of GLP-1 (< 2 min). Two strategies have been employed to overcome this obstacle as a treatment of diabetes. One is to use GLP-1 receptor agonists that have a prolonged half-life due to reduced degradation by DPP-4. These GLP-1 mimetics include exenatide and liraglutide. Another strategy is to inhibit the enzyme DPP-4, which prolongs the half-life of endogenously released active GLP-1. Both these strategies have been successful in animal studies and in clinical studies of up to one year's treatment. This review will summarize the background and the current (mid 2004) clinical experience with these two strategies.     

Effects of novel aggregation inhibitors on serotonin uptake by human blood platelets

Novel aggregation inhibitors blocked serotonin uptake by human blood platelets in concentrations ranging from 0.7 +/- 0.1 microM to 237.5 +/- 35.7 microM; a modified procedure, validated by kinetic analysis, was employed in which pH drift was minimized to 0.03 during the active assay period. Structural features in carbamoylpiperidine and nipecotoylpiperazine derivatives which actually constitute molecular probes, and show remarkable specificity for aggregation-inhibitory target sites, disclosed striking differences between the latter and serotonin receptors or other loci affecting serotonin uptake.