Effects of Ca-Ionophore A23187 and Calmodulin Antagonists on Regulatory Mechanisms of Glycolysis and Cell Viability of NIH-3T3 Fibroblasts

We studied here, in NIH-3T3 fibroblasts, the effect of the Ca²⁺-ionophore A23187 (which is known to increase intracellular-free Ca²⁺) on the control of glycolysis and cell viability and the action of calmodulin antagonists. Time-response studies with Ca²⁺-ionophore A23187 have revealed dual effects on the distribution of phosphofructokinase (PFK) (EC 2.7.1.11), the rate-limiting enzyme of glycolysis, between the cytoskeletal and cytosolic (soluble) fractions of the cell. A short incubation (maximal effect after 7 min) caused an increase in cytoskeleton-bound PFK with a corresponding decrease in soluble activity. This leads to an enhancement of cytoskeletal glycolysis. A longer incubation with Ca²⁺-ionophore caused a reduction in both cytoskeletal and cytosolic PFK and cell death. Both the “physiological” and “pathological” phases of the Ca²⁺-induced changes in the distribution of PFK were prevented by treatment with three structurally different calmodulin antagonists, thioridazine, an antipsychotic phenothiazine, clotrimazole, from the group of antifungal azole derivatives that were recently recognized as calmodulin antagonists, and CGS 9343B, a more selective inhibitor of calmodulin activity. The longer incubation with Ca²⁺-ionophore also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two allosteric stimulatory signal molecules of glycolysis. All these pathological changes preceded the reduction in cell viability, and a strong correlation was found between the fall in ATP and cell death. All three calmodulin antagonists prevented the pathological reduction in the levels of the allosteric effectors, ATP and cell viability. These experiments may throw light on the mechanisms underlying the therapeutic action of calmodulin antagonists that we previously found in treatment of the proliferating melanoma cells, on the one hand, and skin injuries, on the other hand.     

Local anesthetics induce a decrease in the levels of glucose 1, 6-bisphosphate, fructose 1,6-bisphosphate, and ATP, and in the viability of melanoma cells

Glycolysis is known to be the primary energy source in cancer cells. We investigated here the effect of local anesthetics, lidocaine and bupivacaine, on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP levels and cell viability in B16 melanoma cells. We found that both drugs induced a significant, dose-dependent reduction in the levels of glucose 1,6-bisphosphate, fructose 1, 6-bisphosphate, ATP, and cell viability. Bupivacaine was more potent than lidocaine. The decrease in glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, induced by the local anesthetics, preceded the reduction in the viability of melanoma cells, indicating that these are early changes and not a result of cell death. Cell viability was reduced in a close correlation with the fall in ATP. These findings suggest that the fall in the levels of the two signal allosteric regulators of glycolysis, induced by the local anesthetics, is one of the mechanisms that causes a reduction in glycolysis and ATP levels, which eventually leads to melanoma cell death. These experiments suggest that local anesthetics, and especially bupivacaine, are most promising agents in the treatment of melanoma.     

Modulation of 6-phosphofructo-1-kinase oligomeric equilibrium by calmodulin: formation of active dimers

Muscle 6-phospho-1-kinase (PFK) is the key regulatory enzyme of the glycolytic pathway and is a calmodulin-binding protein binding two calmodulin molecules per PFK protomer. This enzyme is characterized by a complex regulation that involves its allosteric behavior modulated by several ligands, which modulate the equilibrium between the active tetramers and the inactive dimers of the enzyme. Calmodulin is described to induce the dimerization of PFK, so inhibiting its catalytic activity. Here, we show that binding of calmodulin specifically to its higher-affinity site of PFK induce its dimerization without compromising enzyme catalytic activity forming a hitherto not described active dimmer of PFK. It is also shown that the dimerization is a Ca2+ -dependent event that responds to physiological intracellular Ca2+ concentrations and decrease the interaction of the enzyme to membrane site, which stimulate its catalytic activity. We propose that the effects of calmodulin on PFK reported here are of great physiological significance due to the response to physiological concentrations of Ca2+ and due to be in accordance to the known effects of calmodulin on cell ATP production. We also propose that calmodulin might affect the interaction of PFK to other cellular components as the cytoskeleton.     

Detachment of glycolytic enzymes from cytoskeleton of Lewis lung carcinoma and colon adenocarcinoma cells induced by clotrimazole and its correlation to cell viability and morphology

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. We report here that clotrimazole (l-(alpha-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, induced a dose-dependent detachment of the glycolytic enzymes, phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and aldolase (D-fructose-l,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), from cytoskeleton of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of the cytoskeleton membrane, and would also affect cytoskeleton structure and cell shape. We show here that clotrimazole decreased the viability of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. After 3h of incubation with clotrimazole, complete cell destruction was detected. Ultrastructural cell damage was manifested by disintegration of the outer membrane by scanning electron microscopy (SEM). The detachment of glycolytic enzymes from cytoskeleton, induced by clotrimazole, preceded the decrease in cell viability, which indicates that this is an early effect and not a result of cell death. Since the cytoskeleton is being recognized as an important modulator of cell function, proliferation, differentiation, and neoplasia, detachment of the glycolytic enzymes from cytoskeleton induced by clotrimazole, as well as its reported inhibitory action on cell proliferation, makes this drug the most promising agent in the treatment of cancer.     

Inhibitory effect of water-soluble chitosan on TNF-alpha and IL-8 secretion from HMC-1 cells

Mast cells are known to play an active role as effector cells in allergic inflammation and in diverse immunological and pathological processes. Activated mast cell-derived pro-inflammatory cytokines are important pathologic factors of progression of allergic inflammation. In this study, we investigated whether pro-inflammatory cytokines (TNF-alpha and IL-8) can be induced by calcium stimulation in HMC-1 cells, and high molecular weight water-soluble chitosan (WSC) can inhibit the production of these cytokines. We provided evidence that the secretion of TNF-alpha and IL-8 from HMC-1 cells was induced by Ca2+-ionophore A23187 or Ca2+-ATPase inhibitor TSG. Treatment of WSC (10 microg/ml) prior to stimulation with calcium agonists significantly blocked the secretion of TNF-alpha by 65.1% for A23187 and 87.7% for TSG. IL-8 secretion in response to A23187 or TSG was inhibited by 49.2% for A23187 and 34.1% for TSG, respectively, compared to absence of WSC. These results suggest that WSC has potential regulatory effects on allergic inflammatory diseases by down-modulating Ca2+-induced mast cell activation.     

Role of Ca2+ and Mg2+ for endothelial permeability of water and albumin in vitro

The permeability of endothelial monolayers grown on a polycarbonate filter membrane and continuously exposed to a hydrostatic pressure of 10 cm H2O was studied. The ionophores A23187 and ionomycin dose dependently (0.1-10 microM) enhanced the hydraulic conductivity of sealed endothelial cell monolayers 10 to 15-fold, at the same time the reflection coefficient of albumin dropped from 0.75 to 0.2. The effects of A23187 were dependent on extracellular Ca2+. In the absence of Ca2+, however, Mg2+ could substitute for Ca2+. Phase contrast- and scanning electron-microscopy showed that the A23187-induced effects were accompanied by gap-formation in the intercellular clefts. These gaps probably are the sites which allowed enhanced fluid exchange. Ionophore-induced effects on permeability could be modified by antagonists of calmodulin function and of arachidonate liberation and metabolism. The data suggest that alterations of endothelial Ca2+-homeostasis induce metabolic events which result in an increased permeability of an endothelial monolayer.     

Effects of fructose-1,6-bisphosphate on morphological and functional neuronal integrity in rat hippocampal slices during energy deprivation

D-fructose-1,6-bisphosphate, a high energy glycolytic intermediate, attenuates ischemic damage in a variety of tissues, including brain. To determine whether D-fructose-1,6-bisphosphate serves as an alternate energy substrate in the CNS, rat hippocampal slices were treated with D-fructose-1,6-bisphosphate during glucose deprivation. Unlike pyruvate, an endproduct of glycolysis, 10 mM D-fructose-1,6-bisphosphate did not preserve synaptic transmission or morphological integrity of CA1 pyramidal neurons during glucose deprivation. Moreover, during glucose deprivation, 10-mM D-fructose-1,6-bisphosphate failed to maintain adenosine triphosphate levels in slices. D-fructose-1,6-bisphosphate, however, attenuated acute neuronal degeneration produced by 200 microM iodoacetate, an inhibitor of glycolysis downstream of D-fructose-1,6-bisphosphate. Because (5S, 10R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, an antagonist of N-methyl-D-aspartate receptors, exhibited similar protection against iodoacetate damage, we examined whether (5S, 10R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine and D-fructose-1,6-bisphosphate share a common neuroprotective mechanism. Indeed, D-fructose-1,6-bisphosphate diminished N-methyl-D-aspartate receptor-mediated synaptic responses and partially attenuated neuronal degeneration induced by 100-microM N-methyl-D-aspartate.Taken together, these results indicate that D-fructose-1,6-bisphosphate is unlikely to serve as an energy substrate in the hippocampus, and that neuroprotective effects of D-fructose-1,6-bisphosphate are mediated by mechanisms other than anaerobic energy supply.     

New insights into a mutant of Saccharomyces cerevisiae having impaired sugar uptake and metabolism

Summary A regulatory mutant of Saccharomyces carlsbergensis unable to inactivate fructose-1,6-bisphosphatase was shown to have a normal response to the glucose signal as measured by trehalase and 6-phosphofructose-2-kinase activities. The level of fructose 2,6-bisphosphate, however, was found to be 4- to 5-fold lower than that found in the wild-type strain. A rapid and drastic depletion in ATP was confirmed. A partial revertant for growth on glucose which retained its inability to grow on fructose did not show normal levels of fructose 2,6-bisphosphate; however, ATP levels were restored. Trehalose-6-phosphate synthase activity was found in its phosphorylated, less active form. A high degree of phosphorylation at the level of enzymatic activity and of the sugar phosphorylating systems might be responsible for the impairment of control between hexose transport and metabolism, as well as for the absence of trehalose accumulation.Abbreviations F2,6P₂ fructose 2,6-bisphosphate - PFK1 6-phosphofructose-l-kinase - FBPasel fructose-1,6-bisphosphatase - PFK2 6-phosphofructose-2-kinase - PEP phosphoenolpyruvate     

Calmodulin antagonists suppress gap junction coupling in isolated Hensen cells of the guinea pig cochlea

The effect of calmodulin (CaM) antagonists W7, trifluoperazine (TFP) and a calmodulin inhibitory peptide on gap junction coupling in isolated Hensen cells of the organ of Corti was analysed by the double whole-cell patch-clamp technique. Addition of the conventional antagonists W7 and TFP in the micromolar range caused a rapid decrease of gap junction conductance after a delay of a few minutes in a dose-dependent manner. Fluorescence spectroscopy of cytoplasmic free calcium concentration ([Ca(2+)](i)) by Fura-2 showed no significant change of [Ca(2+)](i) by W7. Chelation of [Ca(2+)](i) by 10 mM BAPTA or use of nominally Ca(2+)-free external bath did not suppress the W7-induced gap junction uncoupling. The results suggest that W7 and TFP induce gap junction uncoupling at unchanged global [Ca(2+)](i) in Hensen cells. To obtain additional evidence for an involvement of CaM in regulating gap junction conductance a calmodulin inhibitory peptide, the MLCK peptide (250 nM), was added to the standard pipette solution. Again gap junction uncoupling was observed, but on a significantly slower time scale. This is the first study of an effect of calmodulin antagonists on gap junction coupling in isolated Hensen cells. The question whether the effect of calmodulin inhibitors is specific and involves CaM-dependent gating of gap junction coupling in Hensen cells is discussed.     

The Ca2+/calmodulin signaling system in the neural response to excitability. Involvement of neuronal and glial cells.

Ca2+ plays a critical role in the normal function of the central nervous system. However, it can also be involved in the development of different neuropathological and neurotoxicological processes. The processing of a Ca2+ signal requires its union with specific intracellular proteins. Calmodulin is a major Ca(2+)-binding protein in the brain, where it modulates numerous Ca(2+)-dependent enzymes and participates in relevant cellular functions. Among the different calmodulin-binding proteins, the Ca2+/calmodulin-dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. We present an overview on different works aimed at the study of the Ca2+/calmodulin signalling system in the neural response to convulsant agents. Ca2+ and calmodulin antagonists inhibit the seizures induced by different convulsant agents, showing that the Ca2+/calmodulin signalling system plays a role in the development of the seizures induced by these agents. Processes occurring in association with seizures, such as activation of c-fos, are not always sensitive to calmodulin, but depend on the convulsant agent considered. We characterized the pattern of expression of the three calmodulin genes in the brain of control mice and detected alterations in specific areas after inducing seizures. The results obtained are in favour of a differential regulation of these genes. We also observed alterations in the expression of the Ca2+/calmodulin-dependent protein kinase II and calcineurin after inducing seizures. In addition, we found that reactive microglial cells increase the expression of calmodulin and Ca2+/calmodulin-dependent protein kinase II in the brain after seizures.     

Inhibition of histamine release from human basophils in vitro by calmodulin antagonists

Calmodulin is a ubiquitous and versatile Ca2+-binding protein that plays a pivoting role in cellular metabolism. We have investigated the possibility that calmodulin plays a role in immediate hypersensitivity reactions by evaluating the effects of two agents, trifluoperazine dihydrochloride (TFP) and the sulfonamide derivative N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) which selectively bind to calmodulin. TFP and W-7 cause a dose-dependent inhibition of histamine secretion from human basophils in vitro induced by several immunological (i.e., antigen and anti-IgE) and nonimmunological (i.e., formyl-methionine-containing peptide and the Ca2+ ionophore A23187) stimuli. These results indicating that two specific calmodulin antagonists are potent inhibitors of the secretory response of human basophils support the hypothesis that calmodulin may play a role in the control of the release of preformed mediators from human inflammatory cells.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Neuronal vulnerability of CLN3 deletion to calcium-induced cytotoxicity is mediated by calsenilin

Calsenilin/DREAM/KChIP3, a neuronal Ca(2+)-binding protein, has multifunctions in nucleus and cytosol. Here, we identified CLN3 as a calsenilin-binding partner whose mutation or deletion is observed in Batten disease. In vitro binding and immunoprecipitation assays show that calsenilin interacts with the C-terminal region of CLN3 and the increase of Ca(2+) concentration in vitro and in cells causes significant dissociation of calsenilin from CLN3. Ectopic expression of CLN3 or its deletion mutant containing only the C-terminus (153-438) and capable of binding to calsenilin suppresses thapsigargin or A23187-induced death of neuronal cells. In contrast, CLN3 deletion mutant containing the N-terminus (1-153) or (1-263), which is frequently found in Batten disease, induces the perturbation of Ca(2+) transient and fails to inhibit the cell death. In addition, the expression of calsenilin is increased in the brain tissues of CLN3 knock-out mice and SH-SY5Y/CLN3 knock-down cells. Down-regulation of CLN3 expression sensitizes SH-SY5Y cells to thapsigargin or A23187. However, additional decrease of calsenilin expression rescues the sensitivity of SH-SY5Y/CLN3 knock-down cells to Ca(2+)-mediated cell death. These results suggest that the vulnerability of CLN3 knock-out or CLN3 deletion (1-153)-expressing neuronal cells to Ca(2+)-induced cell death may be mediated by calsenilin.     

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.     

Effects of mucoid and non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients on inflammatory mediator release from human polymorphonuclear granulocytes and rat mast cells.

Mucoid Pseudomonas aeruginosa causing chronic bronchopulmonary infection in cystic fibrosis (CF) patients may interfere with host defence mechanisms. We investigated 13 P. aeruginosa strains isolated from sputa of CF patients with regard to the induction or modulation of inflammatory mediator release from human neutrophils (PMN) and rat mast cells. The effects of mucoid as compared to non-mucoid bacteria were studied using a mucoid strain and its non-mucoid revertant. The release of leukotrienes (LT) and histamine in response to the majority of the CF strains was insignificant. However, preincubation of PMN with P. aeruginosa caused a dose-dependent decrease (50-95%) of LTB4 and LTC4 generation and LTB4 metabolism induced by the Ca(2+)-ionophore A23187 or opsonized zymosan (ZX) (P less than 0.001). The mucoid strains caused a three- to 10-fold higher impairment of LTB4 release (P less than 0.05) and a concomitant down-regulation of LTB4 receptors on neutrophils. Inhibitory effects were also obtained for mucoid and non-mucoid bacteria when the phorbol-ester or the Ca(2+)-ionophore induced luminol enhanced chemiluminescence response (P less than 0.001) or the histamine release from rat peritoneal mast cells (P less than 0.01) was studied. The bacteria-cell contact with non-mucoid strains was associated with an increased Ca2+ influx into PMN, whereas mucoid bacteria had no effect. In addition, a protein kinase C-dependent decrease of the C3bi receptor was suppressed by the mucoid--and less effectively--by the non-mucoid strain. The results suggest that the impairment of the phagocytic and inflammatory system may contribute to the pathogenesis and persistence of mucoid P. aeruginosa infection in CF.     

TRPM8 acute desensitization is mediated by calmodulin and requires PIP(2): distinction from tachyphylaxis

The cold-sensing channel transient receptor potential melastatin 8 (TRPM8) features Ca(2+)-dependent downregulation, a cellular process underlying somatosensory accommodation in cold environments. The Ca(2+)-dependent functional downregulation of TRPM8 is manifested with two distinctive phases, acute desensitization and tachyphylaxis. Here we show in rat dorsal root ganglion neurons that TRPM8 acute desensitization critically depends on phosphatidylinositol 4,5-bisphosphate (PIP(2)) availability rather than PIP(2) hydrolysis and is triggered by calmodulin activation. Tachyphylaxis, on the other hand, is mediated by phospholipase hydrolysis of PIP(2) and protein kinase C/phosphatase 1,2A. We further demonstrate that PIP(2) switches TRPM8 channel gating to a high-open probability state with short closed times. Ca(2+)-calmodulin reverses the effect of PIP(2), switching channel gating to a low-open probability state with long closed times. Thus, through gating modulation, Ca(2+)-calmodulin provides a mechanism to rapidly regulate TRPM8 functions in the somatosensory system.     

Effects of Ca2+on Erythrocyte Membrane Skeleton-Bound Phosphofructokinase,ATP Levels,and Hemolysis

Erythrocyte Ca²⁺overload is known to occur in several different disease states, and to affect the erythrocyte membrane deformability. We show here that an increase in intracellular Ca²⁺concentration in erythrocytes, induced by ionomycin, caused a reduction in ATP levels. Concomitant to the fall in ATP, a marked activation of phosphofructokinase (PFK) (EC 2.7.1.11), the rate-limiting enzyme in glycolysis, in the membrane skeleton fraction occurred. The increase in the membrane skeleton-bound PFK activity was most probably mediated by Ca²⁺, as direct addition of Ca²⁺to the membrane skeleton fraction from the erythrocyte induced an enhancement of the bound PFK activity. Time-response curves revealed that erythrocyte hemolysis did not occur during the first 30 min of incubation with ionomycin, when the membrane skeleton-bound PFK was activated. Longer incubation time resulted in solubilization of the membrane skeleton-bound PFK and a concomitant hemolysis of the erythrocytes. These results suggest that the Ca²⁺-induced activation of membrane skeleton-bound PFK, and thereby glycolysis, the sole source of energy in erythrocytes, may be a defense mechanism to surmount the damage induced by high Ca²⁺levels.     

Neurotoxicity of calcium ionophore A23187 in immature rat cerebellar slices

The causal role of Ca2+ in neuronal necrosis is controversial and it has been suggested that neuronal Ca2+ uptake is only a secondary effect to cell death. Here, I address this question directly by studying the morphological effects of calcium ionophore A23187 on immature cerebellar slices. Parasagittal slices were prepared and incubated for 30, 90 or 120 min in physiological saline with or without A23187. In some cases Ca2+ was omitted from the incubation medium. Slices were processed for light microscopy. A23187 produced nuclear changes indicative of cell death that encompassed cells of the external granule cell layer at short incubation times (30 min) and more deeply situated cells at longer times (120 min). This indicates that A23187 diffusion is limited in the slice. The histological changes produced by 30 min exposure to the ionophore could not be reversed by incubation for 90 min in normal medium. Necrosis was never observed when slices were exposed to A23187 in Ca2+-free medium. The results demonstrate that influx of excessive amounts of Ca2+ kills cells of the central nervous system.     

Encapsulation, in vitro characterization, and in vivo biocompatibility of Sertoli cells in alginate-based microcapsules

A method for microencapsulation of isolated neonatal porcine Sertoli cells is described. Using a conventional alginate-poli-L-ornithine encapsulation procedure, which has been used in our laboratory for almost two decades to envelop pancreatic islets, we observed significant loss of Sertoli cell viability, possibly due to excessive Ca(2+) ion exposure. Replacing calcium with barium, or shortening the incubation period in the presence of Ca ions, we obtained barium or calcium alginate gel microbeads that did not alter morphology and viability of the encapsulated Sertoli cells. The procedure might permit access to a novel approach to immunologically alter cell graft acceptance.     

The influence of tissue transglutaminase on the function of Fc receptors

In contrast to FcRII the soluble Fc receptor (FcRI) of human peripheral mononuclear blood cells (PMBC) is shed from PMBC following a 4-37 degrees C temperature shift and inhibits rosette formation of nonshed PMBC with antibody-coated erythrocytes (EA). Purified FcR, could be polymerized by tissue transglutaminase as was revealed by SDS-polyacrylamide gel electrophoresis. Comparing the Sephadex G-150 elution profile of the EA rosette inhibitory capacity of FcRI vs FcRI incubated in the presence of transglutaminase, the latter was found in a higher mol. wt region and could inhibit rosette formation by both FcRI and FcRII. Furthermore, the shedding of FcRI could be prevented by the addition of transglutaminase or Ca2+-ionophore A23187 (which leads to the activation of PMBC transglutaminase) to the cell suspension. The function of FcRII was not affected by either the addition of transglutaminase or Ca2+-ionophore to the cells. The results point to the involvement of transglutaminase in the determination of the functional state of the Fc receptor on the cell surface.