Intracellular Ca and cell injury: A paradoxical role of Ca in complement membrane attack

Disturbances in intracellular Ca2+ are known to be important in cell injury caused by a wide range of toxic factors. The complement system is a major effector of immune damage in vivo, and is known to be involved in the pathogenesis of many immune diseases. We present here evidence that the potentially lethal membrane attack complex of complement causes a rapid increase in intracellular free Ca2+ concentration before any other detectable biochemical changes in the cell. In nucleated cells the increased intracellular free Ca2+ concentration initially stimulates recovery processes, allowing the cell to escape mild complement attack and also activates the production of inflammatory mediators, which may amplify an ongoing inflammatory response. More severe complement membrane attack causes a more rapid rise in intracellular free Ca2+ concentration allowing a threshold to be breached above which recovery processes are overwhelmed, and cell death occurs. The importance of non-lytic effects and recovery processes mediated by Ca2+, and the molecular basis of these effects are discussed, and the hypothesis proposed that the cell-injuring effects of other "pore-forming" toxins are also caused by increases in intracellular free Ca2+.     

Increases in cytosolic free Ca2+ induced by ATP, complement and beta-lipoprotein in mouse L fibroblasts

By means of Ca2+- and K+-selective microelectrodes, the changes in intracellular free Ca2+ and K+ were measured during the hyperpolarizing responses induced by ATP, complement and beta-lipoprotein in mouse fibroblastic L cells. The cytoplasmic Ca2+ concentration [( Ca]i) was about 0.4 microM in the resting state. The hyperpolarizing responses always coincided with a phasic increase in [Ca]i. ATP or beta-lipoprotein induced about a 2-fold rise in [Ca]i, and complement did up to 3-fold. Both the hyperpolarizing responses and [Ca]i increases were prevented by removal of external Ca2+ or by application of a Ca-channel blocker, nifedipine. Quinine, a Ca-activated K-channel inhibitor, suppressed the hyperpolarizing responses but not the [Ca]i increases. During the hyperpolarizing response, the intracellular free K+ concentration gradually decreased from about 120 to 110 mM. Thus, it is concluded that ATP, complement and beta-lipoprotein caused a transient elevation of cytoplasmic free Ca2+ due to Ca2+ influxes, thereby inducing electrical membrane responses through activation of Ca-dependent K-channels in the fibroblasts.     

The recovery of human polymorphonuclear leucocytes from sublytic complement attack is mediated by changes in intracellular free calcium.

Using polymorphonuclear leucocyte-erythrocyte ghost hybrids entrapping the calcium-activated photoprotein obelin, we have demonstrated that sublytic amounts of the complement membrane attack complex induce a rapid but transient increase in intracellular free calcium ion concentration ([Ca2+]i). This increase in [Ca2+]i occurs prior to, and is required for, rapid removal of membrane attack complexes from the cell surface. The increase in [Ca2+]i is not only due to increased influx from outside the cell, but also results from mobilization of intracellular stores. The possible mechanism of mobilization of calcium, and the importance of an increase in [Ca2+]i as a mediator of recovery processes in nucleated cells, are discussed.     

Analysis of signal transducing mechanisms in CD3+ CD4- CD8- cells expressing the putative T cell receptor gamma gene product

The signal transducing mechanisms, involved in the activation of CD3+ WT31- cells bearing the putative products of T cell receptor gamma genes, have been investigated. After stimulation with phytohemagglutinin or with monoclonal antibodies directed against CD2 or CD3 surface molecules, a rapid increase in free cytoplasmic Ca2+ concentration rise was detected in one representative CD3+ WT31- clone and in PEER cell line. Experiments performed in the presence of the Ca2+ chelator EGTA indicated that the free cytoplasmic Ca2+ concentration rise was consequent to an early release of Ca2+ from internal stores, followed by a sustained Ca2+ influx from the extracellular compartment. Moreover, increased levels of inositol-3-phosphate (the putative mobilizer of Ca2+ from the intracellular stores) were observed after stimulation, thus suggesting that activation of this cell subset occurs via the classical inositol-lipid metabolic pathway.     

Direct measurement of the increase in intracellular free calcium ion concentration in response to the action of complement.

1. The effect of rabbit anti-(pigeon erythrocyte) antibodies plus human complement on the concentration of intracellular free Ca2+ in sealed pigeon erythrocyte ''ghosts'' was investigated with the photoprotein obelin. 2. The addition of human serum, as a source of complement, to ''ghosts'' coated with antibody caused a rapid increase in intracellular free Ca2+ after a lag of 20-40 s, as detected by an increase in obelin luminescence. 3. The increase in obelin luminescence could not be explained by release of obelin into the medium. It was also Ca2+-dependent in that extracellular EGTA abolished the effect and intracellular EGTA inhibited it and required the complete terminal complex (C56789). No effect was seen with C5678. 4. The concentration of intracellular free Ca2+ before addition of complement was approx. 0.3 microM. This increased to a maximum of 5-30 microM after complement addition and then remained constant for at least 1-2 min. 5. Antibody plus complement induced a rapid increase in 42K+ efflux and an inhibition of cyclic AMP formation. 6. When partially purified complement components (C5b-9) were used in ''reactive lysis'' it was possible to inhibit the release of macromolecules from pigeon erythrocyte ''ghosts'' by extracellular EGTA. 7. It was concluded that the increase in intracellular free Ca2+ concentration caused by anti-cell antibody plus complement occurred before cell lysis and may be involved in the mechanism of complement-induced cell injury.     

Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils.

1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of obelin, coelenterazine, to these cells containing apo-obelin or Obelia mRNA resulted in formation of active Ca2+-activated obelin. 3. The obelin formed within the neutrophils responded to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (1 microM) and to the membrane attack complex of complement (C5B6789n). 4. The formation of the apo-obelin from mRNA within neutrophils was inhibited by over 80% in the absence of added amino acids, and by over 90% by the protein-synthesis inhibitor puromycin (100 micrograms/ml). 5. The translation of Obelia mRNA inside cells provides a method for circumventing consumption of Ca2+-activated photoproteins during cell activation or injury, and for monitoring protein synthesis in living cells.     

CD1 stimulation of human T cell lines induces a rapid increase in the intracellular free Ca2+ concentration and the production of IL-2.

The effect of a battery of CD1 mAb on intracellular free Ca2+ concentration and IL-2 production has been examined on different T cell lines in this study. Both 0249F and NU-T2 two CD1b specific mAb tested, induced a rapid increase in the intracellular Ca2+ concentration on HPBALL T cells whereas only one (L161) among three different CD1c mAb (L161, 10C3, and M241) produced a similar effect. In contrast the addition of four different CD1a mAb directed against two different epitopic groups of this molecule were uneffective in modifying the intracellular Ca2+. Both L161 and 0249F also induced a comparable increase in the intracellular Ca2+ concentration on MOLT 4 and JURKAT, two other T cell lines of similar phenotype. The effect of L161 mAb on the IL-2 production of the IL-2 producing T cell line JURKAT was also examined. The association of the latter with PMA strongly induced the production of IL-2 on this cell model while either L161 or PMA alone had no effect. Although the natural ligand and the function of CD1 molecules are still unknown, the accumulation of these data strongly suggest that CD1b and CD1c might represent two activatory pathways for immature T cells operating before the classical CD2 and CD3 activation pathways.     

Simultaneous measurement of stimulus-induced changes in cytoplasmic Ca2+ and in membrane potential of human neutrophils

The activation of human neutrophils by chemotactic peptides evokes a rapid change in membrane potential and an increase in cytoplasmic Ca2+ levels. These events are followed up to a minute later by detectable levels of microbicidal agents formed by the oxidative burst. Except for the latter, the sequence of events has remained unclear. We report here that a new fluorescent Ca2+ indicator developed by R. Tsien, Indo-1, has allowed us to resolve the temporal relationship between the rapid and transient cytoplasmic Ca2+ rise and the membrane potential change and to do so on very small samples by using a fluorescence-activated cell sorter. We have adapted a FACS 440 for simultaneous single cell membrane depolarization and cytoplasmic [Ca2+] detection in human neutrophils upon stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP). A membrane potential probe, dipentyloxacarbocyanine, allows us to determine that the membrane potential change is fMLP dose-dependent and apparently biphasic. The depolarization is maximal 40 s after stimulation. In contrast, cytosolic [Ca2+], while fMLP-dose dependent, is maximal at 10 s and already decreasing rapidly when the cell has reached its lowest potential. It can be measured with Indo-1 which has a fluorescence emission (lambda ex = 357 nm) maximum at 485 nm when Ca2+-free and 405 nm when Ca2+-liganded. The ratio of these fluorescences may then be calibrated in terms of cytoplasmic Ca2+ levels. Thus, Ca2+ release into the cytoplasm becomes the earliest evidence of neutrophil stimulation by fMLP and occurs in close association with an apparent membrane hyperpolarization.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

Decrease in cytosolic free Ca2+ and enhanced proteoglycan synthesis induced by cartilage derived growth factors in cultured chondrocytes

Cartilage-derived growth factors, enhance proteoglycan synthesis in cultured chick-embryo chondrocytes, and have almost no effect on cell proliferation. Addition of cartilage derived growth factors to cartilage cells loaded with the fluorescent Ca2+ indicator quin 2, caused a rapid, concentration dependent decrease in cytoplasmic free Ca2+. This decrease persisted also in Ca2+-free medium, indicating that it is not mediated by a decrease in the passive permeability of cell membrane to Ca2+. Addition of the Ca2+ ionophore A23187, with or without cartilage derived factors, caused an increase in cytoplasmic free Ca2+ together with inhibition of proteoglycan synthesis and enhanced cell proliferation. The results may indicate that whereas cell proliferation in chondrocytes is signaled by an increase in cytoplasmic Ca2+ ([Ca2+]in), proteoglycan synthesis is signaled by a decrease in [Ca2+]in. The data lead to suggesting a mechanism for antagonistic regulation of cell proliferation and the expression of the differentiated state.     

Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca2+ release and Ca2+ influx: analysis with indo-1

The new Ca2+-probe indo-1 has a high fluorescence intensity, which allows low intracellular dye loadings. Stimulation of indo-1-loaded mouse B cells with anti-Ig antibodies provoked rapid rise of free cytoplasmic Ca2+ from 100 nM to greater than 1 microM, followed by a decline to a plateau at 300-400 nM. The initial rapid rise was not detected in quin2-loaded cells, presumably due to the Ca2+-buffering effects of the dye. The sustained Ca2+ increase was due to influx, whereas the initial rise was caused by release from intracellular stores. The magnitudes of Ca2+ release and inositol trisphosphate release were closely correlated. Concanavalin A does not provoke inositol trisphosphate release in mouse B cells. It did not induce a rapid initial Ca2+ rise in indo-1-loaded B cells either, but only a sustained increase to 200-300 nM. Finally, Ca2+ influx induced by both anti-Ig and concanavalin A were not affected by membrane depolarization.     

Platelet-derived growth factor stimulates non-mitochondrial Ca2+ uptake and inhibits mitogen-induced Ca2+ signaling in Swiss 3T3 fibroblasts

Changes in intracellular free Ca2+ concentration [( Ca2+]i) were used to study the interaction between mitogens in Swiss 3T3 fibroblasts. Platelet-derived growth factor (PDGF) produced an increase in [Ca2+]i and markedly decreased the increases in [Ca2+]i caused by subsequent addition of bradykinin and vasopressin. If the order of the additions was reversed the [Ca2+]i response to PDGF was not inhibited by bradykinin or vasopressin. Inhibition of protein kinase C by staurosporine or chronic treatment of the cells with phorbol 12-myristate 13-acetate prevented the inhibitory effect of PDGF on the [Ca2+]i response to vasopressin but not bradykinin. PDGF did not decrease the receptor binding of bradykinin and produced only a small decrease in the receptor binding of vasopressin. PDGF decreased the rise in [Ca2+]i caused by the Ca2+ ionophores 4-bromo-A23187 and ionomycin and by a membrane perturbing ether lipid, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, both in the presence and absence of external Ca2+. There was no change in cell 45Ca2+ influx caused by PDGF, vasopressin, or bradykinin. 45Ca2+ efflux from cells exposed to PDGF and vasopressin mirrored the changes in [Ca2+]i caused by the agents, that is, PDGF added after vasopressin produced a further increase in 45Ca2+ efflux but vasopressin did not increase 45Ca2+ efflux after PDGF. PDGF but not vasopressin caused an increase in the uptake of 45Ca2+ into an inositol 1,4,5-trisphosphate-insensitive non-mitochondrial store in permeabilized cells. The results suggest that the decreased [Ca2+]i response to mitogens after PDGF represents an action of PDGF at a point beyond the release of intracellular Ca2+ and the influx of external Ca2+, caused by an increase in the rate of removal of cytoplasmic free Ca2+. This increased removal of cytoplasmic Ca2+ by PDGF is not due to the increased export of Ca2+ from the cell but results from increased Ca2+ uptake into non-mitochondrial stores.     

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.     

Calcium pump of the plasma membrane is localized in caveolae

The Ca2+ pump in the plasma membrane plays a key role in the fine control of the cytoplasmic free Ca2+ concentration. In the present study, its subcellular localization was examined with immunocytochemical techniques using a specific antibody generated against the erythrocyte membrane Ca2+ pump ATPase. By immunofluorescence microscopy of cultured cells, the labeling with the antibody was seen as numerous small dots, often distributed in linear arrays or along cell edges. Immunogold EM of cryosections revealed that the dots correspond to caveolae, or smooth invaginations of the plasma membrane. The same technique applied to mouse tissues in vivo showed that the Ca2+ pump is similarly localized in caveolae of endothelial cells, smooth muscle cells, cardiac muscle cells, epidermal keratinocytes and mesothelial cells. By quantitative analysis of the immunogold labeling, the Ca2+ pump in capillary endothelial cells and visceral smooth muscle cells was found to be concentrated 18-25-fold in the caveolar membrane compared with the noncaveolar portion of the plasma membrane. In renal tubular and small intestinal epithelial cells, which have been known to contain the Ca2+ pump but do not have many caveolae, most of the labeling was randomly distributed in the basolateral plasma membrane, although caveolae were also positively labeled. The results demonstrate that the caveolae in various cells has the plasmalemmal Ca2+ pump as a common constituent. In conjunction with our recent finding that an inositol 1,4,5-trisphosphate receptor-like protein exists in the caveolae (Fujimoto, T., S. Nakade, A. Miyawaki, K. Mikoshiba, and K. Ogawa. 1992. J. Cell Biol. 119:1507-1513), it is inferred that the smooth plasmalemmal invagination is an apparatus specialized for Ca2+ intake and extrusion from the cytoplasm.     

Effect of SH-reagents on Ca2+ level in lymphocyte cytoplasm

The effect of SH-reagents on cytoplasmic free Ca2+ concentration [( Ca2+]i) in rat thymocytes and B lymphoma Raji cells has been studied by means of fluorescent Ca2+ indicator quin-2. N-ethylmaleimide and ethylmercurythiosalicylate have been found to induce a dose-dependent increase of Ca2+ concentration from about 100 nM in the control cells up to 1000 nM. The effect is weakened with a decrease of the external Ca2+ concentration and is not observed already with Ca2+ concentration in the medium less than 0.2 mM. Reduction of the level of intracellular ATP does not suppress the Ca2+ response to SH-reagents. The effect of SH-reagents is weakened with a decrease of the temperature from 37 to 24 degrees C. Addition of 1 mM Mn2+ or Ca2+ into the standard medium containing 1 mM CaCl2 prevents Ca2+ concentration increase in the cytoplasm under the action of SH-reagents. The conclusion is made that in lymphocytes Ca2+ permeability is regulated by a protein(s) sensitive to the SH-reagent and that a high level of SH-group oxidation is necessary to maintain the low Ca2+ permeability of lymphocyte plasma membrane. Mechanisms of SH-reagents action on the Ca2+ level in the cell are discussed.     

Inositol trisphosphate and calcium mobilisation in permeabilised enterocytes

Saponin-permeabilised epithelial cells isolated by hyalurodinase incubation from chicken small intestine were used to study 45Ca uptake into intracellular stores. At low (6.7 X 10(-7) M) free Ca2+ concentration most of the Ca2+ appears to be taken up into non-mitochondrial stores, whilst the mitochondria seem to play a major role at high (2 X 10(-5) M) Ca2+ concentration. Addition of inositol trisphosphate (IP3) causes a rapid and reversible release of 45Ca from non-mitochondrial stores, with a half-maximal effect of approximately 1 microM.     

Spontaneous Ca^2＋ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca^2＋ pools

INTRODUCTION In vascular smooth muscle, as in other types of muscle,an increase in intracellular Ca2 is the immediate triggerfor contraction, which ultimately determines vascular toneand peripheral resistance. In the past 12 years, investiga-tors have …     

Electrophysiological responses to bradykinin and microinjected inositol polyphosphates in neuroblastoma cells. Possible role of inositol 1,3,4-trisphosphate in altering membrane potential

Addition of bradykinin to mouse N1E-115 neuroblastoma cells evokes a rapid but transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i rise is accompanied by a transient membrane hyperpolarization, due to a several-fold increase in K+ conductance, followed by a prolonged depolarizing phase. Pretreatment of the cells with a Ca2+-ionophore abolishes the hormone-induced hyperpolarization but leaves the depolarizing phase intact. The transient hyperpolarization can be mimicked by iontophoretic injection of IP3(1,4,5) or Ca2+, but not by injection of IP3(1,3,4), IP4(1,3,4,5) or Mg2+ into the cells. Instead, IP3(1,3,4) evokes a small but significant membrane depolarization in about 50% of the cells tested. Microinjected IP4(1,3,4,5) has no detectable effect, nor has treatment of the cells with phorbol esters. These results suggest that, while IP3(1,4,5) triggers the release of stored Ca2+ to hyperpolarize the membrane, IP3(1,3,4) may initiate a membrane depolarization.     

Fluorescence and differential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells

This report describes an optical system for microspectrophotometry in a single cardiac cell from which the sarcolemma has been removed by microdissection (skinned cardiac cell). This system is attached to the high power inverted microscope used for the microdissection and includes (a) a single variable wavelength microspectrophotometer used to define the spectrum of a given dye or Ca2+ probe; and (b) a dual wavelength, differential microspectrophotometer used to record differentially between the optimum wavelength and a wavelength separated by 25--30 nm. Results are presented using the following optical methods: (a) fluorescence measurements with chlorotetracycline to monitor the amount of Ca2+ bound to the inner face of the sarcoplasmic reticulum (SR) membrane; (b) differential absorption measurements with arsenazo III to measure changes of myoplasmic [Ca2+]free resulting from Ca2+ release from the SR; (c)fluorescence and (or) differential absorption measurements with the potential-sensitive dyes merocyanine 540, NK 2367, and di-S-C3(5) to monitor changes of charge distribution on the SR membrane during Ca2+ accumulation in the SR, as well as before and during Ca2+-induced release of Ca2+ from the SR. A small and rapid signal is observed which precedes the Ca2+-induced release of Ca2+ from the SR. It is detected as an increase of CA2+ binding inside the SR with chlorotetracycline and as a "hyperpolarization" with potential-sensitive dyes, while no transient change of myoplasmic [Ca2+]free is detected with arsenazo III. This small and rapid signal preceding the Ca2+ release may be a first hint to an understanding of the mechanism whereby a small increase of [Ca2+]free outside the SR triggers Ca2+ release from the SR.     

Changes in cytoplasmic free calcium caused by halothane. Role of the plasma membrane and intracellular Ca2+ stores

Malignant hyperthermia is a muscle disease characterized by an abnormal response to anaesthetics, stress, and exercise. It is typified by muscle contracture and a dramatic elevation in body temperature. A defect in the regulation of the concentration of cytoplasmic free calcium, [Ca2]i, is thought to underlie this disease, but the actual [Ca2+]i was not measurable until recently. We have shown that the anaesthetic halothane increases [Ca2+]i in isolated lymphocytes from malignant hyperthermia-susceptible humans and pigs but not in the normal counterparts. In this report we extend these observations to a larger number of cases and analyze the molecular mechanisms responsible for the increase. The halothane-mediated rise in [Ca2+]i required external Ca2+ and was prevented by nifedipine, an inhibitor of the voltage-sensitive Ca2+ channels of the cell membrane. In addition, the effect of halothane on the releasable Ca2+ from intracellular stores was determined by measuring the size of the releasable pool before and after addition of the anaesthetic. After addition of halothane, about 73% of this Ca2+ pool was still available for release by the Ca2+ ionophore ionomycin in cells from normal humans and pigs. In contrast, only about 45% of the free Ca2+ in intracellular stores was left after treatment with halothane in cells from malignant hyperthermia-susceptible humans and swine. These results indicate that halothane acts both at the cell membrane and at intracellular organelles, and that this action results in a net increase in [Ca2+]i in malignant hyperthermia, but not in normal cells. The action at the cell membrane appears to be on the voltage-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)