thapsigargin and Carcinoma--Ehrlich-Tumor

Inhibition of respiration by glucose, known as the Crabtree effect, has been observed in several tumours and some other highly glycolytic cells and tissues. Among mechanisms proposed to explain this effect were: competition between glycolysis and respiration for ADP or for inorganic phosphate, change of intracellular pH, change in the permeability of mitochondrial membranes, specific regulatory behavior of glycolytic enzymes, and specific enzyme topography within the cell. None of these proposals alone seems satisfactory. The present article describes the research carried out in the author's laboratory, pointing to the role of Ca2+ in the mechanism of the Crabtree effect. This supposition is based on the following observations: (1) in Ehrlich ascites tumour cells glucose elicits a steady increase of the cytoplasmic concentration of free Ca2+; (2) isolated Ehrlich ascites mitochondria and mitochondria within digitonin-permeabilised cells, preloaded with Ca2+, exhibit a depression of State 3 respiration and lowering of the rate of ATP synthesis; (3) ATPase activity of toluene-permeabilised Ehrlich ascites mitochondria becomes substantially inhibited at micromolar concentrations of Ca2+; (4) Ca2+ potentiates the effect of the inhibitory subunit of F1F0-ATPase. These results allow to hypothesize on the following sequence of events: (1) glucose elevates the cytoplasmic concentration of Ca2+; (2) this elicits an increased accumulation of Ca2+ in mitochondria; (3) loading of mitochondria with Ca2+ leads to an increased association of the inhibitory subunit with F1F0 which results in (4) the inhibition of coupled respiration. The importance of these mechanisms for glycolytic and rapidly proliferating cells is discussed.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Calcium; Carcinoma, Ehrlich Tumor; Deoxyglucose; Glucose; Oxygen Consumption; Submitochondrial Particles; Thapsigargin

Stimulation of single Ehrlich ascites tumor cells with agonists (bradykinin, thrombin) and with arachidonic acid (AA) induces increases in the free intracellular Ca2+ concentration ([Ca2+]i) in the presence and absence of extracellular Ca2+, measured using the Ca2+-sensitive probe fura 2. Sequential stimulation with two agonists elicits sequential increases in [Ca2+]i, unlike addition of the same agonist twice. Bradykinin and thrombin have additive effects on [Ca2+]i in Ca2+-free medium. The phosphoinositidase C inhibitor U-73122 inhibits the agonist-induced increases in [Ca2+]i, whereas ryanodine has no effect. Pretreatment of cells in Ca2+-free medium with thapsigargin abolishes the bradykinin-induced increase in [Ca2+]i but not the response to thrombin. The AA-induced response is not inhibited by U-73122 and cannot be mimicked by the inactive structural analog trifluoromethylarachidonyl ketone. Pretreatment of the cells with 50 microM AA (but not with 10 microM AA) abolishes the agonist-induced increase in [Ca2+]i. Thus bradykinin, thrombin, and AA induce increases in [Ca2+]i in Ehrlich cells due to Ca2+ entry and release from intracellular stores. Thrombin causes release of Ca2+ from an intracellular store that is insensitive to bradykinin and is not depleted by thapsigargin but is depleted by AA.

Topics: Animals; Arachidonic Acid; Bradykinin; Calcium; Calcium Signaling; Carcinoma, Ehrlich Tumor; Enzyme Inhibitors; Estrenes; Intracellular Membranes; Mice; Mice, Inbred Strains; Osmolar Concentration; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Pyrrolidinones; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Thrombin; Tumor Cells, Cultured

The distribution of Ca(2+) in intact cells was monitored with fluorescent probes: fura-2 for cytosolic [Ca(2+)] and rhod-2 for mitochondrial [Ca(2+)]. It was found that in neoplastic cells, such as Ehrlich ascites tumour and Zajdela hepatoma, but not in non-malignant cells, such as fibroblasts, glucose and deoxyglucose elicited release of Ca(2+) from endoplasmic reticulum stores and an increase in Ca(2+) concentration in the cytosol. Parallel to this, a decrease in the rate of Ca(2+) extrusion from the cell and an enhanced uptake of Ca(2+) by mitochondria were observed. The increase in mitochondrial [Ca(2+)] was accompanied by an increase in the mitochondrial membrane potential and the reduction state of nicotinamide nucleotides. F(1)F(o)-ATPase in submitochondrial particles of Zajdela hepatoma was strongly inhibited in the presence of micromolar Ca(2+) concentrations, whereas this activity in submitochondrial particles from rat liver appeared to be less sensitive to Ca(2+). Indications of glycosylation of Ehrlich ascites tumour cell proteins were also obtained. These data strengthen the proposal [Bogucka, K., Teplova, V.V., Wojtczak, L. and Evtodienko, Y. V. (1995) Biochim. Biophys. Acta 1228, 261-266] that the Crabtree effect is produced by mobilization of cell calcium, which is subsequently taken up by mitochondria and inhibits F(1)F(o)-ATP synthase.

Topics: Adenosine Triphosphate; Animals; Calcium; Carcinoma, Ehrlich Tumor; Deoxyglucose; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Glucose; Humans; Kinetics; Liver; Liver Neoplasms, Experimental; Mitochondria; Rats; Rats, Wistar; Spectrophotometry; Thapsigargin; Time Factors; Tumor Cells, Cultured

Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2',7',-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca2+]i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na+/H+ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca2+]i. In contrast, a smaller, slower increase in [Ca2+]i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na+/H+ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca2+]i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na+/H+ exchanger. Thus, activation of the exchanger by the Ca2+-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH4Cl-induced intracellular alkalinization resulted in an increase in [Ca2+]i, apparently via stimulation of Ca2+ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca2+ influx. Thus, cell shrinkage appears to inhibit the Ca2+ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca2+ influx was inhibited by cell shrinkage.

Topics: Ammonium Chloride; Animals; Bradykinin; Calcium; Carcinoma, Ehrlich Tumor; Cell Size; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Hypertonic Solutions; Sodium-Hydrogen Exchangers; Thapsigargin; Thrombin

The mechanisms, by which the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca2+]i) and in intracellular pH (pHi), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca2+]i evoked by ATP or UTP is abolished after depletion of intracellular Ca2+ stores with thapsigargin in Ca2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca2+]i is primarily due to release from intracellular, Ins(1,4,5)P3-sensitive Ca2+ stores. ATP also activates a capacitative Ca2+-entry pathway. ATP as well as UTP evokes a biphasic change in pHi, consisting of an initial acidification followed by alkalinization. Suramin and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pHi, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5'-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na+/H+ exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca2+ sensitive K+ and Cl- efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K+ concentration, or by adding the K+-channel blocker, charybdotoxin, does not affect the increase in [Ca2+]i, but abolishes the activation of the Na+/H+ exchanger, indicating that activation of the Na+/H+ exchanger is secondary to the Ca2+-induced cell shrinkage.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Amiloride; Animals; Calcium Signaling; Carcinoma, Ehrlich Tumor; Cell Size; Enzyme Inhibitors; Estrenes; Hydrogen-Ion Concentration; Mice; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyrrolidinones; Receptors, Purinergic P2; Signal Transduction; Sodium-Hydrogen Exchangers; Suramin; Thapsigargin; Type C Phospholipases; Uridine Triphosphate

Changes in free cytoplasmic Ca2+ ([Ca2+]i) in Ehrlich ascites tumour cells were studied under the effect of deoxyglucose and agents which modify transmembrane Ca2+ fluxes. It was shown that the reason for deoxyglucose-induced [Ca2+]i increase in Ca2+ release from internal stores and the influx from the external medium. Mitochondrial metabolic inhibitors (oligomycin+KCN, oligomycin++uncouplers of oxidative phosphorylation) induce themselves some rise in [Ca2+]i and increase the deoxyglucose effect on [Ca2+]i significantly. The conclusion was made that mitochondria can participate in cytosolic Ca2+ regulation and Ca2+ redistribution in tumour cells.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Carcinoma, Ehrlich Tumor; Cell Membrane; Deoxyglucose; Ion Transport; Oligomycins; Oxidative Phosphorylation; Potassium Cyanide; Terpenes; Thapsigargin; Tumor Cells, Cultured

Incorporation of [14C]serine into phosphatidylserine by the base exchange reaction in Ehrlich ascites tumor cells is inhibited by 30% by glucose and by 60% by 2-deoxyglucose (10 mM each). The inhibition by thapsigargin (0.2 microM) amounts to 80%. This inhibition is interpreted as being due to depletion of calcium stores in the endoplasmic reticulum and is compatible with the previous observation [Teplova, V. V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszyński, J., and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154] that glucose and deoxyglucose elicit an increase of cytoplasmic [Ca2+] at the expense of intracellular Ca2+ stores and with the finding [Barańska, J. (1989) FEBS Lett. 256, 33-37] that the base exchange reaction requires high concentration of Ca2+ within the endoplasmic reticulum lumen.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Carcinoma, Ehrlich Tumor; Deoxyglucose; Endoplasmic Reticulum; Glucose; Kinetics; Mice; Oxygen Consumption; Phosphatidylserines; Phospholipids; Serine; Terpenes; Thapsigargin

To investigate the presence and the size of different non-mitochondrial Ca2+ pools of Ehrlich ascites tumor cells (EATCs), digitonin-permeabilized cells were allowed to accumulate Ca2+ in the presence of mitochondrial inhibitors and treated with the reticular Ca(2+)-ATPase inhibitor thapsigargin, IP3 and the Ca2+ ionophore A23187. Emptying of thapsigargin-sensitive Ca2+ stores prevented any Ca2+ release by IP3, and, after IP3 addition, little or no Ca2+ was released by thapsigargin. In both instances, a further Ca2+ release was accomplished by A23187. The IP3-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondrial stored Ca2+, respectively. In intact EATCs, IP3-dependent agonists and thapsigargin discharged Ca2+ pools almost completely overlapping, and A32187 released a minor residual Ca2+ pool. The IP3-insensitive pool appeared to have a relatively low affinity for Ca2+ (below 600 nM). The high affinity, IP3-sensitive Ca2+ pool was discharged in a 'quantal' manner following step additions of sub maximal [IP3], and the IP3-induced fractional Ca2+ release was more marked at higher concentrations of stored (luminal) Ca2+, The IP3-sensitive Ca2+ pool appeared to be devoid of the Ca(2+)-activated Ca2+ release channel since caffeine did not released any Ca2+ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.

Topics: Animals; Caffeine; Calcium; Calcium-Transporting ATPases; Carcinogens; Carcinoma, Ehrlich Tumor; Endoplasmic Reticulum; Humans; Inositol 1,4,5-Trisphosphate; Male; Mice; Terpenes; Thapsigargin; Tumor Cells, Cultured

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane-fractions. Agents that increase intracellular cAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system by cGMP. In fact, permeant analogs of cGMP, dibutyryl cGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition of cGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca2+ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca2+ ions.

Topics: Animals; Bucladesine; Calcimycin; Calcium; Calcium-Transporting ATPases; Carcinoma, Ehrlich Tumor; Cell Membrane; Cyclic GMP; Dibutyryl Cyclic GMP; Enzyme Inhibitors; Female; Kinetics; Mice; NADH, NADPH Oxidoreductases; Neomycin; Oxidation-Reduction; Second Messenger Systems; Signal Transduction; Sphingosine; Thapsigargin

Concentration of free cytoplasmic Ca2+ ([Ca2+]i) in Ehrlich ascites tumour cells loaded with fura-2 was measured in single cells applying a video imaging system. In resting cells [Ca2+]i amounted to 60-340 nM and was increased after addition of 10 mM D-glucose or D-2-deoxyglucose by 80-200 nM. This increase occurred within 30-60 s following addition of the sugars and lasted for several minutes. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose and, vice versa, thapsigargin added after the sugars mobilized less Ca2+ than when added before. A possible relation of the [Ca2+]i rise evoked by glucose and deoxyglucose to the Crabtree effect is discussed.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Carcinoma, Ehrlich Tumor; Cytoplasm; Deoxyglucose; Female; Glucose; Mice; Terpenes; Thapsigargin

Concentration of free cytoplasmic Ca2+ ([Ca2+]i) in Ehrlich ascites tumor cells, measured using fura-2, amounted to 170-300 nM and was increased by 50-160 nM after addition of 10 mM D-glucose or D-2-deoxyglucose but not 3-O-methylglucose at pH 7.4. In the range of external pH between 6.8 and 7.8 the increase was higher at higher pH. This increase occurred within 30-60 s after addition of hexose and lasted for at least 10 min. This [Ca2+]i rise was observed both in presence and virtual absence of Ca2+ in the external medium. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose. The mechanism of [Ca2+] in the external medium. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose. The mechanism of [Ca2+]i rise evoked by glucose and deoxyglucose and its importance in switching cell metabolism from oxidative to glycolytic are discussed.

Topics: 3-O-Methylglucose; Animals; Calcium; Calcium-Transporting ATPases; Carcinoma, Ehrlich Tumor; Cytoplasm; Deoxyglucose; Female; Glucose; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Methylglucosides; Mice; Spectrometry, Fluorescence; Terpenes; Thapsigargin