Malca Chen-Zion

Sequence of insulin effects on cytoskeletal and cytosolic phosphofructokinase, mitochondrial hexokinase, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels, and the antagonistic action of calmodulin inhibitors, in diaphragm muscle

1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.

Treatment of frostbite with the calmodulin antagonists thioridazine and trifluoperazine

1. Thioridazine and trifluoperazine, which have been previously found in this laboratory to be the most effective calmodulin antagonists in treatment of burns, are shown here to be also effective in the treatment of frostbite. 2. Electron microscopic studies have revealed a complete reversal of both the vascular and skin tissue damage induced by frostbite. 3. The reversal of the vascular damage was also demonstrated by the ability of these compounds to abolish the increase in hemoglobin content in the skin. 4. The reversal of the skin tissue damage was also revealed by the ability of these compounds to raise the decreased ATP level and the reduced activities of 6-phosphogluconate dehydrogenase and mitochondrial and soluble hexokinase in skin, induced by frostbite, to normal control levels.

Therapeutic and prophylactic treatment of skin burns with several calmodulin antagonists

1. Several calmodulin antagonists abolished the decrease in ATP level and in the activities of 6-phosphogluconate dehydrogenase and mitochondrial and soluble hexokinase, induced by burns in the rat skin. 2. These antagonists had also a protective action on the blood capillaries and erythrocyte membrane, as judged by the electron microscopic appearance, as well as the abolishment of hemoglobin increase and burn edema. 3. Of all the compounds investigated here, the most effective were trifluoperazine and thioridazine, which are also known as the more potent calmodulin antagonists. 4. The present experiments suggest that calmodulin antagonists may be effective drugs in treatment of burns, having both therapeutic and prophylactic action.

Ca2+-ionophore A23187 and the Ca2+-mobilizing hormones serotonin, vasopressin, and bradykinin increase mitochondrially bound hexokinase in muscle

We show that a rise in cytosolic-free Ca2+ in muscle, induced by Ca(2+)-ionophore A23187 or by the Ca(2+)-mobilizing hormones serotonin, vasopressin, and bradykinin, increases the binding of hexokinase to mitochondria in muscle. This increase could be prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a novel, potent, and selective inhibitor of calmodulin activity) which strongly suggests that calmodulin is involved in the Ca(2+)-induced binding of the enzyme to muscle mitochondria.

Effect of the calmodulin antagonist CGS 9343B on skin burns

1. CGS 9343B is a novel, potent and selective inhibitor of calmodulin activity, which unlike other known calmodulin antagonists, does not inhibit protein kinase C activity and does not possess potential antidopaminergic activity. Here we show that CGS 9343B, like other calmodulin antagonists reported previously, is most effective in treatment of burns. 2. The effectiveness of CGS 9343B on skin burns was evaluated by electron microscopic studies, as well as by measurements of hemoglobin, ATP and enzymes which are markedly changed in the burned skin. 3. As CGS 9343B is a more selective probe for calmodulin function than other inhibitors, the similarity of its effects on burns to that of other calmodulin antagonists, strongly suggest that their action on burns is mediated through calmodulin inhibition.

Serotonin-induced decrease in brain ATP, stimulation of brain anaerobic glycolysis and elevation of plasma hemoglobin; the protective action of calmodulin antagonists

1. Injection of serotonin (5-hydroxytryptamine) to rats, induced a dramatic fall in brain ATP level, accompanied by an increase in P(i). Concomitant to these changes, the activity of cytosolic phosphofructokinase, the rate-limiting enzyme of glycolysis, was significantly enhanced. Stimulation of anaerobic glycolysis was also reflected by a marked increase in lactate content in brain. 2. Brain glucose 1,6-bisphosphate level was decreased, whereas fructose 2,6-bisphosphate was unaffected by serotonin. 3. All these serotonin-induced changes in brain, which are characteristic for cerebral ischemia, were prevented by treatment with the calmodulin (CaM) antagonists, trifluoperazine or thioridazine. 4. Injection of serotonin also induced a marked elevation of plasma hemoglobin, reflecting lysed erythrocytes, which was also prevented by treatment with the CaM antagonists. 5. The present results suggest that CaM antagonists may be effective drugs in treatment of many pathological conditions and diseases in which plasma serotonin levels are known to increase.

Rapid stimulatory effect of insulin on binding of glycolytic enzymes to cytoskeleton of C-6 glial cells, and the antagonistic action of calmodulin inhibitors

Insulin was shown in our previous experiments to induce an increase in binding of glycolytic enzymes to muscle cytoskeleton. We show here the same stimulatory effect of insulin in C-6 glial cells in culture. In these cells, like in muscle, a short time of incubation with insulin (1–10 min) induced an increase in cytoskeleton bound phosphofructokinase and aldolase. This stimulatory effect of insulin could be prevented by treatment with calmodulin antagonists trifluoperazine, thioridazine or CGS 9343 B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that calmodulin is involved in this action of insulin. Our previous experiments have shown that growth factors and Ca2+ also induce a rapid, calmodulin-mediated stimulation of binding of glycolytic enzymes to cytoskeleton. The present and previous results suggest that the rapid binding of glycolytic enzymes to cytoskeleton, may be a general mechanism, in different cells, in signal transduction of insulin, growth factors and other Ca2+ -mobilizing hormones. The accelerated cytoskeletal glycolysis will supply local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following the binding of hormone to its receptor.

Effects of carbamylcholine and pyridostigmine on cytoskeleton-bound and cytosolic phosphofructokinase and ATP levels in different rat tissues

1. The effects of carbamylcholine (CaCh) (acetylcholine agonist) and pyridostigmine (Pyr) (acetylcholinesterase inhibitor), on the activity of cytoskeleton-bound and cytosolic phosphofructokinase (PFK), the rate-limiting enzyme in glycolysis, and ATP levels, were studied in rat tibialis anterior (TA) muscle, heart, and brain. 2. In the TA muscle, a marked (about three-fold) increase in the allosteric activity of cytosolic (soluble) PFK was found, 3-5 min following the injection of CaCh or Pyr. The intracellular distribution of the enzyme was not affected by both drugs. Stimulation of glycolysis in this muscle was also expressed by a significant increase in the concentrations of glycolytic intermediates and lactate. Glucose 1,6-bisphosphate (Glc-1,6-P2) levels were unchanged, whereas fructose-2,6-bisphosphate (Fru-2,6-P2) was increased. Glycogenolysis was also stimulated, as deduced from the decrease in glycogen content. The stimulation of glycolysis, induced by both drugs, was accompanied by an increase in ATP level in the TA muscle. 3. In contrast to the stimulatory action of CaCh or Pyr on glycolysis in the TA muscle, both drugs had no effect on cytosolic and cytoskeletal PFK in heart and brain. However, ATP content in both heart and brain was markedly reduced by these drugs, most probably due to their reported harmful effects on mitochondrial function, leading to tissue damage. 4. Electron microscopic studies of TA muscle and heart from rats treated with CaCh or Pyr, revealed severe damage of heart but no harmful effects on TA muscle, which is a muscle with high glycolytic and low oxidative capacity. The present experiments suggest that the accelerated glycolysis in this muscle induced by both drugs, supplies ATP, thus preventing muscle damage.

Opposite changes with age in liver and muscle in the mitochondrial and soluble glucose-1,6-bisphosphate and 6-phosphogluconate dehydrogenase

Glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was markedly decreased in liver of adult rats (2 months of age) as compared to young rats (1-2 weeks of age). This regulator was found to be present in both the mitochondrial and soluble fractions of liver. Its concentration in both these fractions was decreased with age. Concomitant to the decrease in Glc-1,6-P2, which is a potent inhibitor of 6-phosphogluconate dehydrogenase, the activity of this enzyme was markedly increased with age in both the mitochondrial and soluble fractions. However, the increase in this enzymes activity was more pronounced in the mitochondrial fraction. The mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 as compared to the soluble enzyme, and this may explain the greater enhancement in its activity with age in this fraction. The tibialis anterior muscle exhibited changes with age opposite to those found in liver; Glc-1,6-P2 concentration, in both the mitochondrial and soluble fractions of muscle increased with age, and this increase was accompanied by a concomitant reduction in the activity of the mitochondrial and soluble 6-phosphogluconate dehydrogenase. Similar to liver, the mitochondrial enzyme was more affected by age, as it also exhibited a greater susceptibility to inhibition by Glc-1,6-P2.