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.

Reversible shift between two states of Ca2+-ATPase in human erythrocytes mediated by Ca2+ and a membrane-bound activator.

The (Ca2+ + Mg2+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation. The A-state showed low maximum activity (V) and the Ca2+ activation was characterized by a Hill coefficient, nH, of about 1 and a Michaelis constant, KCa, about 30 micron. The B-state showed high V, a nH above 1, which indicates positive cooperativity of Ca2+ activation, and KCa of about 1 micron. With varying ATP concentrations, both the A-state and B-state showed negative cooperativity and slightly different values of Km. The B-state was shifted to A-state when the membranes were exposed to low Ca2+ concentration. The shift reached 50% at approx. 0.5 micron Ca2+. At the low Ca2+ concentrations an activator was released from the membranes. The A-state was shifted to the B-state when the membranes were exposed to Ca2+ in the presence of the activator. The shift reached 50% at about 30 micron Ca2+. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca2+ and activator accelerated the recovery. It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may respresent a resting and an active state, respectively, of the calcium pump.

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations.

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.

Rate constants for calmodulin binding to Ca2+-ATPase in erythrocyte membranes.

The Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes, which is part of the Ca2+ pump, can be activated by binding of calmodulin. Rate constants (k1) for association of calmodulin and enzyme, which depends on the Ca2+ concentration, have been determined by the aid of an enzyme model. k1 increased from 0.25 . 10(6) to 17.3 . 10(6) M-1 . min-1 (70 times) when the free Ca2+ concentration was raised from 0.7 to 20 microM. The binding of calmodulin to the Ca2+-ATPase is reversible. The rate constants (k-1) for dissociation of enzyme-calmodulin complex decreased from 6.0 to 0.044 min-1 (135 times) when the free Ca2+ concentration was increased from 0.1 to 2-20 microM. The apparent dissociation constant Kd = k-1/k1 accordingly increased from 2.5 nM to 25 microM (or higher) when the Ca2+ concentration was reduced from 20 to 0.1 microM. Therefore, at 10(-7) M free Ca2+ most of the Ca2+-pump enzyme will not bind calmodulin. For the intact cell the time dependences of activation and deactivation of the Ca2+-pump enzyme have been estimated from the rate constants above. The results suggest that the Ca2+ pump is well suited to maintain a cytosolic concentration of 10(-7) M free Ca2+ (or lower) in the unstimulated cell and, when the cell is stimulated, to allow transient Ca2+ signals up to approx. 10(-5) M in the cytosol.

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.

Hysteretic activation of the Ca2+ pump revealed by calcium transients in human red cells.

The enzymatic basis for the Ca2+ pump in human red cells is an ATPase with hysteretic properties. The Ca2+-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca2+ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133-143). In order to study whether the Ca2+ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Cai), induced by the divalent cation ionophore A23187. The time-dependent changes of Cai were measured by use of radioactive calcium (45Ca2+) and analysed with the aid of a mathematical model, based partly on the Ca2+-dependent parameters obtained from Ca2+-ATPase experiments, partly on the A23187-induced Ca2+ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca2+ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca2+ pump in human red cells responds hysteretically. It is suggested that Ca2+ pumps in other types of cell also have hysteretic properties.

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.

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.

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.

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.

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.

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.

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.

Activator-associated Ca2+-ATPase in erythrocyte membranes from cystic fibrosis patients.

Erythrocyte membranes were prepared by a method which should ensure binding of an activator protein (calmodulin) to the calcium dependent membrane ATPase involved in calcium transport. The level of enzyme activity, assayed at optimum conditions, was 5-400 times higher than that found in previous investigations on cystic fibrosis patients. The Ca2+-ATPase activity of the cystic fibrosis patients was reduced by 15% compared to control subjects, whereas patients suffering from chronic pulmonary diseases did not deviate from controls. Even if a reduction of Ca2+ pumping activity occurs in other cells, a 15% decrease could hardly be the only cause of the changed calcium concentrations in secretions from cystic fibrosis patients.

Assessment of the influence of thyroglobulin (Tg) autoantibodies and other interfering factors on the use of serum Tg as tumor marker in differentiated thyroid carcinoma.

The purpose of the study was to examine the value of a commercial immunoradiometric (IRMA) method for measuring serum thyroglobulin as a tumor marker after treatment for differentiated thyroid carcinoma. A prospective analysis of consecutive serum samples from 53 patients was performed using the IRMA method and a traditional double antibody radioimmunoassay (RIA). The results were compared with those of 100 healthy control subjects and furthermore the method was validated by investigating sera from 24 patients with Hashimoto's thyroiditis positive for thyroglobulin autoantibodies. Finally, in vitro studies of the influence of thyroglobulin autoantibodies on the method were done. The IRMA method had an acceptable analytical precision and was more sensitive than the RIA. It was furthermore less sensitive to the presence of thyroglobulin autoantibodies but it was affected by them, and it showed less unspecific serum effect. Both methods had limitations as tumor marker when the patients had a thyroid remnant, when serum thyrotropin was not suppressed, and in cases of local recurrence. The highest predictive value was found in patients with distant metastases. Thus, in cases of only slightly elevated serum thyroglobulin, the strongest indication for recurrence is still an increasing serum thyroglobulin level within the same patient rather than a single value.

Decreased (Ca2+ + Mg2+)-stimulated ATPase activity in erythrocyte membranes from polycythemia vera patients.

Erythrocytes were hemolyzed in hypotonic phospate buffer containing 0.5 mmol/l Ca2+ and the membranes subsequently washed twice in hypotonic tris buffer. The centrifugation was performed in a continuous flow system, which was necessary to obtain maximal ATPase activity. The Mg2+-dependent Ca2+-stimulated ATPase activity of 14 patients with polycythemia vera was only 67 per cent (P less than 0.001) of the activity of a control material consisting of 10 donors and 11 bank blood specimens. Five patients with secondary polycythemia and four patients with an increased erythrocyte fraction did not differ significantly from the controls. The polycythemia vera patients with the highest leukocyte count showed the lowest ATPase activity. The apparent calcium dissociation constant of the ATPase in polycythemia vera was about 10(-6) mol/l, as in controls. The relation between the reduced ATPase activity and the abnormal hemopoiesis of polycythemia vera patients is discussed.

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.