Calcium-independent pathway of tumor necrosis factor-mediated lysis of target cells

The role of Ca2+ in cell-mediated cytotoxicity has been the subject of many investigations and both Ca2+-dependent and -independent pathways have been reported. TNF was suggested to play a role in NK and macrophage cell-mediated cytotoxicity. We assumed that its role in target cell lysis might take place by a Ca2+-independent mechanism. This hypothesis was investigated in assays of rTNF-mediated lysis of tumor target cells. Extracellular Ca2+ depletion by the calcium chelator EGTA (2 mM and 5 mM) and blocking of intracellular Ca2+ mobilization by 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride did not inhibit TNF-mediated tumor cell lysis. Furthermore, blocking of Ca2+ influx in the presence of the Ca2+ channel blocker Verapamil did not inhibit TNF-mediated tumor cell lysis. Previous reports showed that lysis of sensitive tumor cells by TNF is preceded by binding of TNF to TNF receptors, internalization, and DNA degradation. These events were tested in the absence of Ca2+. Treatment with Ca2+ inhibitors did not affect binding of 125I-TNF to target cells. Also TNF induced the fragmentation of cellular DNA in target cells without extracellular or intracellular Ca2+. These findings demonstrate that the mechanism of TNF-mediated tumor cell lysis does not depend on intracellular or extracellular Ca2+ and that events associated with target cell lysis can also function in the absence of Ca2+. Thus, our findings support the contention of a Ca2+-independent lytic pathway in which secreted or membrane-bound TNF may interact with the target cells and ultimately result in DNA degradation and target cell lysis.     

Calcium-activated DNA fragmentation kills immature thymocytes

Glucocorticoid hormones kill immature thymocytes by activating a self-destructive process that involves extensive DNA fragmentation. It has been demonstrated that thymocyte suicide is dependent on an early, sustained increase in cytosolic Ca2+ concentration, and new protein synthesis, but the biochemical lesion that leads to cell death has not been established. To determine whether endonuclease activation or activation of another Ca2+-dependent process could mediate cell killing, we treated thymocytes with the glucocorticoid methylprednisolone in the presence of inhibitors of various Ca2+-dependent degradative enzymes. The role of poly(ADP-ribose) polymerase, an enzyme known to be activated by DNA damage, was also assessed. Glucocorticoid-induced chromatin cleavage and cell killing were blocked by the endonuclease inhibitor aurintricarboxylic acid, whereas inhibitors of other Ca2+-dependent degradative processes or of poly(ADP-ribose) polymerase did not abrogate cell death. In addition, stimulation of thymocyte DNA fragmentation by the Ca2+ ionophore A23187 resulted in cell killing that could be blocked by the endonuclease inhibitor. Together, our results suggest that thymocyte suicide is caused by extensive Ca2+-stimulated DNA fragmentation.     

Survival of mammalian B104 cells following neurite transection at different locations depends on somal Ca2+ concentration

We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long-standing observations that somal survival decreases with more-proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine- or thapsigargin-induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+-induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i.     

The role of Ca2+ in dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Cultured Friend cells can be induced by dimethyl sulfoxide (Me2SO) and several other agents to mature along the erythroid pathway. Evidence has been presented that an increase in Ca2+ influx is an early and necessary prelude to the commitment to maturation by these cells (Levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5948-5952). The simplest hypothesis supporting all the available data is that Me2SO and other inducers elevate the cytosolic Ca2+ concentration. We have now measured cytosolic Ca2+ using the fluorescent indicator quin-2, and find, contrary to expectation, a small decrease upon treatment of cells with Me2SO. Cytosolic Ca2+ was increased by raising the Ca2+ in the medium, but was not dramatically altered by addition of ouabain or monensin or by incubation in Na+-free medium. Measurement of total cell Ca2+ by a triple-labeling technique using 3H2O and 125I-albumin to determine cell water and extracellular space, respectively, revealed no significant change upon treatment with Me2SO for up to 40 h. A decrease in the initial rate of 45Ca2+ influx was observed in Me2SO-treated cells, when measured at 4 degrees C. These data do not support the hypothesis that an increase in cell Ca2+ is necessary for the induction of Friend cell differentiation or that Na+/Ca2+ exchange is a significant regulator of cytosolic Ca2+ in Friend cells.     

Regulation of intracellular calcium in epithelial cells

The role of [Ca2+]i as a second messenger in non-excitable cells has been appreciated for almost 3 decades. The advent of fluorescent Ca2+ indicators has allowed the monitoring of Ca2+ signalling in suspensions of these cells. Agonist mediated changes in [Ca2+]i usually show an initial Ca2+ transient followed by a maintained increase. The former has been shown to be due to Ca2+ release from one or more intracellular stores, the latter due to activation of receptor operated Ca2+ entry (ROCE). More recently it has been recognized that many cells show distinct maintained oscillatory behavior when examined by single cell optical methods. It is proposed here that these oscillations are the consequence of IP3 and Ca2+ stimulation of Ca2+ release and ligand activation of ROCE followed by Ca2+ inhibition of Ca2+ and ROCE as Ca2+ pumps are activated. These oscillations allow more exact regulation of a pump/leak controlled second messenger such as [Ca2+]i.     

Arachidonic acid activates Ca2+ extrusion in macrophages

Stimulation of macrophages with platelet-activating factor (PAF) elicits an increase of intracellular calcium concentration, Ca2+i, which was monitored here at the single cell level with the calcium-sensitive dye Fura-2. The sustained component of this Ca2+i increase reflects the dynamic balance achieved between enhanced Ca2+ influx and efflux. In macrophages where a steady increase of Ca2+i has been evoked by 50 nM thapsigargin (a molecule known to empty Ca2+ stores and elevate Ca2+i in various cell types), PAF activates Ca2+ efflux, without causing a preceding increase in Ca2+i. This result shows that in this case, Ca2+ extrusion is not merely a consequence of a Ca2+i increase. PAF-evoked Ca2+ extrusion does not result from the activation of the Na+/Ca2+ exchanger. Exogenous arachidonic acid (10-100 microM) elicits Ca2+ efflux in macrophages where Ca2+i has been previously elevated by either PAF or thapsigargin. PAF-induced Ca2+ extrusion is blocked by 4-bromophenacylbromide, an inhibitor of arachidonic acid production by phospholipase A2. Together, these results suggest that arachidonic acid, which is produced in PAF-stimulated macrophages, contributes to the regulation of a Ca2+ extrusion system, which is presumably a Ca2(+)-ATPase.     

Characterization of the calcium signaling system in the submandibular cell line SMG-C6

Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG-C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)-Ca2+ signaling system in this cell line. Inositol 1,4,5-trisphosphate(1,4,5-IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, alpha1-adrenergic, P2Y-nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha). Consistently, 1, 4,5-IP3 formation was dramatically stimulated by ACh and ATP. ACh-stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+-induced Ca2+ release mechanism is involved in the ACh-elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3-sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store-operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) both the IP3-sensitive and -insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store-operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.     

Specific epidermal protein markers are modulated during calcium-induced terminal differentiation

Extracellular calcium concentration has been shown to be an important determinant of proliferation rate in a number of cell culture models. Recently, the role of calcium as a regulator of cellular differentiation has also become apparent. This effect of calcium was exemplified by the discovery that keratinocytes of mouse or human origin grew as a proliferating monolayer in medium with a calcium concentration of 0.02-0.09 mM but that proliferation ceased and cells stratified and cornified when calcium was increased greater than 0.1 mM. While the morphological and biological effects of changes in calcium concentration are dramatic in keratinocyte cultures, it has been difficult to identify specific protein changes associated with the modulation of maturation. In vivo, however, several proteins that are markers for stratified squamous epithelia have been identified by specific autoimmune sera. Pemphigoid antigen is a 220-kdalton protein found in the basement membrane and closely associated with the plasma membrane of the basal cell. Pemphigus antigen is a 130-kdalton glycoprotein found on the cell surface of stratifying epithelial cells. Immunofluorescence staining of cells cultured in low Ca2+ or cells switched to high Ca2+ for 48 h before staining demonstrated that pemphigoid antigen was detected in low Ca2+ cultures but was diminished or absent in high Ca2+ cultures and that pemphigus antigen was seen only in high Ca2+ cultures. The synthesis of each antigen was studied in immunoprecipitates of cell lysates radiolabeled with 14C-amino acids or D-[1-14C]glucosamine. Pemphigoid antigen was synthesized mainly by proliferating cells in low Ca2+ medium and its synthesis was decreased by greater than 90% in cells switched to high Ca2+ medium. In contrast, synthesis of pemphigus antigen was detected only in stratifying cells cultured in high Ca2+ medium. These studies indicate that extracellular calcium concentrations which modulate the transition between proliferating and stratifying epidermal cells also modulate, in parallel, the synthesis of specific marker proteins for these cell types.     

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.     

Ca2+-dependent phosphorylation of tyrosine hydroxylase in PC12 cells

Ca2+-dependent protein phosphorylation has been detected in numerous tissues and may mediate some of the effects of hormones and other extracellular stimuli on cell function. In this paper we demonstrate that a Ca2+/calmodulin-dependent protein kinase similar to the enzyme previously purified and characterized from rat brain is present in PC12, a rat pheochromocytoma cell line. We show that Ca2+ influx elicited by various forms of cell stimulation leads to increased 32P incorporation into tyrosine hydroxylase (TH), a major phosphoprotein in these cells. Several other unidentified proteins are either phosphorylated or dephosphorylated as a result of Ca2+ influx. Acetylcholine stimulates TH phosphorylation by activation of nicotinic receptors. K+-induced depolarization stimulates TH phosphorylation in a Ca2+-dependent manner, presumably by opening voltage-dependent Ca2+ channels. Ca2+ influx that results from the direct effects of the ionophore A23187 also leads to TH phosphorylation. Phosphorylation of TH is accompanied by an activation of the enzyme. These Ca2+-dependent effects are independent of cyclic AMP and thus implicate a Ca2+-dependent protein kinase as a mediator of both hormonal and electrical stimulation of PC12 cells.     

Participation of Ca-dependent systems in thrombocyte aggregation induced by the action of staphylococcal toxin and ADP

The mechanism of ADP and staphylococcal toxin effect on the platelet aggregation has been studied on the rabbit's platelet-rich plasma. Ca2+-channels blockade of the cell membrane by verapamil resulted in considerable inhibition of aggregation induced by ADP and some weakening of toxin action. Binding of extracellular calcium EDTA inhibited sharply or blocked the aggregation of both inductors. It has been concluded that Ca2+ transport into cell is necessary chain in ADP and staphylococcal toxin effect but under the action of toxin transport Ca2+ into platelet is brought through a verapamil-resistant Ca2+-channels forming in the membrane under the interaction with toxin.     

Evidence that lanthanum ions stimulate calcium inflow to isolated hepatocytes.

LaCl3 stimulated the initial rate of 45Ca2+ exchange measured under steady-state conditions in isolated liver cells. Cu2+ greater than La3+ = Fe3+ greater than Fe2+ = Zn2+ greater Ni2+ greater than Mn2+ also stimulated 45Ca2+ exchange. Compartmental analysis of 45Ca2+-exchange curves obtained in the presence or absence of La3+, and in the presence or absence of adrenaline, showed that the predominant effect of La3+ is to stimulate the inflow of Ca2+ to the cell from the medium. No evidence for an inhibition of Ca2+ outflow from the cell was obtained. In the presence of La3+, adrenaline caused no further stimulation of Ca2+ inflow to the cell. In the absence of adrenaline, La3+ increased the uptake of Ca2+ (measured by atomic-absorption spectroscopy) by isolated hepatocytes incubated at 1 degree C. The proposal that La3+ stimulates Ca2+ inflow to the liver cell by inducing a conformational change in the Ca2+-inflow transporter of the plasma membrane is briefly discussed.     

T-type calcium channels and tumor proliferation

The role of T-type Ca2+ channels in proliferation of tumor cells is reviewed. Intracellular Ca2+ is important in controlling proliferation as evidenced by pulses, or oscillations, of intracellular Ca2+ which occur in a cell cycle-dependent manner in many tumor cells. Voltage-gated calcium channels, such as the T-type Ca2+ channel, are well suited to participate in such oscillations due to their unique activation/inactivation properties. Expression of the T-type Ca2+ channels has been reported in numerous types of tumors, and has been shown to be cell cycle-dependent. Overexpression of the alpha1 subunit of T-type Ca2+ channels in human astrocytoma, neuroblastoma and renal tumor cell lines enhanced proliferation of these cells. In contrast, targeting of the alpha1 subunit of the T-type calcium channel via siRNA decreased proliferation of these cells. A Ca2+ oscillatory model is proposed involving potassium channels, Ca2+ stores and Ca2+ exchangers/transporters. A review of T-type channel blockers is presented, with a focus on mibefradil-induced inhibition of proliferation. The development of newer blockers with higher selectivity and less potential side effects are discussed. The conclusion reached is that calcium channel blockers serve as a potential therapeutic approach for tumors whose proliferation depends on T-type calcium channel expression.     

The Na+/Ca2+ exchanger regulates cytolysin/perforin-induced increases in intracellular Ca2+ and susceptibility to cytolysis.

The Ca2+ dependency of NK cell-mediated and cytolysin-mediated cytolysis may be related to increases in target cell intracellular Ca2+. In a previous study we hypothesized that the Na+/Ca2+ exchanger can act as a counter-lytic mechanism by regulating the damaging increases in intracellular free calcium ([Ca2+]i) produced by cytolysin. We found that conditions said to inhibit Ca2+ extrusion by Na+/Ca2+ exchange, namely low extracellular Na+ or the presence of certain amiloride analogs which block Na+/Ca2+ exchange, enhanced the cytolysin-mediated cytolysis of YAC-1 lymphoma cells. In the present work we have confirmed the above hypothesis by measuring the [Ca2+]i of fura-2- or aequorin-labeled YAC-1 cells treated with cytolysin and low Na+ medium or amiloride analogs. YAC-1 cells appear to have a Na+/Ca2+ exchange system: low Na+ medium caused gradual increases in [Ca2+]i, and this effect was reversed in Na(+)-replete medium. Cytolysin purified from NK cell granules caused rapid dose-dependent increases in [Ca2+]i, and low Na+ medium enhanced these cytolysin-mediated increases. The Na+/Ca2+ exchange system appeared to be more active in cytolysin-challenged cells: amiloride analogs, which inhibit Na+/Ca2+ exchange in other systems, acted synergistically with cytolysin to cause large increases in [Ca2+]i, but had little effect, if any, on their own. 5-(N-4-Chlorobenzyl)-2',4'-dimethylbenzamil, the amiloride analog which has the greatest specificity for the Na+/Ca2+ exchanger and which previously was found to be the most potent enhancer of cytolysin-mediated cytolysis, was the most potent enhancer of cytolysin-mediated increases in [Ca2+]i. The above results suggest that Na+/Ca2+ exchange may be one of the target cell mechanisms of resistance to cytolysin and NK cell-mediated cytolysis.     

Calcium and pH homeostasis in neurons during hypoxia and ischemia

One of the important events during hypoxia or ischemia in the brain (or other organs for that matter, including the myocardium) is the accumulation of Ca2+ ions intracellularly. Although various studies have shown various sources of and routes for Ca2+ entry and accumulation, it is clear now that it is likely that there is a multitude rather than a single mechanism for this accumulation. In this review, we highlight this Ca2+ accumulation during low O2 states and discuss some of the mechanisms leading to accumulation for two main reasons: (a) an accumulation of Ca2+ in the cytosol has been proven to be deleterious for cell function although this accumulation of Ca2+ and consequences represent only a limited view of events that can lead to cell injury during such stress and (b) developing therapeutic strategies involving the reduction or elimination of this accumulation depends, by and large, on the mechanism of entry. In addition to reviewing some of these Ca2+ events, we will also review the relation between pH (H+) and Ca2+ since these two ions and their regulation are tied to each other in a major way. For example, extracellular acidosis, which can occur during ischemia, has a remarkable effect on the function of some of the Ca2+ entry routes.     

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering,sore-operated Ca2+ entry,and Ca2+ store refilling

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.     

Inositol trisphosphate receptor calcium release is required for cerebral artery smooth muscle cell proliferation

Vascular damage signals smooth muscle cells to proliferate, often exacerbating existing pathologies. Although the role of changes in "global" Ca2+ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role of specific Ca2+ signals in determining VSM phenotype remains relatively unexplored. Earlier work with cultured VSM cells suggests that inositol 1,4,5-trisphosphate receptor (IP3R) expression and sarcoplasmic reticulum (SR) Ca2+ release may be linked to VSM cell proliferation in native tissue. Thus we hypothesized that SR Ca2+ release through IP3Rs in the form of discrete transient signals is necessary for VSM cell proliferation. To investigate this hypothesis, we used mouse cerebral arteries to design an organ culture system that permitted examination of Ca2+ dynamics in native tissue. Explanted arteries were cultured in normal medium with 10% FBS, and appearance of individual VSM cells migrating from explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca2+ waves in myocytes in the arteries that were blocked by the IP3R antagonist 2-aminoethoxydiphenylborate (2-APB). Inhibition of IP3R opening (via 100 microM 2-APB, 10 microM xestospongin C, or 25 microM U-73122) dramatically reduced outgrowth cell number compared with untreated or ryanodine-treated (10 microM) arteries. Consistent with this finding, 2-APB inhibited cell proliferation, as measured by reduced proliferating cell nuclear antigen immunostaining within 48 h of culture but did not inhibit cell migration. These results indicate that activation of IP3R Ca2+ release is required for VSM cell proliferation in these arteries.     

SR Function in malignant hyperthermia

Malignant hyperthermia (MH) is a genetic disease in man and other animal species that predisposes to a catastrophic hypermetabolic syndrome that is triggered by certain anesthetic agents. A working hypothesis is that a defect in regulation of muscle cell calcium is the primary mechanism that initiates the MH syndrome. This paper reviews the evidence for a defect in muscle cell calcium as regulated by the sarcoplasmic reticulum membrane system. Skeletal muscle biopsied from MH man, pigs and dogs has abnormal in vitro contracture response to halothane and caffeine and these responses can be altered by lowering calcium content of the bathing solution and/or the muscle. Measurements of MH muscle cell Ca2+ by Ca2+-specific microelectrodes in vivo and fura-2 in vitro have demonstrated abnormal Ca2+ levels in resting and in caffeine-stimulated states. The SR membrane system is the primary calcium regulating organelle in skeletal muscle and a likely site for the defect in MH muscle. Two Ca2+ regulating functions of the SR have been explored in SR isolated from MH muscle. An abnormality of the 100K Ca2+-ATPase protein that functions to transport Ca2+ from myoplasm to inside the SR does not appear to be responsible for MH. The most probable defective site in the SR appears to be Ca2+ release channels and a Ca2+-induced Ca2+ release pathway has been shown to be abnormal in SR from MH human and pig muscle.     

Membrane IgM and IgD molecules fail to transduce Ca2+ mobilizing signals when expressed on differentiated B lineage cells

We have measured Ca2+ mobilization in a panel of B lineage cell lines after stimulation with anti-Ig to assess whether membrane Ig transduces a functional signal in cells that are representative of immature, mature, or terminally differentiated stages. For these studies, three transfected cell lines which express the same IgM molecule (300-19 microns lambda 36/8, K46-17 microns lambda, and J558L microns lambda 3) as well as two lines expressing an identical IgD molecule (K46 delta m2.6 and J558L delta m8.8) were used. Cross-linking of membrane Ig on IgM+ or IgD+ lymphomas (K46-17 microns lambda or K46 delta m2.6) resulted in a Ca2+ mobilization response that is similar to that seen in mature, resting B cells. Both intracellular release and extracellular influx of Ca2+ were observed. In contrast, ligation of membrane Ig on an IgM+ pre-B cell line (300 - 19 microns lambda 36/8) induced extracellular influx of Ca2+ but no detectable intracellular release. Finally, cross-linking of membrane Ig on IgM+ or IgD+ plasmacytomas (J558L microns 3 or J558L delta m8.8) or an IgD+ B cell hybridoma (B1.8.delta 1) expressing an endogenous Ig gene, did not result in a detectable Ca2+ mobilization response. Importantly, stimulation of cells with the GTP-binding protein activator, aluminum fluoride, resulted in a comparable Ca2+ mobilization response in all cell lines. In view of the fact that aluminum fluoride induced a Ca2+ response in the terminally differentiated B cell lines, J558L microns 3, J558L delta m8.8, and B1.8.delta 1, it is likely that there is an alteration in the signal transduction cascade at some point proximal to GTP binding protein activation. This finding suggests that differentiation of the B cell is accompanied by the loss or alteration of one or more components that couple membrane Ig to subsequent signal transduction elements. Finally, it has previously been demonstrated that the IgM+ cell lines described above, express the recently described membrane Ig-associated protein, B34. Thus, it is apparent based on the fact that the J558L microns 3 cell line does not mobilize Ca2+ after stimulation with anti-Ig, that coexpression of B34 in association with membrane Ig does not constitute a functional receptor complex capable of activating GTP-binding proteins that in turn regulate Ca2+ mobilization.