Presence Of Calcium Channel Blockers Research Articles

Do calcium channel blockers applied to cardiomyocytes cause increased channel expression resulting in reduced efficacy?

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug’s efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug’s inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug’s overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

A computational framework for pharmaco‐mechanical interactions in arterial walls using parallel monolithic domain decomposition methods

AbstractA computational framework is presented to numerically simulate the effects of antihypertensive drugs, in particular calcium channel blockers, on the mechanical response of arterial walls. A stretch‐dependent smooth muscle model by Uhlmann and Balzani is modified to describe the interaction of pharmacological drugs and the inhibition of smooth muscle activation. The coupled deformation‐diffusion problem is then solved using the finite element software FEDDLib and overlapping Schwarz preconditioners from the Trilinos package FROSch. These preconditioners include highly scalable parallel GDSW (generalized Dryja–Smith–Widlund) and RGDSW (reduced GDSW) preconditioners. Simulation results show the expected increase in the lumen diameter of an idealized artery due to the drug‐induced reduction of smooth muscle contraction, as well as a decrease in the rate of arterial contraction in the presence of calcium channel blockers. Strong and weak parallel scalability of the resulting computational implementation are also analyzed.

Stimulation of Erythrocyte Membrane Blebbing by Bifenthrin Induced Oxidative Stress.

BackgroundBifenthrin is an insecticide and anti-estrogenic compound primarily used to control residential pests by depolarizing sodium gated voltage channels in the nervous system. Eryptosis, the suicidal death of erythrocytes, featured by PS exposure, membrane blebbing and cell shrinkage. Anemia is an outcome of uncontrolled eryptosis.Research DesignIn this study, erythrocytes were treated with different concentrations (.5-1-1.5 μM) of bifenthrin over a period of 48 hours. In order to investigate the oxidative stress induced by bifenthrin, catalase, superoxide dismutase, and glutathione peroxidase activities were investigated.ResultsObtained data indicated the decrease in the enzymes (superoxide dismutase, glutathione peroxidase, and catalase) activities in bifenthrin treated cells at 1 μM concentration. In addition, measurement of cell size and confirmation of the role of calcium in the stimulation of the eryptotic activity of bifenthrin were performed. A significant increase in mean cell volume was found in the presence of bifenthrin and a decrease in mean cell volume in the presence of calcium channel blocker was observed. Similarly, there was also a significant increase in the percentage of hemolysis indicating the necrotic activity of bifenthrin.ConclusionsIt is concluded that the indicated doses of bifenthrin triggered oxidative stress which may lead to early cell death by eryptosis and hemolysis.

366 Role of calcium ions released by a calcium alginate dressing on NK cell activation

Wound healing is a physiological process involving 4 major stages, among which an inflammatory phase to eradicate causal aggression. The wound bed is infiltrated by inflammatory cells from the innate response, including natural killer (NK) cells detected within the first 24 hours. Calcium alginate dressings including Algosteril® (Algo) are widely used in wound management. They release calcium ions in the exudates and enrich its level locally. Calcium ion is a key factor in this process and a well-known activator of NK cells. Our aim was to study the effects of Algo on the activation of the innate response through the analysis of the cytotoxic capacities and the activation of NK cells. NK cells from healthy donors’ blood were incubated with conditioned media of the Algo dressing (ISO 10-993 standard) in vitro for 18h. A control 3 mM CaCl2 solution was used as a supply of exogenous calcium. The cytotoxicity and activation of NK cells were assessed by flow cytometry using respectively the death rate of target K562 cells, the expression levels of the degranulation marker CD107a and of intracytoplasmic cytokines (ex: IFN-γ). The mode of action of Algo-released calcium on NK cells and the calcium channels involved were evaluated using a calcium probe in the presence of calcium channel blockers. Algo dressing significantly stimulated the cytotoxicity of NK cells against K562 cells (x1.3 at 10 NK/1 K562 ratio). It also induced a significant increase in the percentage of cytotoxic CD56dim/CD107a+ NK cells and in the expression of IFN-γ in CD56bright NKs. Incubation with EDTA (calcium chelator) inhibited the stimulatory effect of Algo on NK cytotoxicity by 69% at the 10/1 ratio. SOCE calcium channels are the major one involved in NK stimulation by the dressing-released calcium ions. Altogether, these results demonstrate the major role of calcium in innate response during wound healing and the interest in using the calcium alginate dressing Algo to control infection.

Effect of 3-substituted 1,4-benzodiazepin-2-ones on maximal normalized rate of bradykinin-induced smooth muscle contraction in the presence of calcium channel blockers

The development of modern organic chemistry and molecular modeling technologies simplify the search for potential inhibitors of various receptor systems and biological processes. The one of the directions is the development of analgesics of broad spectrum and low toxicity. It is important to search for inhibitors of the kinin-kallikrein system that regulates many functions: inflammation, pain, carcinogenesis, vascular tone, smooth muscle contraction and other. Derivatives of 3-substituted 1,4-benzodiazepine-2-ones have a unique spatial conformation that allows one to simulate β-structures of bioactive peptides. The functional activity of compounds is determined by properties of their peripheral chemical radicals. We analyzed the effect of 3-substituted 1,4-benzodiazepin-2-ones derivatives on the normalized maximal rate of bradykinin-induced smooth muscle contraction and relaxation of the stomach in the presence of calcium channel blockers: verapamil (1 μM), gadolinium (300 μM) and 2-aminoethyl diphenylborinate (0.1 μM). The levels of bradykinin and 3-arylamino-1,2-dihydro-3H-1,4-benzodiazepine-2-ones in incubation solution were 10–6 M. Data processing on dynamics of contraction was performed according to the method of Burdyha and Kosterin. Compounds MX-1775 and MX-1925 reduced maximal normalized rate (Vn) of bradykinin-induced smooth muscle contraction in the presence of Gd3+ by 21.2% and 31.0% respectively. Compound MX-1925 increased Vn of relaxation by 11.6%. A similar effect is typical for MX-2011, where there is an increase by 34.6%. In the presence of verapamil this compound additionally decreased Vn contraction by 20.5%. Substances MX-1775, MX-2004 and MX-1925 restored maximal normalized rate of relaxation to original values of bradykinin-induced contraction. In the presence of 2-aminoethyldiphenylborinate MX-1775 additionally reduced Vn of contractions by 7.5%. 3-substituted 1,4-benzo­diazepine-2-ones did not change the maximal normalized rate of contraction and relaxation of carbachol- and potential-induced smooth muscle contraction. Based on the results and previous investigations, the MX-1775 is a potential blocker of kinin B2-receptors. Effects obtained for other compounds require additional research.

Ultrasound Enhanced PEI-Mediated Gene Delivery Through Increasing the Intracellular Calcium Level and PKC-δ Protein Expression

Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. It has also been shown that combining ultrasound (US) with PEI could enhance and prolong in vitro and in vivo transgene expression. However, the role US in the enhancement of PEI uptake is poorly understood. This study investigates the impact of US on PEI-mediated gene transfection. Specific endocytosis pathway siRNA, including clathrin HC siRNA, caveolin-1 siRNA and protein kinase C-delta (PKC-δ) siRNA, are used to block the corresponding endocytosis pathways prior to the transfection of luciferase DNA/PEI polyplexes to cultured cells by 1-MHz pulsed US with ultrasound contrast agent SonoVue®. Transgene expression was found not to be enhanced by US treatment in the presence of the PKC-δ siRNA. We further demonstrated that PKC-δ protein could be enhanced at 6h after US exposure. Moreover, intracellular calcium levels were found to be significantly increased at 3h after US exposure, while transgene expressions were significantly reduced in the presence of calcium channel blockers both in vitro and in vivo. Our results suggest that US enhanced PEI-mediated gene transfection specifically by increasing PKC-δ related fluid phase endocytosis, which was induced by increasing the intracellular calcium levels.

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.

Effect of Amlodipine in Comparison to Nifedipine on Vascular Perfusion Pressure of Isolated Rat Kidney

Objective(s) This study aimed to investigate and to compare the effects of nifedipine and amlodipine, dihydropyridine (DHP) calcium channel blockers (CCBs) on perfusion pressure of isolated perfused rat kidney. Materials and Methods Following the establishment of renal perfusion with a constant baseline pressure of 85-95 mmHg, the renal vasculature was constricted by phenylephrine (PE) injection. Changes in the baseline perfusion pressure were recorded. Then nifedipine and amlodipine prepared in perfusion medium was fed to the kidney for 30 min. Finally alterations in the baseline pressure arising from PE administrations in the presence of CCBs were recorded and data analyses were done. Results PE-induced increases in perfusion pressure attenuated significantly in the presence of 5 and 10 µM of nifedipine and 1, 5, and 10 µM of amlodipine. Increases in perfusion pressure arising from PE (100 and 200 µM) in the presence of amlodipine (1, 5, and 10 µM) was significantly less than that in the presence of nifedipine (1, 5, and 10 µM). Calculated EC50 value of amlodipine for inhibition was significantly lower than that of nifedipine. Based on the EC50 values, the potency of amlodipine in inhibiting PE-induced responses is significantly higher compared to nifedipine. Conclusion

Calcium channel blocking activities of Withania coagulans

The effects of crude methanolic extract of Withania coagulans (Wc.cr) were studied for possible calcium channel blocking activities in rabbit’s jejunum preparations at different doses. Potassium chloride (KCl) induced contractions were also inhibited by the Wc.cr extract suggesting calcium channel blocking activity. Calcium channel blocking activity was confirmed by the right shift in the dose response curves of the tissues in calcium free tyrode’s solution. The dose response curves of the Wc.cr resemble the curves produced by verapamil, a standard calcium channel blocker. The results confirm the presence of calcium channel blocker(s) in the extract and the plant species could be a potential target for activity-guided isolation of the active moieties. Key words: Withania coagulans, spasmolytic, verapamil, calcium channel-blocking activity.

Adrenalectomy potentiates the anti-inflammatory activity of a calcium channel blocker

Calcium movement is important in the activation of inflammatory cells. Prior studies have shown that calcium channel blockers (CCBs) inhibit carrageenan-induced paw edema in rats by acting at pituitary and hypothalamic levels. Here we evaluated the role of calcium channel blockers and their regulation via the hypothalamus-pituitary-adrenal (HPA) axis in acute and chronic models of rat paw inflammation, using both carrageenan (acute) and formalin (chronic) as inflammation inducers. Adrenalectomized (ADX) and intact Sprague Dawley rats (n = 4 per group) weighing 150-250 each were treated with an intraperitoneal injection of nifedipine (400 microg/kg) or vehicle. Edema was assessed plethysmometrically by evaluating paw volume changes. The results show a significant contradiction between acute and chronic inflammation data. In intact animals, nifedipine demonstrated a significant anti-inflammatory effect in the acute inflammatory model but not in the chronic model. In adrenal-ectomized animals, the anti-inflammatory effect of nifedipine was significantly enhanced both in acute and chronic models. We conclude that adrenalectomy plays a significant role in modulating the inflammatory pathway in the presence of calcium channel blockers.

Fast pollen tube growth in Conospermum species.

An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.

Paracrine Intercellular Communication by a Ca 2+- and SNARE-Independent Release of GABA and Glutamate Prior to Synapse Formation

GABA and glutamate receptors are expressed in immature “silent” CA1 pyramidal neurons prior to synapse formation, but their function is unknown. We now report the presence of tonic, spontaneous, and evoked currents in embryonic and neonatal CA1 neurons mediated primarily by the activation of GABA A receptors. These currents are mediated by a nonconventional release of transmitters, as they persist in the presence of calcium channel blockers or botulinium toxin and are observed in Munc18-1-deficient mice in which vesicular release is abolished. This paracrine communication is modulated by glutamate but not GABA transporters, which do not operate during this period of life. Thus, a Ca 2+- and SNARE-independent release of transmitters underlies a paracrine mode of communication before synapse formation.

Immune Mechanisms in Pain Control

Immune Mechanisms in Pain Control

Intracellular free calcium in the neutrophils of maintenance haemodialysis patients.

Chronic renal failure has on occasion been referred to as a state of calcium toxicity. The aim of this study was to investigate the status of intracellular free Ca2+ in the neutrophils of chronic renal failure patients on maintenance haemodialysis treatment. Factors previously suggested to influence intracellular free Ca2+ were investigated including PTH levels, oxidative stress and recombinant human erythropoietin administration. The study involved 14 chronic renal failure patients on the haemodialysis programme of the Pretoria Academic hospital. Intracellular free Ca2+ and transmembrane Ca2+ fluxes were investigated by fluorescence spectrophotometry. Increases above control values were found in intracellular free Ca2+ (P-value 0.0242) and in the transmembrane Ca2+ flux upon fMLP stimulation (P-value 0.0002). The results showed significant differences in intracellular free Ca2+ between patients on rHuEPO and patients not on rHuEPO. The apparently rHuEPO-induced increase in intracellular free Ca2+ persisted in the presence of calcium channel blockers. No overt indications of oxidative stress could be detected by the antioxidant vitamin levels. It is concluded that factors other than those associated with uraemia, such as rHuEPO administration, might contribute to the often reported increase in intracellular free Ca2+ in these patients. Further studies to investigate the relationship between intracellular free Ca2+, rHuEPO and calcium channel blockers are suggested.

Lipid Metabolism and Cardiovascular Morbidity and Mortality in Hemodialysis Patients: Role of Factors Modulating Cytosolic Calcium.

Animal studies indicate that insulin resistance and glucose intolerance leading to dyslipidemia in uremic rats are associated with increased cytosolic calcium ([Ca++ i]). The resistance and intolerance are reversed with verapamil, but recur after its discontinuation. This finding suggests that hyperparathyroid-induced [Ca ++ i] increase is responsible for the metabolic derangement. We retrospectively examined, over a 12-year period, the effects of factors that lower [Ca ++ i] on total serum cholesterol and triglycerides in 332 hemodialysis (HD) patients. Because the study was retrospective, detailed lipid profiles were not available. We therefore relied on morbidity and mortality outcomes related to atherosclerotic vascular disease. Patients with diabetes mellitus were excluded, because their dyslipidemia and vascular disease are mediated via a different mechanism. Four groups emerged: group I [high parathormone (PTH) in the absence of calcium channel blockers (CCBs), n = 107], representing the highest [Ca++ i]; group II (high PTH in the presence of CCBs, n = 76) and group III (lower PTH in the absence of CCBs, n = 66), representing intermediate [Ca ++ i]; and group IV (lower PTH in the presence of CCBs, n = 83) representing the lowest [Ca ++ i]. The theoretically lower [Ca ++ i] was achieved via CCB therapy or lower PTH, or both. The mean serum cholesterol in group I was 322 ± 24 mg/dL and the level of triglycerides was 398 ± 34 mg/dL. Group II had mean serum cholesterol of 196 ± 16 mg/dL and triglycerides of 157 ± 17 mg/dL. Group III had a mean serum cholesterol of 202 ± 19 mg/dL and triglycerides of 160 ± 15 mg/dL. Group IV had a mean serum cholesterol of 183 ± 9 mg/dL and triglycerides of 94 ± 6 mg/dL. The differences in cholesterol and triglyceride levels among four groups were significant (p < 0.001) by one-way analysis of variance (ANOVA). The incidence of cardiovascular morbidity and mortality events was 61% in group I, 24% in group II, 28% in group III, and 18% in group IV (χ 2 = 47.7, p < 0.001). We conclude that, in non diabetic HD patients, hyperparathyroidism, especially in the absence of CCBs, is associated with severe dyslipidemia and increased risk of cardiovascular morbidity and mortality. Dyslipidemia may be related to a hyperparathyroid-induced increase in cytosolic calcium [Ca++ i]. Lowering [Ca++ i] by decreasing PTH or by blocking calcium entry into cells (via CCBs), or both, is associated with less dyslipidemia and improved long-term cardiovascular morbidity and mortality. Prospective randomized studies, with actual measurement of [Ca ++ i], are needed to verify the results of this study.

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin.

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.

Characterization of the antiseptic-resistance gene qacEΔ 1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacEΔ 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacEΔ 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacEΔ 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacEΔ1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacEΔ 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.

Characterization of the antiseptic-resistance gene qacE delta 1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1.

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.

Differential membrane interactions of calcium channel blockers. Implications for antioxidant activity.

Lipid peroxidation causes cellular damage during aging and various diseases, including atherosclerosis. Chronic administration of highly lipophilic calcium channel blockers (CCB) may reduce lipid peroxidation as a result of concentration in cell membranes and altering physico-chemical properties of the lipid bilayer. In the study, small angle X-ray scattering was used to examine reconstituted cardiac membrane lipid bilayers in the presence of CCB with various antioxidant activities, including nisoldipine, nifedipine, and diltiazem. Analysis of one-dimensional electron density profiles demonstrated that these compounds have different molecular distributions relative to the center of the membrane: diltiazem (+/- 14-22 A), nifedipine (+/- 12-22 A), and nisoldipine (+/- 7-22 A). The overall hydrocarbon core width for control samples was 44 A and was unaffected by the addition of drugs at these concentrations (< 1% by mass). High resolution differential scanning calorimetry indicated that CCB markedly perturbed the thermotropic properties of liposomes, including thermal phase transition temperature and enthalpy, relative to control samples. The effects of these compounds on membrane thermotropic properties correlate with their reported antioxidant activities. These data support the hypothesis that calcium channel blockers have potent physico-chemical interactions with the membrane lipid bilayer, which may underlie their antioxidant activity.

The role of calcium influx in cellular proliferation induced by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. It was previously shown that the B subunit had no effect on cAMP, protein kinase C or phosphoinositide turnover, but did cause an increase in the influx of calcium from extracellular sources (Spiegel, S. and Panagiotopoulos, C. (]988) Exp. Cell Res. [77, 414–427). In contrast to the action of known growth factors, the B subunit induced significant DNA synthesis after only a l–3 h treatment. We utilized this unique property to determine whether the increase in calcium influx plays a role in B subunit-induced mitogenicity. Cells were briefly treated with the B subunit in the presence of calcium channel blockers, followed by removal of the blockers and further incubation in B subunit-free medium for the remaining time required to measure DNA synthesis. When 1 mM cobalt was only present during the first 3 h incubation, DNA synthesis induced by either the B subunit or fetal bovine serum was completely abolished. However, both nickel (1 mM) and the L-type voltage-gated calcium channel inhibitor nicardipin (10 μM) inhibited B subunit-induced cell proliferation without abrogating the response to fetal bovine serum. Using a gel retardation assay, we found that the B subunit markedly stimulated specific DNA-binding activity of the transcription factor, activator protein- 1 (AP-1), which functions as a major convergence point coupling early events induced by a variety of mitogens to long term growth responses. Presence of c-Fos protein in the AP-1 complex was demonstrated as a supershift band in the gel mobility assay using c-Fos polyclonal antibody. Cobalt, which markedly inhibited B subunit-induced DNA synthesis, also completely abolished AP-1 DNA-binding activity stimulated by the B subunit. In sharp contrast, cobalt had no effect on DNA-binding activity of AP-1 induced by the tumor promoter, 12- O-tetradecanoylphorbol 13-acetate. Our results suggest that calcium influx is a key element for both DNA-binding activity of AP-1 and cell proliferation induced by binding of the B subunit of cholera toxin to cell surface ganglioside GM1.