Anticalmodulin Drugs Research Articles

Effect of intracellular free Ca2+ on leucine transport in Chang liver cells.

Addition of Ca2+ ionophore (A23187) to the medium stimulated the Na+-independent leucine transport in Chang liver cells, increasing the cytoplasmic free Ca2+ concentration, irrespective of the presence or absence of extracellular Ca2+. Anticalmodulin drugs, such as chlorpromazine, trifluoperazine, and W-7, significantly inhibited the leucine transport in the cells. The stimulatory effect of A23187 on leucine transport was completely blocked in the presence of the anticalmodulin drug. Two microtubule disrupting drugs, colchicine and colcemid, significantly stimulated leucine transport. On the other hand, taxol, a microtubule stabilizing agent, decreased the stimulatory effect of colchicine on the leucine transport. These results strongly suggest the involvement of Ca2+ and calmodulin in regulation of Na+-independent leucine transport, possibly through control of assembly and disassembly of the microtubule network.

The stimulation by the tumour promoters 12-O-tetradecanoylphorbol-13-acetate and phenobarbital of the growth of primary neonatal rat hepatocytes.

A single exposure to a wide range of concentrations (10(-15)-10(-4) mol/l) of the tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital (PB) significantly stimulated the flow into DNA synthesis and mitosis of neonatal rat hepatocytes in 4-day-old primary cultures kept in high-calcium (1.8 mmol/l) Eagle's FBS-MEM. Maximal effects were observed in the dose range 10(-12)-10(-6) mol/l. Moreover, both xenobiotics retained their full mitogenic effectiveness when given to hepatocytes incubated in calcium-deficient (0.01 mmol/l) FBS-MEM, thereby evoking a neoplastic phenotype in otherwise normal (i.e., non-initiated) cells. Proliferation kinetic studies showed that TPA and PB acted according to the actual cell cycle setting of the cells: they committed a fraction of the quiescent (GO) hepatocytes to grow, and enabled hepatocytes previously poised at the G1/S and G2/M boundaries to start cycling again, but exerted no influence on liver cells already engaged by themselves in active cycling. These diverse activities of TPA and PB were independent of serum (growth) factors; they were also fully elicited in hepatocytes grown in synthetic media (Eagle's MEM or HiWoBa2000), and were not changed by the addition of a mitogenically effective dose (10(-10) mol/l) of epidermal growth factor/urogastrone (EGF) with or without FBS. Conversely, the addition of the specific plasmalemmal calcium chelator, EGTA, or displacer, La3+, or of the anticalmodulin drugs, W-13 and calmidazolium (or R24571: an agent hardly entering cells) fully inhibited the mitogenic activities of tumour promoters in quiescent and intra-cycle-blocked hepatocytes, while having no effect on untreated or xenobiotic-treated liver cells already cycling by themselves. Such inhibitory effects were in all instances independent of the actual extracellular calcium concentration and took place according to time-related double kinetics. Hence, xenobiotic-activated processes taking place at the plasmalemmal level and involving the activation of calcicalmodulin-dependent enzymes were of critical importance for both the G0/G1 and G1/S transitions in primary hepatocytes.

Calcium thresholds in the activation of DNA and RNA synthesis in cultured planarian cells: relationship with hormonal and DB cAMP effects

DNA and RNA syntheses were reduced to a basal level in dissociated planarian cells grown for 48 h in a Ca 2+-free medium. These syntheses could be triggered anew by raising the Ca 2+ concentration in the medium. Serotonin could be substituted for Ca 2+ in stimulating DNA synthesis by Ca 2+-depleted cells, while dopamine greatly enhanced RNA synthesis in these cells. When Ca 2+ concentration was raised in hormone-treated cultures, DNA synthesis was again slightly increased but RNA synthesis was depressed. Both hormonal effects were completely inhibited by the anticalmodulin drug trifluoperazine. As serotonin and dopamine are both known to stimulate the adenylate cyclase system, it was further investigated whether the hormonal effects were mediated by cAMP. Indeed, a DB cAMP concentration of 1 μM increased DNA labelling when applied for 8 h to Ca 2+-depleted cultures. However, when Ca 2+ was present, the 8-h treatment with 1 μM DB cAMP was inhibiting. A 4-h pulse with 1 μM DB cAMP just after Ca 2+ addition was a condition for a high stimulation of DNA labelling. The other DB cAMP concentrations used, 0.1 and 10 μM, reduced DNA labelling. In the absence of Ca 2+, RNA labelling was only slightly increased by 0.1 μM DB cAMP, but was highly stimulated by a 4-h treatment of 1 μM DB cAMP in the presence of Ca 2+. The noted effects with 1 or 0.1 μM DB cAMP on DNA or RNA labelling corresponded to true changes in synthesis rather than alterations of the specific activity of the nucleotide pool by DB cAMP. Besides, it was precluded that these effects were due to butyrate issued from DB cAMP degradation. It was further shown that DB cAMP at 1 μM increased Ca 2+ uptake in planarian cells, whereas the other concentration reduced it. This observation might explain the stimulating effect on nucleic acid synthesis of 1 μM DB cAMP applied at the appropriate moment. Based on these results it seems that, for triggering RNA synthesis, the threshold value of Ca 2+ was lower than for DNA synthesis. These Ca 2+ thresholds might be reached, in the absence of Ca 2+ in the medium, by treatments with DB cAMP or hormones at the appropriate doses and periods. This interpretation is in agreement with the succession of biochemical events described in regenerating planarians and suggests that these events might be causally related.

Potentiation of bleomycin lethality by anticalmodulin drugs: a role for calmodulin in DNA repair.

Treatment of exponentially growing Chinese hamster ovary cells with bleomycin causes a dose-dependent decrease in cell survival due to DNA damage. This lethal effect can be potentiated by the addition of a nonlethal dose of the anticalmodulin drug N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide ( W13 ) but not its inactive analog N-(4-aminobutyl)-2-naphthalenesulfonamide ( W12 ). By preventing the repair of damaged DNA, W13 also inhibits recovery from potentially lethal damage induced by bleomycin. These data suggest a role for calmodulin in the DNA repair pathway.

An anticalmodulin drug, W-7, inhibits the voltage-dependent calcium current in Paramecium caudatum.

The anticalmodulin drug, W-7 [N-(6 aminohexyl)-5-chloro-1-naphthalenesulphonamide] specifically inhibits the voltage-dependent Ca-current of Paramecium as well as the behavioural consequence of Ca2+ influx, backward swimming. The dechlorinated analogue, W-5, is four to five times less effective. Analysis of membrane currents under voltage clamp shows that W-7 not only reversibly inhibits the voltage-dependent Ca-current but also shifts the voltage sensitivity of this Ca-current towards less negative voltages in a concentration-dependent manner. We suggest that Paramecium can be used as a system to screen behaviourally for other Ca-channel blockers as well as to study the mechanism of action of these drugs.

Agonist and antagonist properties of calmodulin fragments.

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.

Calmodulin in starfish oocytes: II. Trypsin treatment suppresses the trifluoperazine-sensitive step

The 1-methyladenine-induced oocyte maturation in starfish is reversibly inhibited by the anticalmodulin drug, trifluoperazine (TFP). However, when oocytes are exposed for 10 min to trypsin, they lose their sensitivity to TFP. Trypsin does not alter the length of the hormone-dependent period (1-methyladenine minimal contact time) or the 1-methyladenine concentration requirements. Trypsin-treated oocytes remain sensitive to other maturation inhibitors such as procaine, theophylline, caffeine, and D-600. Trypsin exposure modifies the protein pattern composition of the oocyte cortex (breakdown of a 140-kDa protein). TFP binding site localization was studied using fluorescence microscopy: in addition to a general diffuse fluorescence, staining is localized to probably acidic granules located in the cortex. Results are discussed in relation to calmodulin and plasma membrane calmodulin-dependent enzyme involvement in the stimulation of starfish oocyte maturation.

Volume-induced increase of K+ and Cl- permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+.

Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers , but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl-, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca2+- activated K+ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca2+ ionophore A23187 in the presence of external Ca2+, and also by a sudden increase in external Ca2+. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca2+ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl- permeability of the cell membrane there was no change in cell volume or Cl- content when the K+ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca2+ ionophore A23187 in isotonic medium promoted Cl- loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl- permeability was increased in parallel with and even more than the K+ permeability. It is proposed that separate conductive K+ and Cl- channels are activated during regulatory volume decrease by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin.

Changes in calmodulin and its mRNA accompany reentry of quiescent (G0) cells into the cell cycle.

Release of CHO-K1 cells from plateau or stationary phase and reentry into the cell cycle is specifically and reversibly blocked at two distinct sites by the anticalmodulin drug W13. The first block occurs early during release while the cells are still at G0/G1, whereas the second occurs later in reentry during early S phase. As determined by radioimmunoassay, calmodulin levels undergo changes at three distinct steps in plateau-phase entry and release. First, the entry of exponentially growing cells into plateau phase is accompanied by an increase in the calmodulin level. The second change is a reduction in the calmodulin content of cells within the first hour following release from plateau phase. The third change is the subsequent increase in calmodulin levels, which precedes entry of the cells into S phase. Analysis of calmodulin mRNA levels by dot-blot hybridization demonstrates that the changes in calmodulin protein are preceded by changes in calmodulin mRNA. Furthermore, whereas a decrease in CaM mRNA is observed within the first hour following plateau release, no such decrease is observed for beta-actin mRNA, suggesting that this decrease may be selective for calmodulin. This selectivity is further substantiated by the fact that identical changes in calmodulin and calmodulin mRNA are observed in cells released from plateau by two different techniques. Taken together, these data suggest that calmodulin may play an important role in the reentry of cells into the cell cycle.

Calmodulin-binding proteins in a cloned rat insulinoma cell line.

Calmodulin concentrations were measured in isolated hamster islets or in a cloned rat insulin-secreting cell line RIN-m-5F treated with high glucose. There was no change in the cellular calmodulin content of either islets or RIN-m-5F cells despite increases of insulin concentrations in the media. Treatment of cells with the anti-calmodulin drug W13 inhibited insulin-stimulated glucose release, whereas a small effect on insulin accumulation in the media was observed with W12, the dechlorinated, less active analogue of W13. At the lowest dose tested (30 microM) the effect of W13 on insulin accumulation in the media was completely reversible. To further investigate the possible role of calmodulin in insulin secretion, calmodulin-binding proteins in subcellular fractions of the RIN-m-5F cells were identified using a gel overlay technique. Ca2+-dependent binding of 125I-calmodulin was observed to cytosolic proteins with apparent Mr = 125, 110, 56, 52, and 34 k (kilodalton). This binding was completely displaceable with unlabeled calmodulin, whereas only partial displacement was observed when the homologous Ca2+ binding protein of skeletal muscle, troponin C, was used. Four proteins with Mr similar to the histones bind calmodulin in a Ca2+-independent manner. 125I-calmodulin:calmodulin binding protein interactions were inhibited in a dose-dependent manner by the anti-calmodulin drug, W13. Little effect was observed with the analogue W12.(ABSTRACT TRUNCATED AT 250 WORDS)

Compound [formula omitted] is a selective and powerful inhibitor of calmodulin-regulated functions

Compound 48 80 , a condensation product of N-methyl- p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound 48 80 was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca 2+-transport ATPase, with IC 50 values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca 2+ transport into inside-out red blood cell vesicles by compound 48 80 follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca 2+-transport ATPase induced by calmodulin is inhibited by compound 48 80 according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound 48 80 bind to calmodulin in a Ca 2+-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound 48 80 is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca 2+-transport ATPase that has been described hitherto. In addition, compound 48 80 was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca 2+-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound 48 80 , respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca 2+-transport ATPase or calf cardiac sarcolemma (Na + + K +)-transport ATPase are far less influenced by compound 48 80 as compared with trifluoperazine and calmidazolium. Because of its high specificity compound 48 80 is proposed to be a promising tool for studying calmodulin-dependent processes.

A high-affinity, calmodulin-dependent Ca2+ pump in the basal-lateral plasma membranes of kidney cortex.

A purified preparation of kidney basolateral membrane vesicles is capable of ATP-dependent Ca2+ uptake. The reaction has high affinity for Ca2+ (Km about 0.1 microM) and a V of 5.8 nmol Ca2+ X mg-1 protein X min-1 in the predominantly right-side-out vesicular preparation used. It is inhibited by vanadate (K0.5 about 5 microM) and by anti-calmodulin drugs. A stimulatory effect of calmodulin is visible in membranes depleted of the activator. Exposure of basolateral membranes to 125I-azido-modified calmodulin results in the specific labeling of a membrane protein of Mr 141 000, which is tentatively suggested to be the Ca2+-pumping ATPase.

Inhibition of Ca 2+-dependent protein kinase and [formula omitted] in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca 2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca 2+-dependent Mg 2+-ATPase ( Ca 2+ Mg 2+ -ATPase ). Some of their properties have been compared: their affinity for Ca 2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca 2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca 2+ Mg 2+ -ATPase . R24571 decreased the affinity of the Ca 2+ Mg 2+ -ATPase for Ca 2+ ions (K Ca 0.35 versus 0.75 μM) and exerted an inhibition non-competitive with Ca 2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca 2+ Mg 2+ -ATPase , appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13–20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.

Inhibition of measles virus budding by phenothiazines

HeLa cells infected with measles virus show an accumulation of virus-specific strands at the plasma membrane after addition of the anticalmodulin drugs trifluoperazine (TFP) and chlorpromazine (CPZ), whereas spherical virus particles are almost completely absent. At low drug concentrations (10–15 μ MTFP; 30–40 μ MCPZ) the inhibitory effect is dependent on the presence of extracellular calcium. The strands complete the budding process after removal of the drugs. Restoration of virus budding is not sensitive to cycloheximide and immunoprecipitation experiments give evidence that the viral protein synthesis is not qualitatively altered in the presence of TFP. The data indicate that both drugs arrest the budding process at an intermediate stage at the plasma membrane. The inability of the strands to comigrate with cytochalasin B-induced actin patches suggests that the inhibition of budding is probably the result of an impaired interaction of viral structures with the cytoskeleton.

Calcium regulation of the actin-mediated cytoskeletal transformation of sea urchin coelomocytes.

Coelomocytes from several echinoderm species undergo an actin-mediated cytoskeletal transformation once subjected to hypotonic shock. In this study, coelomocytes from the sea urchins Lytechinus variegatus and Arbacia punctulata were induced to "transform" by treatment with greater than 5 microM of the calcium ionophore A23187 in the presence of external Ca++. The dependence of ionophore transformation on external Ca++ and the lack of chlorotetracycline staining indicates that these cells rely on external Ca++ sources. NBD-phallacidin (7-Nitrobenz-2-oxa-1,3-diazole-phallacidin) staining of lysolecithin permeabilized cells and whole-mount transmission electron microscopy (TEM) show that similar reorganizations of the actin cytoskeleton take place during hypotonic shock and ionophore transformation, although actin filament bundling is less apparent in A23187-treated cells. As has been shown with hypotonic shock transformation, the ionophore elicited shape change is inhibited by anticalmodulin drugs. Greater than 10 microM concentrations of W 13 inhibit filopod formation, while this drug's less active structural analogue, W 12, exhibits no effects. W 13 also appears to disrupt actin filament-membrane associations in the cells. Fluorescent localization of calmodulin using a photooxidized derivative of trifluoperazine indicates a general cytoplasmic distribution with some concentration in filopod core bundles. Coelomocyte transformation may be an example of a cellular shape change regulated by Ca++ through the action of calmodulin modulation of actin-membrane interactions.

Purified red blood cell Ca 2+-pump ATPase: Evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca 2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca 2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca 2+-pump ATPase and/or its lipid environment is suggested.

Calcium uptake in isolated hepatic plasma-membrane vesicles.

A liver plasma-membrane fraction capable of Ca2+ uptake was isolated. The fraction exhibited high Na+, K+-ATPase, low glucose-6 phosphatase activity, and transported alanine in a Na+-dependent fashion. The uptake of Ca2+ was ATP-dependent; UTP, GTP, or CTP did not substitute for ATP. The presence of oxalate did not significantly alter the rate of uptake. The pH optimum of the reaction was basic (no uptake was visible at pH 6.8). These properties are at variance with those of the endoplasmic reticulum Ca2+ uptake system, which is oxalate-dependent, and has an acid pH optimum. The ATP-dependent Ca2+ uptake has a Km(Ca2+) of 1.4 X 10(-8) M and a Vmax of transport of 30 nmol X mg protein-1 X min-1. No conclusive results were obtained on the calmodulin-sensitivity of the process: addition of calmodulin to the vesicles did not stimulate uptake, and the anti-calmodulin drug trifluoperazine had no inhibitory effect. However, another anti-calmodulin drug (R24571) had a limited, but statistically significant, inhibitory action. A partial release of the accumulated Ca2+ from the vesicles could be induced by the addition of Na+, and incubation of the vesicles in a high Na+ medium (as compared to high K+ medium) resulted in lower (about 25%) calcium uptake. Partial release of the accumulated Ca2+ could be induced also by the addition of H+. The releasing effect of H+, taken together with the absence of Ca2+ uptake at acid pH, suggests the possibility of a H+/Ca2+ exchange.

Drugs that block calmoduLin activity inhibit cell-to-cell coupling in the epidermis of Tenebrio molitor.

In many cell systems, the permeability of membrane junctions is modulated by the cytoplasmic level of free Ca++. To examine whether the calcium-dependent regulatory protein calmodulin is involved in this process, the ability of anticalmodulin drugs to influence the cell-to-cell passage of injected current and an organic tracer was tested using standard intracellular glass microelectrode techniques. Several antipsychotics and local anesthetics were found to block junctional communication in the epidermis of the beetle Tenebrio molitor. Treatment of the epidermis with chlorpromazine (0.25 mM) raised intercellular resistance two- to threefold within 20 to 25 min; cell-to-cell passage of electrical current was abolished within 41 +/- 5 min. Loss of electrotonic coupling was accompanied by a block in the cell-to-cell movement of the organic tracer carboxyfluorescein. The reaction is fully reversible, with normal electrotonic coupling being restored within 2 to 4 hr. Other antipsychotics and local anesthetics had similar effects on cell coupling. The order of potency found was: trifluoperazine greater than thioridazine greater than D-butaclamol greater than chlorprothixine = chlorpromazine greater than L-butaclamol greater than dibucaine greater than tetracaine. The relative uncoupling potencies of these drugs correlate well with their known ability to inhibit calmodulin-dependent phosphodiesterase activity. Other anesthetic compounds, procaine and pentobarbital, did not block cell-to-cell communication. Altering the extracellular Ca++ concentration did not affect the rate of uncoupling by antipsychotics, while chelation of extracellular Ca++ with EGTA raised electrotonic coupling. The effect of three metabolic inhibitors on coupling was also examined. Iodoacetate uncoupled the epidermal cells while DNP and cyanide did not. These results are discussed in terms of possible mechanisms by which calmodulin may control junctional communication in this tissue.

The anticalmodulin drugs trifluoperazine and R24571 remove the activation of the purified erythrocyte Ca 2+-ATPase by acidic phospholipids and by controlled proteolysis

The anticalmodulin drugs trifluoperazine and R24571 remove the activation of the purified erythrocyte Ca 2+-ATPase by acidic phospholipids and by controlled proteolysis

Calmodulin antagonists and hormonal control of meiosis in starfish oocytes

Drugs, including chlorpromazine, fluphenazine, trifluoperazine, procaine, dibucaine, theophylline, propranolol, vinblastine, vincristine and W7 [ N-(6-aminohexyl)5-chloro-1-naphthalene-sulfonamide] which have been shown to prevent formation of the ternary activated complex of Ca 2+-calmodulin with several soluble or membrane proteins, inhibit meiosis reinitiation in starfish oocytes when applied from outside. Inhibition occurs only if drugs are present in the medium during the hormone-dependent period. Inhibition can be overcome by raising external concentration of 1-MeAde. In contrast, direct intracellular microinjection of these drugs, even at concentrations exceeding by far I 50 for external application, as well as microinjection of potent and specific anticalmodulin antibodies, has no effect on hormone-induced meiosis reinitiation. These findings support the view that anticalmodulin drugs act at the level of the plasma membrane by interfering either with recognition or with transduction of the hormonal message, rather than by preventing the formation of a ternary activated complex of Ca 2+-calmodulin with some hypothetical intracellular target protein which would be essential for meiosis reinitiation. The implication is that Ca 2+, which is released from the plasma membrane in cytoplasm following 1-MeAde addition, is perhaps not the second messenger of the hormone in meiosis reinitiation of starfish oocytes.