CGS 9343B Research Articles

Inhibition of Autophagy in Cardiomyocytes by Calmodulin Antagonists

Macroautophagy (hereafter referred to as autophagy) is a catabolic process that regulates important aspects of myocardial metabolism. While numerous studies have indicated involvement of signaling via Ca2+ in autophagic flux, the precise mechanisms remain unknown. Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca2+ signals by interacting with an estimated network of 300 cellular proteins. We have begun to examine the role of CaM in autophagic flux in cardiomyocytes. A basal level of autophagy is required for normal myocardial function. Chloroquine dose‐dependently reduces basal autophagic flux in H9c2 cardiomyocytes, as indicated by LC3 immunoblotting, p62 immunofluorescence, and autophagic flux assays. Competitive binding assays using purified CaM and FRET‐based CaM biosensors demonstrate direct Ca2+‐dependent CaM binding to chloroquine. The structurally distinct CaM antagonists W‐7, trifluoperazine, and CGS 9343B also dose‐dependently inhibit basal autophagic flux by reducing autophagosome degradation. Most CaM‐dependent processes depend at the front end on the amplitude and dynamics of intracellular Ca2+ signals. Interestingly, chloroquine, W‐7, trifluoperazine, and CGS 9343b all mobilize intracellular Ca2+. The effects of these pharmacological agents to inhibit autophagic flux are thus associated with their inhibition of CaM and not of initial Ca2+ signals produced in cells. These data indicate a role for CaM in the control of autophagic flux in cardiomyocytes.Support or Funding InformationAmerican Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (Eric Wauson); NIH HL112184 and DMU IOER‐090717 (Quang‐Kim Tran)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K+ (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) value of 0.81μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77±0.04μM−1s−1 and 2.55±1.50s−1, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition.

CGS 9343B and W7 (calmodulin antagonists) inhibit KCl-induced increase in cytosolic free calcium and insulin secretion of RINm5F cells.

CGS 9343B:1,3-Dihydro-1-[1-[4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1] benzoxazepin-4-yl)methyl)-4-piperidinyl]-2H-benzimidazol-2-o ne maleate and W7:N-6(aminohexyl)-5-chloro-1-naphthalenesulfonamide) are calmodulin antagonists with different specificities. The effects of CGS 9343B and W7 on cytosolic free calcium concentration ([ Ca2+]i) and insulin release were investigated in rat insulinoma cells (RINm5F). As measured with the Quin-2 technique, preincubation with CGS 9343B (0.3-10 microM) and W7 (5-50 microM) concentration dependently decreased KCl (25 mM)-mediated accumulation of cytosolic calcium. Both, CGS 9343B (10 microM) and W7 (50-100 microM) almost abolished the alanine- and KCl-induced increase in [Ca2+]i and significantly inhibited KCl (25 mM)- and alanine (10 mM)-mediated insulin release. W5 (100 microM), the chlorine-deficient analogue of W7 with decreased affinity for calmodulin, did not inhibit the KCl-induced increase in [Ca2+]i and enhanced basal and KCl-mediated insulin release by 56% and 189%, respectively. Our data suggest that CGS 9343B and W7 inhibit the depolarization-induced calcium uptake and subsequent increase in [Ca2+]i.

Interaction of calmodulin- and PKC-dependent contractile pathways in cat lower esophageal sphincter (LES).

We have previously shown that, in circular muscle cells of the lower esophageal sphincter (LES) isolated by enzymatic digestion, contraction in response to maximally effective doses of acetylcholine (ACh) or Inositol Triphosphate (IP3) depends on the release of Ca2+ from intracellular stores and activation of a Ca2+-calmodulin (CaM)-dependent pathway. On the contrary, maintenance of LES tone, and response to low doses of ACh or IP3 depend on a protein kinase C (PKC) mediated pathway. In the present investigation, we have examined requirements for Ca2+ regulation of the interaction between CaM- and PKC-dependent pathways in LES contraction. Thapsigargin (TG) treatment for 30 min dose dependently reduced ACh-induced contraction of permeable LES cells in free Ca2+ medium. ACh-induced contraction following the low level of reduction of Ca2+ stores by a low dose of TG (10(-9) M) was blocked by the CaM antagonist, CGS9343B but not by the PKC antagonists chelerythrine or H7, indicating that the contraction is CaM-dependent. After maximal reduction in intracellular Ca2+ from Ca2+ stores by TG (10(-6) M), ACh-induced contraction was blocked by chelerythrine or H7, but not by CGS9343B, indicating that it is PKC-dependent. In normal Ca2+ medium, the contraction by ACh after TG (10(-9) M) treatment was also CaM-dependent, whereas the contraction by ACh after TG (10(-9) M) treatment was PKC-dependent. We examined whether PKC activation was inhibited by activated CaM. CGS 9343B inhibited the CaM-induced contraction, but did not inhibit the DAG-induced contraction. CaM inhibited the DAG-induced contraction in the presence of CGS 9343B. This inhibition by CaM was Ca2+ dependent. These data are consistent with the view that the switch from a PKC-dependent pathway to a CaM dependent pathway can occur and can be regulated by cytosolic Ca2+ in the LES.

Inhibition of mechanotransducer currents in crayfish sensory neuron by CGS 9343B, a calmodulin antagonist

The effects of CGS 9343B (zaldaride maleate), a calmodulin antagonist, on mechanosensitive channels were examined in crayfish slowly adapting sensory neurons using the two-electrode voltage clamp technique. In addition to its inhibition of voltage-gated Na + and K + currents, CGS 9343B (<30 μM) blocked reversibly the receptor current in a dose-dependent and voltage-dependent manner with a dissociation constant ( K d) of 26.8 μM. The time course of the block was 265 s. Within the extension range of 3–30%, the reduction in receptor current was stimulus-independent and the gating mechanisms were not affected. Extracellular Ca 2+ was not necessary for its blocking effects. No changes in passive muscle tension were observed in the presence of 20 μM CGS 9343B. These results suggest that CGS 9343B, as a calmodulin antagonist, can also block mechanosensitive channels, possibly by being incorporated into the lipid membrane and/or interacting with the channel protein.

Effects of Ca2+-Ionophore A23187 and Calmodulin Antagonists on Regulatory Mechanisms of Glycolysis and Cell Viability of NIH-3T3 Fibroblasts

We studied here, in NIH-3T3 fibroblasts, the effect of the Ca2+-ionophore A23187 (which is known to increase intracellular-free Ca2+) on the control of glycolysis and cell viability and the action of calmodulin antagonists. Time-response studies with Ca2+-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 Ca2+-ionophore caused a reduction in both cytoskeletal and cytosolic PFK and cell death. Both the “physiological” and “pathological” phases of the Ca2+-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 Ca2+-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.

Influence of probes for calcium–calmodulin and protein kinase C signalling on the plasma membrane Ca2+–ATPase activity of rat synaptosomes and leukocyte membranes

The influence of selected inhibitors of calcium signalling on the plasma membrane Ca2+-ATPase activity of rat synaptosomes and peritoneal leukocyte membranes was studied. The calmodulin inhibitor calmidazolium was an efficient inhibitor (50%) of the synaptosomal Ca2+-ATPase activity in a manner competitive with phosphatidylserine. The inhibition by CGS 9343B (30%) was not counteracted by phosphatidylserine. The intracellular calcium antagonist TMB-8 and the protein kinase inhibitor staurosporine and the derivatives CGP 41251 and CGP 42700 hardly affected the synaptosomal Ca2+-ATPase activity. The flavonoid quercetin was a more effective inhibitor of the ATPase activity of synaptosomal than of leukocyte membranes. Phloretin, at relatively high concentrations, caused only a modest inhibition of synaptosomes. The protein kinase C inhibitor sphingosine was a weak inhibitor of the synaptosomal but an effective inhibitor of the leukocyte membrane Ca2+-ATPase activity. The antineoplastic ether phospholipids BM 41.440 (ilmofosine) and ET-18-OCH3 (edelfosine) effectively inhibited the leukocyte membranes whereas the ATPase activity of synaptosomes was significantly increased by 20 μM and slightly inhibited by higher concentrations of these agents. The analogue hexadecylphosphocholine (miltefosine) did not affect the ATPase activity of the synaptosomes and only inhibited that of the leukocyte membranes at concentrations above 20 μM. These results show that several test substances of current interest affect the activity of the plasma membrane Ca2+-ATPase. The effects depend on the origin of the membranes. The investigation does not permit a distinction between direct effects on the enzyme and an interference with its membrane environment although the latter is indicated for the ether phospholipids.

Effects of ethylene glycol tetraacetic acid, A23187 and calmodulin, calcium activated neutral proteinase antagonists on melatonin secretion in perifused chick pineal gland

We have recently described, using perifused pineal glands, that calcium influx participates in the activation of chick pineal gland. This study shows that the loss of perifused chick pineal gland activity is a complex process which seems to involve the release of calcium from intracellular stores, calmodulin and calcium-activated neutral protease (CANP). Pineal glands were perifused with Krebs medium (controls) or with Krebs medium plus the drugs ethylene glycol tetraacetic acid (EGTA; calcium chelator), A23187 (calcium ionophore), EGTA plus A23187 (extra-intra cellular calcium chelation), trifluoperazine and CGS9343B (calmodulin inhibitors), and E-64 (CANP inhibitor) at the time of the natural peak of melatonin release. When EGTA or A23187 were added to the perifusion medium, no effects were observed. On the other hand, when the calcium chelator EGTA plus A23187 (free extra and intracellular calcium levels were dramatically decreased), trifluoperazine, CGS 9343B or E-64 were added to the perifusion medium melatonin synthesis increased significantly and was sustained for 8 h. We propose a prominent role for calcium output from intracellular stores in regulating melatonin production primarily by acting on Ca-calmodulin and calcium-activated neutral protease.

Involvement of calmodulin in the activation of store-operated Ca 2+ entry in rat hepatocytes

The possible participation of calmodulin in the activation of store-operated Ca 2+ entry (SOC) in single rat hepatocytes was investigated microspectrofluorimetrically. SOC was triggered after discharging intracellular Ca 2+ stores using the endoplasmic reticulum Ca 2+-ATPase inhibitor thapsigargin in the absence of external Ca 2+. Re-admission of bath Ca 2+ caused a rapid and pronounced Ca 2+ entry. The calmodulin antagonists calmidazolium or CGS 9343B applied before the thapsigargin treatment inhibited SOC, whereas they were ineffective when added after the thapsigargin-induced Ca 2+ transient. This study suggests that activation of calmodulin after the elevation of cytosolic Ca 2+ associated with the emptying of Ca 2+ stores is involved in the triggering of SOC in hepatocytes.

Calmodulin antagonists decrease glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP and viability of melanoma cells

Glycolysis is known to be the primary energy source in cancer cells. We investigated here the effect of four different calmodulin antagonists: thioridazine (10-[2-(1-methyl-2-piperidyl)ethyl]-2-methylthiophenothiazine), CGS 9343B (1,3-dihydro-1-[1-[(4-methyl-4 H,6 H-pyrrolo[1,2- a][4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2 H-benzimidazol-2-one (1:1) maleate), clotrimazole (1-(α-2-chlorotrityl)imidazole) and bifonazole (1-(α-biphenyl-4-ylbenzyl)imidazole), on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP content and cell viability in B16 melanoma cells. We found that all four substances significantly reduced the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP, in a dose- and time-dependent manner. Cell viability was reduced in a close correlation with the fall in ATP. The decrease in glucose 1,6-bisphosphate and fructose 1,6-bisphosphate did not result from the cytotoxic effects of the calmodulin antagonists, since their content was already reduced before any cytotoxic effect was observed. These findings suggest that the fall in the levels of the two signal molecules of glycolysis, induced by the calmodulin antagonists, causes a reduction in glycolysis and ATP levels, which eventually leads to cell death. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, these substances are most promising agents in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cell growth.

Effects of CGS 9343B (a Putative Calmodulin Antagonist) on Isolated Skeletal Muscle: DISSOCIATION OF SIGNALING PATHWAYS FOR INSULIN-MEDIATED ACTIVATION OF GLYCOGEN SYNTHASE AND HEXOSE TRANSPORT

The role of calmoudulin in control of carbohydrate metabolism in the absence and presence of insulin in isolated mouse soleus muscle was investigated. The calmodulin antagonist CGS 9343B had no effect on basal glycogen synthase activity, the contents of high energy phosphates, glucose-6-P, or glycogen synthesis. However, CGS 9343B inhibited the basal rates of 2-deoxyglucose uptake and 3-O-methylglucose transport by 30% (p < 0.05) and 40% (p < 0.001), respectively. Insulin activated glycogen synthase by almost 40% (p < 0.01) and this increase was not altered in the presence of CGS 9343B. Insulin increased the muscle content of glucose-6-P (approximately equal to 2-fold), as well as glycogen synthesis (approximately equal to 8-fold), 2-deoxyglucose uptake (approximately equal to 3-fold), and 3-O-methylglucose transport (approximately equal to 2-fold), and these increases were inhibited by CGS 9343B. In additional experiments on isolated rat epitrochlearis muscle, it was found that the hypoxia-mediated activation of 3-O-methylglucose transport was also inhibited by CGS 9343B. These data demonstrate that: 1) hexose transport, both in the absence and presence of external stimuli (insulin and hypoxia), requires functional calmodulin; and 2) insulin-mediated activation of glycogen synthase does not require functional calmodulin, nor can it be accounted for by increases in glucose transport or glucose-6-P.

Platelet-Derived Growth Factor (PDGF) Rapidly Stimulates Binding of Glycolytic Enzymes to Muscle Cytoskeleton, Prevented by Calmodulin Antagonists

Glycolytic enzymes are known to be controlled by reversible binding to cytoskeleton. Our previous experiments have shown that insulin, epidermal growth factor (EGF), and Ca2+ induce a rapid and transient stimulation of binding of glycolytic enzymes to muscle cytoskeleton. We show here that platelet-derived growth factor (PDGF) exerts a similar action. Incubation of rat diaphragm muscle in the presence of PDGF resulted in rapid and transient stimulation of binding of phosphofructokinase (EC 2.7.11) and aldolase (EC 4.1.2.13) to muscle cytoskeleton. The increase in cytoskeleton-bound glycolytic enzymes induced by PDGF was prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that Ca(2+)-calmodulin is involved in this effect of PDGF. Similarly, we previously found that stimulation of cytoskeleton-bound glycolytic enzymes exerted by insulin, EGF, or Ca2+, was also calmodulin mediated. The present and previous results suggest that the rapid, Ca(2+)-calmodulin-mediated increase in cytoskeleton-bound glycolytic enzymes, may be a general mechanism in the cell, in signal transduction of insulin, growth factors, and other Ca(2+)-mobilizing hormones. The accelerated cytoskeletal glycolysis will provide local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following binding of growth factor or hormone to its receptor.

Calmodulin-dependent modulation of pH sensitivity of the amino acid transport system L in human placental choriocarcinoma cells

The JAR human placental choriocarcinoma cells express the amino acid transport system L. The activity of this system is Na +-independent and is stimulated by acidic extracellular pH. Treatment of cells with the calmodulin antagonist CGS 9343B results in a marked stimulation of the system L activity. At a CGS 9343B concentration of 50 μM, the stimulation of activity measured at pH 7.5 is about 75–100%. This effect is not blocked by cycloheximide, actinomycin D, colchicine or cytochalasin D suggesting that the stimulation is not due to de novo synthesis of the carrier protein or recruitment of the carrier protein from an intracellular pool. The stimulatory effect of CGS 9343B is reproducible with other calmodulin antagonists. Treatment with CGS 9343B significantly modifies pH sensitivity of the system. The stimulatory effect of H + is markedly reduced in treated cells compared to control cells. The stimulation of activity at pH 5.5 vs. pH 7.5 is 55% in control cells but only 8% in treated cells. Similarly, the stimulatory effect of CGS 9343B is reduced by H +. The stimulation of activity seen with 50 μM CGS 9343B is 80% at pH 8.0, but only 26% at pH 5.5. In addition, H + and CGS 9343B affect the kinetic parameters of system L in a similar manner, the stimulation in both cases being primarily due to an increase in the maximal velocity. The apparent competitive nature between the effects of H + and CGS 9343B is also observed with other calmodulin antagonists. These results show that the transport function and pH sensitivity of the amino acid transport system L in placental choriocarcinoma cells are modulated by calmodulin by processes which do not involve de novo synthesis nor recruitment of the carrier protein.

Calmodulin-dependent regulation of the catalytic function of the human serotonin transporter in placental choriocarcinoma cells.

We investigated the involvement of calmodulin-dependent cellular processes in the regulation of the human serotonin transporter in placental choriocarcinoma cells. Treatment of JAR and BeWo cells with the selective calmodulin antagonist 1,3-dihydro-1-[1-((4-methyl-4H, 6H-pyrrolo[1,2-a][4,1]-benzoxapin-4-yl)-methyl)-4-piperindinyl+ ++]- 2H-benzimidazol-2-one (CGS93-43B (CGS)) for 1 h decreased the imipramine-sensitive serotonin transport activity markedly. The inhibitory effect was specific and was reproducible with other calmodulin antagonists. The basal serotonin transport activity as well as the activity that was stimulated by cholera toxin were inhibited to a similar extent. The CGS-induced inhibition was accompanied by a decrease in the maximal velocity and in the affinities of the transporter for Na+ and Cl- and an increase in the affinity for serotonin. There was, however, no change in the Na+/Cl-/serotonin stoichiometry. The inhibition of the transport activity induced by the treatment of intact cells with CGS was observable in plasma membrane vesicles isolated from these cells. Treatment with CGS had no effect on steady state levels of the serotonin transporter mRNAs nor on the transporter density in the plasma membrane. We conclude that the serotonin transporter is regulated by calmodulin-dependent processes in human placental choriocarcinoma cells involving posttranslational modification, most likely phosphorylation/dephosphorylation, of the transporter protein.

Elongation factor-2 kinase: effective inhibition by the novel protein kinase inhibitor rottlerin and relative insensitivity towards staurosporine.

The elongation factor-2 (eEF-2) is selectively phosphorylated by the eEF-2 kinase (calmodulin-dependent kinase III). This phosphorylation can be inhibited by calmodulin antagonists, such as CGS 9343B (IC50 = 4 microM). The novel protein kinase inhibitor rottlerin is shown to suppress eEF-2 phosphorylation with an IC50 of 5.3 microM. By contrast, the eEF-2 kinase is rather resistant towards the potent but non-selective protein kinase inhibitor staurosporine (IC50 > 50 microM) and thus can be differentiated from most other protein kinases that are suppressed by staurosporine in the nM range.

Effects of various calmodulin antagonists on Na/Ca exchange current of single ventricular cells of guinea-pig.

The effects of various calmodulin inhibitors were examined on the Na/Ca exchange current in single cardiac ventricular cells of the guinea-pig using the whole-cell patch-clamp technique. External application of W-7 and trifluoperazine inhibited Na/Ca exchange current in a dose-dependent manner with IC50 values of 13 and 7 microM, respectively. W-5 inhibited the exchange current but less potently than W-7. More specific calmodulin inhibitors such as CGS 9343B and calmidazolium did not, however, decrease the current as significantly as expected. All these drugs inhibited the Na current more strongly than the Na/Ca exchange current. Ruthenium red (RR), another type of calmodulin inhibitor, did not decrease the exchange current by internal application. Neither mastoparan or melittin (calmodulin-binding peptides) inhibited the exchange current appreciably. RR and the peptides did not affect the Na current either. These results indicate that calmodulin may not involved in the activation of cardiac Na/Ca exchange or the Na current. Internal application of chymotrypsin inhibited the blocking effect of W-7 on the Na/Ca exchange current but not that on the Na current. These results indicate that W-7 blocks the Na/Ca exchange current not by binding to calmodulin but possibly by directly affecting an internal site of the exchanger itself and that the inhibitory action of W-7 is different on the Na/Ca exchange current and the Na current.

Stimulatory effect of epidermal growth factor on binding of glycolytic enzymes to muscles cytoskeleton and the antagonistic action of calmodulin inhibitors.

We report here on a novel mechanism involved in epidermal growth factor (EGF) action, which shows that EGF rapidly stimulates binding of the glycolytic enzymes, phosphofructokinase (EC 2.7.1.11), and aldolase (EC 4.1.2.13) to muscle cytoskeleton. This effect was demonstrated both in vivo, in the tibialis anterior muscle from rats injected with EGF, and in vitro, in the isolated rat diaphragm muscle incubated with EGF. The increase in cytoskeleton-bound glycolytic enzymes induced by EGF was prevented, in both the in vivo and in vitro experiments, by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that Ca2+ and calmodulin are involved in this effect of EGF. Our previous findings have revealed that insulin or Ca2+ exert a similar rapid stimulation of cytoskeletal glycolysis, which is also calmodulin mediated. We now hypothesize that this may be a general mechanism of signal transduction in the cell, involving Ca(2+)-mobilizing hormones and growth factors, and supplying local ATP, in the vicinity of cytoskeleton-membrane, which is required for the rapid cytoskeletal-membrane rearrangements upon membrane-induced events.

Treatment of muscle damage, induced by high intracellular Ca 2+, with calmodulin antagonists

1. 1. Incubation of rat diaphragm muscles in the presence of Ca 2+-ionophore A23187, which causes accumulation of free intracellular Ca 2+, 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. 2. The CaM antagonists raised the decreased glucose-1,6-bisphosphate levels, induced by high Ca 2+, with a concomitant activation of the reduced cytosolic phosphofructokinase (the rate limiting enzyme of glycolysis) and thereby cytosolic glycolysis. 3. 3. All four CaM inhibitors also prevented solubilization of cytoskeleton-bound glycolytic enzymes by high Ca 2+. 4. 4. The protective effect of these compounds on cytosolic and cytoskeletal glycolysis, was also expressed by their action in preserving muscle ATP levels. 5. 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 Ca 2+.

The dual effects of Ca2+ on binding of the glycolytic enzymes, phosphofructokinase and aldolase, to muscle cytoskeleton.

We show here that in rat diaphragm muscle, a short time of incubation with the Ca(2+)-ionophore A23187 induced an increase in cytoskeleton-bound phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13), whereas a longer period of incubation, which causes a pathological rise in intracellular Ca2+, induced a decrease in bound enzymes. Lactate concentration correlated with both phases of Ca2+ action on the binding of the enzymes. The increase in cytoskeleton-bound enzymes could be prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a novel, potent, and selective inhibitor of calmodulin activity). These results suggest that calmodulin is involved in the Ca(2+)-induced binding of the enzymes to muscle cytoskeleton.

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. 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 eytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin, (c) Lactate concentration correlated with the stimulation of cytoskeletal and eytosolic glycolysis. 2. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source ofATP for the rapid actions of insulin, whereas eytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.