thapsigargin and maitotoxin

Liver cells possess store-operated Ca2+ channels (SOCs) with a high selectivity for Ca2+ compared with Na+, and several types of intracellular messenger-activated non-selective cation channels with a lower selectivity for Ca2+ (NSCCs). The main role of SOCs is thought to be in refilling depleted endoplasmic reticulum Ca2+ stores [Cell Calcium 7 (1986) 1]. NSCCs may be involved in refilling intracellular stores but are also thought to have other roles in regulating the cytoplasmic-free Ca2+ and Na+ concentrations. The ability of SOCs to refill the endoplasmic reticulum Ca2+ stores in hepatocytes has not previously been compared with that of NSCCs. The aim of the present studies was to compare the ability of SOCs and maitotoxin-activated NSCCs to refill the endoplasmic reticulum in rat hepatocytes. The experiments were performed using fura-2FF and fura-2 to monitor the free Ca2+ concentrations in the endoplasmic reticulum and cytoplasmic space, respectively, a Ca2+ add-back protocol, and 2-aminoethyl diphenylborate (2-APB) to inhibit Ca2+ inflow through SOCs. In cells treated with 2,5-di-t-butylhydroquinone (DBHQ) or vasopressin to deplete the endoplasmic reticulum Ca2+ stores, then washed to remove DBHQ or vasopressin, the addition of Ca2+ caused a substantial increase in the concentration of Ca2+ in the endoplasmic reticulum and cytoplasmic space due to the activation of SOCs. These increases were inhibited 80% by 2-APB, indicating that Ca2+ inflow is predominantly through SOCs. In the presence of 2-APB (to block SOCs), maitotoxin induced a substantial increase in [Ca2+](cyt), but only a modest and slower increase in [Ca2+](er). Under these conditions, Ca2+ inflow is predominantly through maitotoxin-activated NSCCs. It is concluded that SOCs are more effective than maitotoxin-activated NSCCs in refilling the endoplasmic reticulum Ca2+ stores. The previously developed concept of a specific role for SOCs in refilling the endoplasmic reticulum is consistent with the results reported here.

Topics: Animals; Boron Compounds; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Cytoplasm; Endoplasmic Reticulum; Epinephrine; Fura-2; Hepatocytes; Hydroquinones; Inositol 1,4,5-Trisphosphate; Ion Channels; Kinetics; Male; Marine Toxins; Microscopy, Fluorescence; Oxocins; Rats; Rats, Wistar; Thapsigargin; Vasopressins

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Antineoplastic Agents; Calcium; Calcium Channel Agonists; Calpain; Carbachol; Diazomethane; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Ionomycin; Ionophores; Marine Toxins; Muscarinic Agonists; Oligopeptides; Oxocins; Receptors, Retinoic Acid; Retinoid X Receptors; Ribavirin; Thapsigargin; Transcription Factors; Transglutaminases; Tumor Cells, Cultured

The marine toxin maitotoxin (MTX) induces stimulation of ciliary beating in primary cultures of rabbit tracheal epithelial cells. The response is time- and concentration-dependent. External calcium is an absolute requirement, although at a very low concentration (50 microM for maximal effect). Pretreatment of the cells with MTX induces an early (5 min) and sustained ( > or = 24 h) homologous desensitization. The response to MTX is strongly inhibited by trifluoperazin (an inhibitor of Ca-calmodulin-dependent enzymes) and by chelation of [Ca]i with BAPTA. However, the magnitude and kinetics of [Ca]i rise elicited by MTX do not correlate with those of the ciliary beat frequency (CBF) increase: the CBF increase is transient (with a peak at 5-10 min) while the [Ca]i rise is sustained; the CBF increase occurs at concentrations of MTX which are without an effect on [Ca]i; the CBF increase is not inhibited by 200 microM verapamil, genistein or okadaic acid, which inhibit the MTX-induced [Ca]i rise. The CBF increase is strongly inhibited by antagonists of arachidonic acid metabolism, mepacrine and nordiguaiaretic acid. However, MTX does not stimulate cAMP synthesis. These results suggest that calcium is not the only factor involved in the biological effects of MTX and even suggest that MTX may primarily stimulate phospholipid breakdown in the cell membrane.

Topics: Animals; Arachidonic Acids; Biological Transport, Active; Bradykinin; Calcium; Calcium Channel Blockers; Chelating Agents; Cilia; Cyclic AMP; Cyclooxygenase Inhibitors; Egtazic Acid; Enzyme Inhibitors; Epithelium; Indomethacin; Inositol Phosphates; Ionomycin; Kinetics; Lipoxygenase Inhibitors; Marine Toxins; Masoprocol; Motion; Oxocins; Phospholipases A; Quinacrine; Rabbits; Signal Transduction; Stimulation, Chemical; Terpenes; Thapsigargin; Trachea; Trifluoperazine; Verapamil

We investigated whether maitotoxin activates non-selective cation channels, as was recently proposed [Soergel, Yasumoto, Daly and Gusovsky (1992) Mol. Pharmacol. 41, 487-493]. Stimulation of dibutyryl cyclic AMP-differentiated HL-60 cells with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 0.1 microM), the Ca(2+)-ATPase inhibitor thapsigargin (0.1 microM) or maitotoxin (25 ng/ml) resulted in an increase in cytoplasmic free calcium concentration ([Ca2+]i). Unlike fMLP and thapsigargin, maitotoxin produced no increase in [Ca2+]i in the absence of extracellular Ca2+. The increase in [Ca2+]i induced by fMLP was blocked by pretreatment with pertussis toxin (100 ng/ml for 24 h) but not that induced by maitotoxin. Similarly, the increase in [Ca2+]i produced by fMLP but not that produced by maitotoxin was inhibited by pretreatment with phorbol myristate acetate (100 ng/ml). Both fMLP- and maitotoxin-induced increases in [Ca2+]i were blocked by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl)-1H-imid azole hydrochloride (SKF 96365) in a concentration-dependent manner. However, the maitotoxin-induced increase in [Ca2+]i was more sensitive to inhibition by SKF 96365 than the fMLP-induced increase. fMLP-induced increases in [Ca2+]i were blocked by cations with Gd3+ being more effective than Cd2+, whereas for maitotoxin Cd2+ was more effective than Gd3+. Both fMLP and thapsigargin stimulated quenching of Fura-2 fluorescence in the presence of extracellular Mn2+, whereas maitotoxin produced no Mn2+ quenching. Taken together these results suggest that maitotoxin does not stimulate the nonselective cation channel activated by fMLP, but instead activates Ca2+ influx by a different mechanism.

Topics: Bucladesine; Cadmium; Calcium; Cations; Fluorescent Dyes; Fura-2; Gadolinium; Humans; Imidazoles; Ion Channels; Manganese; Marine Toxins; N-Formylmethionine Leucyl-Phenylalanine; Oxocins; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

Lead (Pb2+) is known to alter the permeability of brain capillaries. A possible mechanism for this alteration may be related to the ability of Pb2+ to substitute for Ca2+. Products derived from phospholipid metabolism, namely eicosanoids and diacylglycerol, control endothelial permeability, are partly regulated by intracellular Ca2+, and thus may be sensitive to Pb2+. We asked in this study whether Pb2+ increased arachidonic acid release or stimulated phosphatidylcholine breakdown in an in vitro model of brain capillaries, namely cultured bovine retinal endothelial (BRE) cells. Pb2+ stimulated arachidonic acid release and phosphatidylcholine and phosphatidylinositol metabolism in the presence of ionomycin, but not by itself. More arachidonic acid was released than phosphorylcholine in BRE cells stimulated with ionomycin and Pb2+, but the magnitudes of these responses were similar in cells exposed to ionomycin plus Ca2+. Ionomycin plus Pb2+ or plus Ca2+ resulted in the activation of phospholipase A2, since an increase in lysophosphatidylcholine and arachidonic acid was observed. Protein kinase C was not required for arachidonic acid release because release was observed in cells with a down-regulated enzyme. Ionomycin plus other metals (La3+, Cd2+, or Mg2+) did not result in arachidonic acid release, but Cd2+ or Co2+ inhibited arachidonic acid release by more than 80% when cells were exposed to ionomycin with either Pb2+ or Ca2+. Thapsigargin or maitotoxin plus Ca2+ increased arachidonic acid release that was inhibited by the receptor-dependent calcium channel antagonist SK&F 96365 but not by the voltage-dependent calcium channel antagonist nifedipine. However, thapsigargin or maitotoxin plus Pb2+ failed to stimulate arachidonic release. Since in this in vitro model Pb2+ stimulated phospholipid metabolism solely in the presence of an ionophore, the increase in permeability observed in Pb(2+)-exposed animals is probably not due to a release of metabolites of arachidonic acid.

Topics: Amino Acid Sequence; Animals; Arachidonic Acid; Brain; Calcium; Capillaries; Cattle; Cells, Cultured; Endothelium, Vascular; Imidazoles; In Vitro Techniques; Inositol Phosphates; Ionomycin; Lead; Marine Toxins; Molecular Sequence Data; Oxocins; Phospholipids; Phosphorylcholine; Protein Kinase C; Terpenes; Thapsigargin

A role for the ganglioside GM1 in arachidonic acid release in bovine aortic endothelial cells (BAEC) was investigated. [3H]Arachidonic acid-labeled BAEC were preincubated with GM1 and incubated with one of four different stimulators. GM1 inhibited arachidonic acid release when stimulated with maitotoxin or melittin but not with ionomycin or thapsigargin. A 10 microM GM1 concentration achieved a 50% and 100% inhibition of the maitotoxin and melittin responses, respectively. The selective inhibition displayed by GM1 on the maitotoxin and melittin responses was not due to its ability to bind calcium since all four drugs, maitotoxin, melittin, ionomycin, and thapsigargin, required extracellular calcium. The effect of GM1 was not specific to arachidonic acid release. GM1 at 50 microM inhibited phosphatidylinositol polyphosphate (PIP) hydrolysis mediated by melittin, but did not affect hydrolysis mediated by ionomycin. Moreover, the activity of GM1 was not restricted to phospholipid metabolism since it also inhibited calcium influx that was stimulated by maitotoxin or melittin but not by ionomycin. We conclude that GM1 is not a specific inhibitor of phospholipases in bovine aortic endothelial cells, but rather its activity is dependent on the type of stimulant used to activate the cell.

Topics: Animals; Aorta; Arachidonic Acid; Calcium; Cattle; Cells, Cultured; Culture Media; Endothelium, Vascular; G(M1) Ganglioside; Ionomycin; Marine Toxins; Melitten; Oxocins; Phosphatidylinositol Phosphates; Terpenes; Thapsigargin