calcimycin and glucose-1-6-bisphosphate

We studied here, in NIH-3T3 fibroblasts, the effect of the Ca(2+)-ionophore A23187 (which is known to increase intracellular-free Ca(2+)) on the control of glycolysis and cell viability and the action of calmodulin antagonists. Time-response studies with Ca(2+)-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 Ca(2+)-ionophore caused a reduction in both cytoskeletal and cytosolic PFK and cell death. Both the "physiological" and "pathological" phases of the Ca(2+)-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 Ca(2+)-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.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Benzimidazoles; Calcimycin; Calcium; Calmodulin; Cell Survival; Clotrimazole; Cytoskeleton; Fructosediphosphates; Glucose-6-Phosphate; Glycolysis; Ionophores; Mice; Phosphofructokinase-1; Solubility; Thioridazine; Time Factors

1. Incubation of rat diaphragm muscles in the presence of Ca(2+)-ionophore A23187, which causes accumulation of free intracellular Ca2+, induced severe myofibrils damage. Electron microscopic studies have revealed that calmodulin (CaM) antagonists, trifluoperazine, thioridazine, pimozide and CGS 9343B, were most effective in preserving muscle structure. 2. The CaM antagonists raised the decreased glucose-1,6-bisphosphate levels, induced by high Ca2+, with a concomitant activation of the reduced cytosolic phosphofructokinase (the rate limiting enzyme of glycolysis) and thereby cytosolic glycolysis. 3. All four CaM inhibitors also prevented solubilization of cytoskeleton-bound glycolytic enzymes by high Ca2+. 4. The protective effect of these compounds on cytosolic and cytoskeletal glycolysis, was also expressed by their action in preserving muscle ATP levels. 5. The present experiments suggest that CaM antagonists may be effective drugs in treatment of muscle damage and various muscle diseases, which are characterized by a high pathological increase in intracellular Ca2+.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Calmodulin; Cytosol; Glucose-6-Phosphate; Glucosephosphates; In Vitro Techniques; Microscopy, Electron; Muscular Diseases; Myofibrils; Phosphofructokinase-1; Rats; Respiratory Muscles

ATP, added externally to the incubation medium of rat diaphragm muscles, abolished the decrease in the levels of glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, induced by phospholipase A2, local anesthetics, Ca2+ ionophore A23187, or lithium. Concomitantly to the changes in Glc-1,6-P2, the potent activator of phosphofructokinase (the rate-limiting enzyme in glycolysis) and phosphoglucomutase, the activities of these enzymes were reduced by the myotoxic agents and restored by exogenous ATP, when assayed under conditions in which these enzymes are sensitive to regulation by Glc-1,6-P2. These findings suggest that ATP may have broad therapeutic action, as it may stimulate the impaired glycolysis in muscle induced by various drugs and conditions which cause muscle weakness or damage.

Topics: Adenosine Triphosphate; Anesthetics, Local; Animals; Calcimycin; Glucose-6-Phosphate; Glucosephosphates; Lithium; Male; Phosphofructokinase-1; Phosphoglucomutase; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Respiratory Muscles