Depletion of calcium stores by thapsigargin induces membrane depolarization by cation entry in human neutrophils.

The ability of various cations to change the electrical potential of the plasma membrane was examined in human neutrophils by the use of the fluorescent cationic dye 3,3'-dipropylthiadicarbocyanine. When the cells were suspended in 140 mM KCl, the fluorescence was high, indicating depolarized neutrophils. Suspension in 145 mM N-methyl-D-glucamine chloride (NMG), replacing sodium and potassium chloride, resulted in hyperpolarized neutrophils. After depletion of the intracellular calcium stores of the NMG-suspended cells with thapsigargin and EDTA or EGTA, the addition of cations depolarized the neutrophils, suggesting the existence of pathways for cation entry. Besides Na+ and K+, several divalent cations were effective in the sequence: Ca2+ > Mn2+ > Ba2+ > Cd2+ > Mg2+ > Co2+ > Zn2+ > Ni2+. Pretreatment of the neutrophils with 0.5 or 1 mM CaCl2, resulting in loading of calcium stores, reduced the ability of some of the cations to depolarize the NMG-suspended cells. From the depolarizing effects of the cations it is concluded that the entries of Ca2+, Mg2+, Mn2+, Ba2+, probably Co2+, to some extent Na+ and K+, but hardly Cd2+, Zn2+, or Ni2+, are regulated by the filling state of the intracellular calcium stores in human neutrophils. The store-regulated entry pathway may contribute to the control of the membrane potential and become active when the neutrophils are stimulated.

Relationship between small-intestinal transit rate and intestinal absorption of (14)C-labelled mannitol and (51)Cr-labelled ethylenediaminetetraacetic acid in healthy subjects.

BACKGROUND: Although the small-intestinal transit rate is generally considered to influence the urinary excretion of markers of intestinal permeability, no study has until now formally addressed the importance of this influence in humans. METHODS: Ten healthy subjects ingested a test solution containing (99m)Tc-labelled diethylenetriaminepentaacetic acid ((99)mTc-DTPA), (14)C-labelled mannitol ((14)C-mannitol), and (51)Cr-labelled ethylenediaminetetraacetic acid ((51)Cr-EDTA). After ingestion, the small-intestinal transit rate of (99)mTc-DTPA was measured with the gamma camera technique. Urine was collected for time periods of 0-2 h, 2-4 h, and 4-6 h to measure the excretion of absorbed (14)C-mannitol and (51)Cr-EDTA. Moreover, the distribution volume and plasma clearance of (14)C-mannitol and (51)Cr-EDTA were determined in each subject. RESULTS: A positive correlation was found between mean small-intestinal transit time and 0- to 6-h urinary excretion of (14)C-mannitol. The study did not show any correlation between small-intestinal transit rate and 0- to 6-h urinary excretion of (51)Cr-EDTA. Urinary excretion of neither (14)C-mannitol nor (51)Cr-EDTA was affected by distribution volume or urine volume. A positive correlation was observed between plasma clearance and 0- to 6-h urinary excretion of (14)C-mannitol, whereas plasma clearance did not influence the urinary excretion of (51)Cr-EDTA. CONCLUSIONS: Small-intestinal transit rate seems to have a significant effect on 0- to 6-h urinary excretion of (14)C-mannitol, whereas small intestinal transit rate does not influence the timed urinary excretion of (51)Cr-EDTA.

Delayed activation of plasma membrane Ca2+ pump in human neutrophils.

The interplay between Ca2+ efflux mechanisms of the plasma membrane (PM) and transient changes of the cytosolic concentration of ionized calcium ([Ca2+]i) was studied in suspensions of human neutrophils loaded with the [Ca2+]i indicator, Fura-2. To reveal Ca2+ efflux through PM the interference of intracellular Ca stores was prevented by preincubating the cells in the presence of EGTA, thapsigargin, and ionomycin. Addition of econazole prevented varying entry of divalent cations regulated by the filling state of Ca stores. The preincubation seemed to empty and permeabilize virtually all Ca stores, ensuring that the monitored changes of [Ca2+]i were caused exclusively by PM Ca2+ transporters. Following preincubation, the addition of CaCl2 induced, mediated by ionomycin, a transient rise of [Ca2+]i, a spike, eventually decreasing to an intermediary [Ca2+]i level. The ATP-dependent decrease of [Ca2+]i terminating the spike was abolished by the calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), but not by the protein kinase C inhibitor, staurosporine, nor by Na(+)-free medium, suggesting that neither activity of protein kinase C nor Na+/Ca2+ exchange was necessary for generation of the Ca2+ spike. In conclusion, the PM Ca2+ pump was responsible for the Ca2+ spike by responding to the rapid rise of [Ca2+]i by a delayed activation, possibly involving calmodulin. This characteristic feature of the PM pump may be important for the generation of cellular [Ca2+]i spikes in general.

[Calcium signals in blood cells]. / Calciumsignaler i blodceller.

Stimulation of blood cells by binding of agonists to receptors initiates cellular calcium signals. The signals appear as transient increases of the cytosolic concentration of ionized calcium. The signals influence secretion, adhesion, phagocytosis, movements and proliferation of the blood cells. The calcium signals are controlled by other signalling systems that regulate calcium transporting devices located in the plasma membrane and the membranes that surround the cellular calcium stores. Calcium ions are transported through channels, pumps, or exchangers located in the cellular membranes. Several calcium transporters are subjected to cellular feedback mechanisms. A few pathological disorders of the calcium signal system are known.

Solitary calcium spike dependent on calmodulin and plasma membrane Ca2+ pump.

Resealed human red cell ghosts were loaded with Fura-2, ATP, Mg2+, and either calmodulin (CaM) or, to prevent CaM activation of the Ca2+ pump, a synthetic peptide that antagonized endogenous CaM (an analogue of the CaM binding domain of protein kinase II, referred to as 'antiCaM'). The ghosts reduced the cytosolic concentration of ionized calcium ([Ca2+]i) to 193 +/- 60 nM (SD, n = 15) in a medium containing 1 mM Ca2+ and to 30 +/- 27 nM (SD, n = 62) in a medium without Ca2+ addition. Without ATP, i.e. no fuelling of the Ca2+ pump, the [Ca2+]i remained high (approx. 5 microM or higher). The simultaneous addition of the ionophore A23187 and Ca2+ rapidly increased the Ca2+ influx, which in the CaM loaded ghosts caused a solitary spike of [Ca2+]i, reaching maximum around 2 microM within 24 +/- 6 s (SD, n = 40). On the contrary, in the ghosts loaded with antiCaM, the addition of A23187 with Ca2+ raised [Ca2+]i during the first 2 min to a high level (2-4 microM) with no preceding spike. Pre-incubation of CaM-ghosts with Ca2+ diminished the height of the Ca2+ spike, and treatment with trypsin even removed the Ca2+ spike. The trypsin treatment activated the Ca2+ pump prior to the rise of [Ca2+]i, making the time-consuming CaM activation unnecessary. In conclusion, the Ca2+ spiking is dependent on a delayed CaM activation of the plasma membrane Ca2+ pump in response to a rapid increase of Ca2+ influx.

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts.

Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30-50 microM) of indo-1 and with high concentrations (3.5-4.5 mM) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca2+]i) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases. aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca2+]i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.

Cytosolic free calcium concentrations in lymphocytes from malignant hyperthermia susceptible patients.

Halothane in concentrations exceeding 1.2 mmol litre-1 increased (P less than 0.05) the apparent intracellular concentration of calcium in lymphocytes from 12 patients being tested for susceptibility to malignant hyperthermia (MH) using the fluorescent Ca2+ indicator, fura2. There was no difference in [Ca2+]i between lymphocytes from patients found to be MH susceptible (n = 5) on in vitro contracture testing with halothane and caffeine and those from MH negative patients (n = 6). Thus determination of [Ca2+]i in lymphocytes after exposure to halothane could not be used as a diagnostic test for MH susceptibility.

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.

Immunodeficiency after allogeneic bone marrow transplantation in man. Effect of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187) in vitro.

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.

Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production, and proliferation.

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbol ester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.

Halothane inhibits hyperpolarization and potassium channels in human red blood cells.

The effect of halothane on the Ca2+-sensitive K+ channel in human erythrocytes has been investigated. The red cells were suspended in buffer-free salt solutions containing Ca2+ or 45Ca2+. The protonophore CCCP was added to bring about a rapid equilibration of protons across the plasma membrane. After addition of the divalent cation ionophore A23187, the cells took up Ca2+ and this caused the K+ channels to open. When the medium contained 1 mM K+, the addition of A23187 induced a transient hyperpolarization of the cells, as monitored by measurement of the pH of the medium. The cellular pH, being buffered by haemoglobin, was virtually constant. Halothane reversibly inhibited hyperpolarization and limited the release of cellular K+ in a dose-dependent way, but did not inhibit the Ca2+-transporting properties of A23187. No stimulatory effects of halothane were observed even at low halothane concentrations. In conclusion, halothane reversibly inhibits the Ca2+-sensitive K+ channel in human erythrocytes with an ED50 of about 0.5 mM.

Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS.

The tumor-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca2+ concentration ([ Ca2+]i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 microM. Half-maximum response was found at about 1 nM of thapsigargin, full response at 100 nM. The effect of thapsigargin on [Ca2+]i exceeded that of phytohaemagglutinin (PHA) which raised [Ca2+]i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas the thapsigargin/PMA treatment could not restore the defective proliferation of AIDS lymphocytes in spite of the increased [Ca2+]i. Thapsigargin or PMA added separately had no stimulatory effects on cell proliferation. The thapsigargin/PMA treatment caused an increase in the interleukin-2 (IL-2) production of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects on lymphocyte proliferation by increasing [Ca2+]i, and that the general defect of AIDS lymphocytes, rather than being ascribed to the initiating signal systems, is associated with later events related to DNA synthesis and proliferation.

The calcium mobilizing tumor promoting agent, thapsigargin elevates the platelet cytoplasmic free calcium concentration to a higher steady state level. A possible mechanism of action for the tumor promotion.

The ability of the platelet agonists thapsigargin (Tg) and thrombin to elevate the cytoplasmic free calcium level ([Ca2+]i) was examined. Both agonists induced a transient increase of [Ca2+]i with a different time-course, however. Thus, the maximal [Ca2+]i was reached 15 sec and 2 min after stimulation with thrombin and Tg, respectively. The thrombin induced rise of [Ca2+]i was reversible, which indicates that active calcium sequestration and/or extrusion is operating. Tg affected [Ca2+]i in a divergent manner, thus, [Ca2+]i was stabilized on a elevated level without initial formation of a pronounced peak. The decline in [Ca2+]i observed after thrombin stimulation was not impaired by the calmodulin binding drug trifluoperazine but it was strongly reduced by vanadate, which suggests the active calcium transport systems to be insensitive to calmodulin. We put forward the hypothesis that the tumor promoting activity of Tg is attributable to its ability to stabilize [Ca2+]i on a new elevated steady state level.

Delayed activation of calcium pump during transient increases in cellular Ca2+ concentration and K+ conductance in hyperpolarizing human red cells.

The net Ca2+ influx was increased in human red cells in suspension by adding moderate concentrations of the Ca2+ ionophore A23187, and due to the increased cellular Ca2+ concentration [( Ca]i) the K+ channels opened (the 'Gardos effect'). At low K+ concentration and with the protonophore CCCP in the buffer-free medium the cells hyperpolarized and the extracellular pH (pH0) increased, enhancing the A23187-mediated net Ca2+ influx. This elicited a prolonged response, viz. a primary transient increase of pH0 and [Ca]i followed by one or more spontaneous pH0 and [Ca]i transients. We explored the pump-mediated Ca2+ efflux by blocking the A23187-mediated Ca2+ flux with CoCl2 at appropriate times during the prolonged response. The Ca2+ pumping was higher during the descendent than during the ascendent phase of the primary transient at equal values of [Ca]i. The data were analyzed using a mathematical model that accounts for the prolonged oscillatory response, including pH0 and [Ca]i. In conclusion, the activation of the Ca2+ pump is delayed due to slow binding of cellular calmodulin, which is a hysteretic response to a rapid increase of the cellular Ca2+ concentration. This mechanism may be important for generation and execution of transient signals in other types of cell.

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 induced calcium transients in human red cells.

A transient increase of cellular calcium was induced by addition of the divalent cation ionophore A23187 to human red cells in the absence or presence of drugs. The peak height of the calcium transient was increased about five times at pH 6.9 and up to eighteen times at pH 7.4 by trifluoperazine (0.30 mM), and two to three times at pH 6.9 by compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). The time-dependent changes of cellular calcium were analysed by the aid of a pump-leak model based partly on the calcium dependent parameters obtained from calcium ATPase experiments, partly on the A23187 induced calcium fluxes determined in experiments with ATP depleted cells. The transient increase of cellular calcium induced within few minutes after the addition of ionophore A23187 could be explained satisfactorily by the model both in the absence and presence of the four drugs, whereas the final level of cellular calcium in the drug experiments was more difficult to predict from the pump-leak model. Comparison of experimental and model calcium transients suggested that trifluoperazine and TMB-8 affected both pump and leak, whereas compound 48/80, probably due to low membrane-permeability, mainly affected the leak and verapamil affected the pump only.

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells.

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on the rate of calmodulin binding to erythrocyte Ca2+-ATPase.

The erythrocyte Ca2+-ATPase shifts reversibly between two states, the calmodulin-deficient A-state and the calmodulin-saturated B-state, dependent on calcium and calmodulin. The effects on this system of the four drugs, trifluoperazine, compound 48/80, TMB-8 and verapamil were studied. All four drugs inhibited the maximum activity of the B -state Ca2+-ATPase and, in addition, trifluoperazine and compound 48/80 in higher doses inhibited the A-state. Furthermore, the four drugs decreased the calmodulin sensitivity of the Ca2+-ATPase in the order of decreasing effect: trifluoperazine greater than compound 48/80 greater than TMB-8 greater than verapamil. In the same order of decreasing effect the drugs increased the time required for full calmodulin activation of the A-state of Ca2+-ATPase, whereas the drugs had only small effects on the rate of deactivation of the B-state, caused by dissociation of calmodulin from the enzyme. It is discussed whether the effects on calmodulin activation were caused by a reduction of free calmodulin due to the formation of drug-calmodulin complexes or whether the drugs, especially trifluoperazine, compound 48/80 and TMB-8, by binding to the Ca2+-ATPase, decreased the rate constants for association of calmodulin and enzyme.