Ca Leak Research Articles

The stoichiometry of the Ca 2+ pump in human erythrocyte vesicles: Modulation by Ca 2+, Mg 2+ and calmodulin

Active Ca 2+ uptake and the associated (Ca 2+ + Mg 2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375–381, 1977). Some preparations were treated with 1 mM EDTA at 30° to further deplete them of endogenous levels of calmodulin. As the Ca 2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca 2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca 2+ uptake and (Ca 2+ + Mg 2+)-ATPase were sensitive to free Ca 2+ over a four log unit concentration range (0.7 μM to 300 μM Ca 2+) at 6.4 mM MgCl 2. Below 24 μM Ca 2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 μM Ca 2+ the stoichiometry approached 1:1. When MgCl 2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca 2+ concentrations examined. In contrast to the results at 6.4 mM MgCl 2, the Ca 2+ pump was maximally activated at about 2 μM free Ca 2+ and significantly inhibited above this concentration at 1 mM MgCl 2. Calmodulin (0.5–2.0 μg/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca 2+ pump in the red blood cell is discussed.

Calcium ions, drug action and the red cell membrane

Intracellular calcium regulates a number of membrane functions in the erythrocyte, including control of shape, membrane lipid composition and cation permeability. Measurement of total red cell calcium has yielded values between 5 and 15 nmol/ml cells, and these low values in part reflect the absence of Ca2+ -containing organelles. Most intracellular Ca2+ is bound and the low cell ionized Ca2+ concentration (approximately 0.2 microM) is maintained by a combination of low membrane permeability and a powerful Ca2+ -pump. This pump has been identified with a (Ca2+ + Mg2+)-stimulated ATPase, and both Ca2+ transport and ATP splitting are stimulated by calmodulin, a low molecular weight protein which binds Ca2+ avidly and activates many Ca2+ -dependent enzymes. Both high and low affinity kinetics for Ca2+ pumping have been demonstrated, depending on the extent of binding of calmodulin to the pump. A stoichiometry of either 1 or 2 Ca2+ ions pumped per ATP molecule split has been shown, and the value may vary with the level of intracellular Ca2+. Phenothiazines, such as chlorpromazine inhibit the Ca2+ -pump by antagonizing the increment in activity produced by calmodulin. The passive inward leak of Ca2+ into erythrocytes can be quantitated by 45Ca2+ uptake into red cells whose Ca2+ -pump has been inhibited. Estimates of the Ca2+ permeability, based on unidirectional influx, yield values many orders of magnitude lower than for nucleated cells. Influx of Ca2+ into human erythrocytes occurs by a facilitated diffusion process, which can be inhibited by phenothiazines and the cinchona alkaloids. Calcium affects many membrane functions including cation permeability, lipid composition and some cytoskeletal interactions which may determine cell shape. Any rise in intracellular Ca2+ activates a specific K+ channel which normally makes little contribution to K+ fluxes. Kinetic studies of this process demonstrate either high or low affinity Ca2+ -activation of K+ efflux, with low affinity of the channel to Ca2+ being the probable state in vivo. Propranolol is the best known activator of Ca2+ -stimulated K+ efflux, although the mechanism of stimulation is unclear. Like other tissues, red cells possess a Ca2+ -activated phosphoinositol phosphodiesterase. Although it has been suggested that the echinocytic shape change induced by Ca2+ is due to the hydrolysis of polyphosphoinositides, it seems more likely that this shape change results from an effect of Ca2+ on the macromolecular interactions of the cytoskeleton. Abnormal Ca2+ permeability may contribute to red cell destruction in a variety of diseases. For example, in sickle cell anemia a large Ca2+ influx occurs when cells are sickled under deoxy conditions, and moreover, the ability of the Ca2+ -pump to extrude the increment of cell Ca2+ is impaired. Thus, red cell Ca2+ is increased 3-7-fold above normal and this may contribute to the short survival of sickle red cells...

The Ca-releasing action of halothane on fragmented sarcoplasmic reticulum.

Ca-releasing action of halothane on fragmented sarcoplasmic reticulum (FSR) from bullfrog and rabbit skeletal muscle was examined to understand the mechanism of Ca release in reference to the etiology of malignant hyperthermia. Halothane has dual action on FSR: the Ca release and the inhibition of Ca uptake. On addition of halothane to loaded FSR, a rapid Ca release was followed by a sluggish Ca leakage which was probably due to a decreased capacity for Ca uptake. The properties of the rapid Ca release by halothane are similar to those of caffeine. It was inhibited by procaine or high concentrations of Mg2+. It was stimulated by a high concentration of ATP. A low temperature was stimulatory for Ca-releasing action on frog FSR while it was inhibitory on rabbit FSR. The result that caffeine shifted the dose response curve for Ca release by halothane to a steeper relation to a range of much lower concentrations suggests that the action of halothane may not be identical with that of caffeine in spite of many similarities.

Selective effects of thiazide therapy on serum 1α,25-dihydroxyvitamin D and intestinal calcium absorption in renal and absorptive hypercalciurias

The effect of long-term thiazide therapy (hydro-chlorothiazide, 50 mg twice/day) on intestinal calcium (Ca) absorption and serum 1α,25-dihydroxy-vitamin D [1α,25-(OH) 2D] concentration was examined in 10 patients with renal hypercalciuria (RH), many of whom had hyperabsorption of Ca, and in 11 cases of absorptive hypercalciuria (AH), all of whom had intestinal hyperabsorption of Ca. In patients with RH, the intestinal Ca absorption decreased significantly during thiazide therapy (mean treatment period of 15 mo) from 0.68 ± 0.09 SD to 0.56 ± 0.10 ( p < 0.01), commmensurate with the “correction” of the renal leak of Ca and secondary hyperparathyroidism. Furthermone, serum 1α,25-(OH) 2D decreased significantly from 5.2 ± 2.2 SD ng/dl to 3.7 ± 0.8 ng/dl ( p < 0.025) during thiazide therapy. In patiens with AH, the intestinal hyperabsorption of calcium persisted during thiazide treatment 0.69 ± 0.07 versus 0.69 ± 0.06), despite restoration of normal urinary Ca. Serum 1α,25-(OH) 2D was virtually unchanged during treatment (4.5 ± 1.4 ng/dl versus 4.7 ± 0.9 ng/dl). The results support the hypothesis that the intestinal hyperabsorption of Ca in RH is a result of increased serum concentration of 1α,25-(OH) 2D secondary to the hyperparathyroid state, while that in AH may not be totally dependent on increased concentrations of 1α,25-(OH) 2D.

Role of Ca2+ in genesis of lower esophageal sphincter tone and other active contractions.

The effects of absent or low Ca2+ (0.5 mM), verapamil, nifedipine, Na nitroprusside, theophylline, La2+, and ethanol on basal active tension (tone), "off" contractions, and carbachol contractions were studied in opossum lower esophageal sphincter strips. Incubation in Ca2+-free Ringer (0.1 mM EGTA) abolished tone and contractions. Low Ca2+, verapamil, nifedipine, and theophylline depressed tone more rapidly than "off" contractions. Only verapamil and nifedipine depressed carbachol contractions. Na nitroprusside rapidly depressed tone but left contractions unchanged. La3+ at 1 X 10(-3) M behaved like Ca2+-free incubation but produced sustained contractions with muscle stimulation. Ethanol depressed "off" contractions more than tone and did not affect carbachol-induced contractions. These results suggest that tone probably results from inward leak of Ca2+, whereas "off" contractions depend on release of Ca2+ sequestered in the cell by a mechanism not immediately dependent on increased Ca2+ influx. Carbachol may increase Ca2+ influx as well as utilize sequestered Ca2+. Nifedipine and verapamil may act to block both resting and stimulated Ca2+ influx. Na nitroprusside may act by increasing Ca2+ efflux. Ethanol may act by decreasing the availability of sequestered Ca2+ or by inhibiting the function of a mediator responsible for "off" contractions.

Triton detergents and the frog neuromuscular end‐plate: An electrophysiological and ultrastructural study

The effects of the nonionic detergents Triton X-45 and Triton X-100 were studied in the frog muscle end-plate, by intracellular recordings of spontaneous miniature end-plate potentials (m.e.p.p.'s) and the potential changes produced by iontophoretic application of acetylcholine (ACh-potentials). In addition, the ultrastructural changes produced by Triton X-100 were studied by transmission electron microscopic and freeze-fracture techniques. It was found that Triton X-45 and Triton X-100 caused a rapidly developing reduction of the amplitude of the m.e.p.p.'s. The response to iontophoretic application of acetylcholine was reduced by Triton X-100. Following return to normal Ringer solution the ACh-potentials recovered, although not completely. The dissociation constant calculated from the rate constants for onset and offset of the reaction (Kp=k2/k1) was 5--50 micron depending on the type of stoichiometric reaction presumed to occur between Triton X-100 and the cholinergic receptor. The ultrastructural changes observed indicate that the nerve terminal plasma membrane and mitochondria are affected by Triton X-100. Leakage of Ca2+ from the latter may therefore be the cause of the increase in m.e.p.p. frequency. It is concluded that the influence on the amplitude of the m.e.p.p.'s and the ACh-potentials can be attributed to a direct effect of the detergent upon the acetylcholine receptor protein.

Selective effects of thiazide on intestinal absorption of calcium in absorptive and renal hypercalciurias

The effect of long-term thiazide therapy on the intestinal Ca absorption was measured in 10 well-defined cases of absorptive hypercalciuria with intestinal hyperabsorption of Ca and 8 with renal hypercalciuria (“renal leal” of CA), many of whom had hyperabsorption of Ca. In most cases of absorptive hypercalciuria, the intestinal hyperabsorption of Ca persisted during treatment, despite restoration of normal urinary Ca. In contrast, the intestinal Ca absorption decreased significantly during thiazide therapy in 7 of 8 patients with renal hypercalciuria commensurate with the “correction” of the renal leak of Ca and secondary hyperparathyroidism. The results support the hypothesis that the intestinal hyperabsorption of Ca in absorptive hypercalciuria may be primary, whereas that in renal hypercalciuria may be associated with the hyperparathyroid state.

The effect of A23187 ionophore on calcium movements and contraction processes in single barnacle muscle fibres.

1. Ca movements were studied in single giant muscle fibres of the barnacle before and after exposure to the Ca ionophore A23187. 2. In fibres micro-injected with the photoprotein aequorin, the resting rate of light emission (resting glow) reversibly increased when the external Ca was augmented in the presence of A23187. This resulted from an increased Ca influx through the outer membrane. In zero external Ca, A23187 induced a delayed increase of the resting glow which was related to Ca leakage from intracellular storage sites. 3. When the experiment was carried out at 5 degrees C instead of 22 degrees C, the resting glow about doubled (temperature-sensitive mechanisms for maintaining a low myoplasmic Ca2+ were thus depressed) but A23187 was much less effective in increasing Ca influx in high external Ca. 4. In 45Ca efflux experiments, A23187 increased both the Ca-Ca exchange diffusion and the net Ca outflux at the outer membrane of the fibre. The Na-Ca exchange process was not affected to a significant extent. 5. The Ca2+ transient and the force of the contraction elicited by imposed membrane depolarizations (below the threshold of the propagated action potential) were markedly increased by A23187, but the electromechanical threshold was not significantly modified. The linear relation between membrane depolarizations and the area of the Ca transient became much steeper in the presence of A23187. However the relation between the area of the Ca transient and the force output was not affected which suggests that A23187 specifically involved the Ca2+ intracellular release, but not the subsequent Ca-troponin reaction. 6. A23187 potentiates by ionophoretic action the Ca movements at the outer membrane, and it also acts inside the intact muscle fibre to intensify the intracellular release of Ca.

The effect of anti-ATPase antibodies upon the Ca ++ transport of sarcoplasmic reticulum

Sheep or guinea pig antisera against the purified Ca ++ transport ATPase of sarcoplasmic reticulum inhibit Ca ++ transport due to a complement-dependent damage of the membrane, which causes massive leakage of Ca ++. The Ca ++-activated ATPase activity is only slightly affected even at ten times higher antibody concentration than that required for inhibition of Ca ++ transport. Antibodies prepared against the Ca ++ binding protein (C 1 protein) have no influence upon either ATPase activity or Ca ++ transport and ferritin-labeled anti-C 1 antibodies do not bind to microsomes.

A potometric determination of the effects of X-irradiation on water, K and Ca flux in isolated roots

A micropotometric technique was used to determine the effects of X-irradiation on the rates of water, calcium and potassium efflux from excised onion roots ( Allium cepa). Calcium and K flux were determined with the aid of an atomic-absorption spectrophotometer. Water efflux was decreased significantly immediately following air exposures of 75 and 100 kR; whereas 50 kR brought about an enhancement of water efflux. Concomitantly, 50 kR appeared to have produced an increase in K and Ca influx and efflux in excised roots. Interestingly, 75 and 100 kR brought about an increase in K efflux from the basal portion as well as an increased K loss into the bathing solution. On the other hand, the higher exposures brought about an increase in Ca efflux from the cut basal end only. There was no leakage of Ca into the bathing solution. The data indicate separate action of irradiation on water and cation flux in root systems.