Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin (original) (raw)
Related papers
Two modes of inhibition of the Ca2+ pump in red cells by Ca2+
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985
Two different and independent modes of inhibition of the Ca 2+ pump by Ca 2+ can be detected measuring active Ca 2÷ extrusion from resealed ghosts of human red cells: one requires extracellular and the other requires intracellular Ca 2+. (2) K i for inhibition by extracellular Ca 2÷ is about 10 raM. (3) Extracellular Mg 2÷ replaces Ca 2+ in inhibiting Ca 2+ transport but with an apparent affinity for inhibition about 3-times less than that for Ca 2+. (4) Inhibition by external Ca 2+ is not affected by Na ÷ or K + at both surfaces of the cell membrane, external EGTA, internal Ca 2+ or ATP. (5) The apparent affinity for external Ca 2÷ progressively raises as pH increases. (6) The effects of extracellular Ca 2÷ and Mg 2÷ are consistent with the idea that for Ca 2 ÷ pumping to proceed, external sites in the pump must be protonated and not occupied by extracellular Ca 2+ or Mg 2+ (6). Inhibition by intraCellular Ca 2+ takes place with a K i of about 1 mM and is independent of external Ca 2 +. (7). The inhibitory effects of intracelhilar Ca 2 ÷ can be accounted for if Ca 2 + and CaATP were competitive inhibitors of the activation of the pump by Mg 2+ and MgATP, respectively.
Journal of Immunological Methods, 2009
In leukocytes, as in many other cell types, cytoplasmic calcium ([Ca 2+ ] i ) changes play a key role in a series of pathways leading to activation. Here we describe a flow cytometric method allowing the simultaneous kinetic analysis of changes in [Ca 2+ ] i in the three types of leukocytes, i.e. monocytes, granulocytes and lymphocytes. Heparinised whole blood was diluted in phosphate buffered saline with Ca 2+ and 1 mM sodium pyruvate and incubated with the Ca 2+ indicator fluo3-acetoxymethyl ester. Leukocytes were identified by labelling with the phycoerythrin-conjugated antibody against CD45, the leukocyte common antigen. Resuspension of the cells in PBS with or without Ca 2+ allowed us to detect the origin of Ca 2+ changes. During flow cytometric analysis only CD45-positive cells were counted and monocytes, granulocytes and lymphocytes were evaluated separately. Baseline fluorescence of the fluo3-Ca 2+ -complex was determined and changes in [Ca 2+ ] i after stimulation with the calcium ionophore A23187 or the chemotactic peptide N-formyl-methionyl-leucylphenylalanine (fMLP) were recorded over a time period of 150 s. Stimulation with A23187 resulted in a rise in [Ca 2+ ] i in all three leukocyte subpopulations. This rise was sustained in the presence of extracellular Ca 2+ (Ca 2+ ex ) but had a transient character in the absence of Ca 2+ ex . For fMLP, [Ca 2+ ] i changes occurred only in monocytes and granulocytes and were transient irrespective of the presence or absence of Ca 2+ ex . In conclusion, the present method is a simple, fast and easy tool to analyse in vitro [Ca 2+ ] i changes over time in leukocytes under physiologically relevant conditions, without the need for their isolation or the lysis of erythrocytes. The whole blood approach allows a continuous interaction between the different leukocyte subpopulations and other blood components and a minimum of preparative manipulations avoids artefactual activation of the cells. A distinction can be made between Ca 2+ release from the intracellular stores and the entry of Ca 2+ from outside the cell. The approach allows to evaluate the effect of various agonists on [Ca 2+ ] i changes in leukocytes, with physiological, patho-physiological or therapeutic purposes.
Properties of the basal calcium influx in human red blood cells
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
The basal 45 Ca 2 + influx in human red blood cells (RBC) into intact RBC was measured. 45 Ca 2 + was equilibrated with cells with t 1/2 = 15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5 F 0.2 Amol/l packed cells (n = 6) at 37 jC. The average value of the Ca 2 + influx rate was 43.2 F 8.9 Amol/l packed cells hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 jC. The basal Ca 2 + influx was saturable with Ca 2 + up to 5 mmol/l but at higher extracellular Ca 2 + concentrations caused further increase of basal Ca 2 + influx. The 45 Ca 2 + influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3V ,4V ,5-tetrachloro-salicylanilide (TCS) 10 À 6-10 À 5 mol/l) inhibited in part the Ca 2 + influx. The results show that the basal Ca 2 + influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca 2 + influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca 2 + are coupled via the RBC H + homeostasis.
Biochemical Society Transactions, 1994
Emptying of the intracellular calcium stores of human neutrophils, by prolonged incubation in Ca2+-free medium, by treatment with low concentrations of the Ca2+ inophore ionomycin, or by activation with cell agonists, increased the plasma-membrane permeability to Ca2+ and Mn2+. The chemotactic peptide formylmethionyl-leucyl-phenylalanine and the natural agonists platelet-activating factor and leukotriene B released different amounts of calcium from the stores and induced Ca2`(Mn2+) uptake, the rate of which correlated inversely with the amount of calcium left in the stores. The increased Mn2+ uptake induced by these agonists was persistent in cells incubated in Ca2+-free medium, but returned to basal levels in cells incubated in Ca2+-containing medium, with the same time course as the refilling of the calcium stores. The calcium-stores-regulated Mn2+ influx, including that induced by agonists, was prevented by cytochrome P-450 inhibitors. We propose that agonist-induced Ca2`(Mn2+) influx in human neutrophils is secondary to the emptying of the intracellular stores which, in turn, activates plasma-membrane Ca2+ channels by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197].
Bioelectrochemistry, 2004
Human red blood cells (RBCs) were loaded with the Ca 2 + -sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E 2 (PGE 2 ) on the intracellular free Ca 2 + concentration ([Ca 2 + ] i ). [Ca 2 + ] i of intact RBCs in a Ca 2 + -containing physiological (high) ionic strength (HIS) solution was 75.1 F 8.3 nM after 5 min incubation, increasing to 114.9 F 9.6 nM after 1 h. In Ca 2 +containing low ionic strength (LIS) solutions, [Ca 2 + ] i was significantly lower than in the Ca 2 + -containing HIS solution ( p = 0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca 2 + ] i was seen after 1 h. In Ca 2 +free (0 Ca 2 + plus 15 AM EGTA) media, [Ca 2 + ] i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE 2 on passive Ca 2 + influx was investigated on ATP-depleted RBCs. Ca 2 + influx was faster during the initial 10 min in comparison with the subsequent time period (10 À 45 min), both in HIS and LIS media, decreasing from 20.3 F 1.9 to 12.9 F 1.3 Amol/(l cells  h) in HIS, and from 36.7 F 5.3 to 8.6 F 1.2 Amol/(l cells  h) in LIS. Prostaglandin E 2 (PGE 2 ; 10 À 7 -10 À 11 M), dissolved in deionised water or in ethanol, did not affect [Ca 2 + ] i in either normal or in ATPdepleted RBCs suspended in Ca 2 + -containing HIS medium. Finally, the addition of carbachol (100 AM) did not affect [Ca 2 + ] i . The present findings suggest that stimulation of the Ca 2 + -activated K + channel by PGE 2 , reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca 2 + ] i .
The Journal of Membrane Biology, 1978
We have measured Ca binding to fragmented human red cell membranes under equilibrium conditions in the presence of low concentrations of EGTA-buffered, ionized Ca. The ionic strength of the assay medium was maintained at 0.16. Two high affinity Ca binding sites were identified: Site I was pH-sensitive. Its apparent dissociation constant (K') increased from 2 x 10-7 to 6 x 10-7 ~ as the pH was shifted from 6.8 to 6.0. Between pH 8.5 and 6.8K' remained constant. The capacity of the same site decreased between pH 8.5 and 6.8 from 0.16 to 0.04 nmoles/mg protein. Site II was insensitive to pH changes between 8.5 and 6.0. It had a K' value of ~3x10-6M and a capacity of ~0.2 nmoles/mg protein. Mg and the local anesthetic propranolol (but not tetracaine) inhibited Ca binding to site I competitively and to site II noncompetitively. The properties of the high affinity Ca membrane binding sites are consistent with the assumption that site I corresponds to the site at which Ca initiates an increase in K permeability in resealed red cell ghosts. Site II is possibly involved in the Ca-mediated resealing of red cell ghosts after osmotic hemolysis. In the presence of MgATR only a single saturable high affinity Ca binding site was observed (K'~ 6 x 10-7 at pH 6.8). The capacity of this site (~1.8 nmoles/mg protein) was almost 10 times higher than the combined capacities of sites I and II under control conditions. The results are discussed in the light of inevitable but severe shortcomings due to the evaluation of binding constants from nonlinear Scatchard plots by a curve-fitting procedure. Good evidence supports the view that membrane-bound Ca-at least in red cells-plays an important role in controlling membrane permeability: (i) Ca can reverse the cation leak developing in red cells which are exposed to media of low ionic strength [3]; (ii) Ca (or Mg) is required for the restitution of a low cation permeability in erythrocyte ghosts ("resealing') after hypotonic hemolysis of red cells [2, 12, 251 ; (iii) Ca can induce a large selective increase in passive K permeability if it has access to the inside of the cell membrane [1, 18, 19, 24, 25, 27, 37-]. A better characterization of the membrane sites which are involved in these Ca-mediated changes of membrane function requires the direct de
2008
Phorbol-14-myristate-13-acetate (PMA) (10 -7 -10 -6 mol/l) inhibited the Ca 2+ -dependent K + efflux (the Gardos effect - GE) induced by Ca 2+ , the hyperpolarisation accompanying the GE, the vanadate-induced 45 Ca 2+ influx, and depolarised the membrane, in vanadate-treated human red blood cells (RBC). The GE induced by propranolol (PLL) was not inhibited by PMA. Both PMA and PLL stimulated the basal 45 Ca 2+ influx. These results suggest that a) protein kinase C activity prevents the activation of GE by vanadate but PLL bypasses this mechanism, b) the stimulation of the Ca 2+ influx by PMA and the GE inhibition are caused by the membrane depolarisation, c) the basal Ca 2+ influx in human RBC is regulated in a complex manner, and d) the effect of vanadate resembles to the activation of agoniststimulated signalling pathway in non-excitable cells.
Physiologic rate of carrier-mediated Ca2+ entry matches active extrusion in human erythrocytes
The Journal of General Physiology, 1991
The intracellular Ca 2+ concentration of nearly all cells is kept at submicromolar levels. The magnitudes of transmembrane Ca ~+ movement that maintain this steady state in the human red blood cell have long been debated. Although there is agreement that the physiologic extrusion of Ca ~÷ by the wellcharacterized Ca ~÷ ATPase amounts to 45 Izmol/liter cells per h (1982. Nature (Lond.). 298:478-481), the reported passive entry rates in physiological saline (2-20 i~mol/liter cells per h) are all substantially lower. This discrepancy could be due to incomplete inhibition of the pump in the previous measurements of Ca 2+ entry. We therefore examined both rate and mechanism of entry after completely inactivating the pump. This required pretreatment with iodoacetamide (to lower the intracellular ATP concentration) and vanadate (to inhibit any residual Ca 2÷ pump activity). The rate of Ca ~+ entry (53 v, mol/liter cells per h) was now found to be comparable to the accepted extrusion rate. Entry closely obeyed Michaelis-Menten kinetics (Vmx = 321 ---17 nmol Ca/g dry wt per h, K m = 1.26 -+ 0.13 mM), was competitively inhibited by external Sr ~÷ (K i = 10.8 ± 1.2 mM), and was accelerated by intracellular Ca ~+. 45Ca2+ efflux from these pump-inactivated cells was also accelerated by either external Ca z+ or Sr 2+. These accelerating effects of divalent cations on the opposite (tram) face of the membrane rule out a simple channel. Substrate-gated channels are also ruled out: cells equilibrated with 45Ca2+ lost the isotope when unlabeled Ca 2+ or Sr 2+ was added externally. Thus, passive Ca 2+ movements occur predominantly by a reversible carrier-mediated mechanism for which Sr 2+ is an alternate substrate. The carrier's intrinsic affinity constants for Ca 2+ and Sr 2+, 1.46 and 0.37 mM -~, respectively, indicate that Ca 2+ is the preferred substrate.
Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody
1. A monoclonal antibody (1G4) was raised against the red-cell Ca2" pump, and it reacted with the pump, as verified by Western blot analysis and by the e.l.i.s.a. method. 2. At 1 mM-ATP and 10 ,sM-Ca2", 1G4 inhibited the activity of the purified Ca2" pump by 40 0. 3. Ca21 pump inhibition by the antibody was noncompetitive with regard to Ca2", calmodulin and the high-affinity portion of the ATP curve. Thus its mechanism was quite different from that of the antibody previously reported [Verbist, Wuytack, Raemaekers, VanLeuven, Cassiman & Casteels (1986) Biochem. J. 240, 633-640], which partially caused inhibition by competition at the ATP site. 4. Antibody 1 G4 reduced the steady-state level of phosphorylated intermediate and increased by 5000 the calmodulin-activated p-nitrophenyl phosphatase activity of the pump. 5. The experimental results are consistent with the hypothesis that 1G4 inhibits the Ca21 pump by decreasing the rate of the transition from the E2 form to the E1 form, causing a higher concentration of E2. 6. Analysis by Western blot of'the pattern of cross-reaction of 1G4 after tryptic digestion of the pump showed that this antibody reacts with bands of Mr 90000, 85000, 50000 and 33000. After chymotryptic digestion, the antibody reacts almost exclusively with a fragment of Mr 105 000 that is fully active but is not responsive to calmodulin. Altogether, the results indicate that 1G4 binds to an epitope involved in the functional properties of the enzyme but which is not related to the calmodulin-binding domain.
Proceedings of the National Academy of Sciences, 1984
The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximat...