Specific Inhibitor Of Calmodulin Research Articles

A possible role for calmodulin in human thyroid cell metabolism.

A protein which shared several characteristics with authentic calmodulin was extracted from human thyroid homogenates. The protein bound to fluphenazine--Sepharose and could be specifically eluted using EGTA. The eluted protein had a u.v. spectrum characteristic of calmodulin and migrated like authentic calmodulin with a calcium-dependent shift on sodium dodecyl sulphate polyacrylamide-gel electrophoresis. Calmodulin in thyroid cell extracts was shown to be biologically active, measured by its ability to activate a calmodulin-deficient cyclic GMP phosphodiesterase; this activation could be inhibited by trifluoperazine. A possible role for calmodulin in the action of TSH on the thyroid was demonstrated by studying the effects of phenothiazines and the naphthalene sulphonamide, W7, a more specific calmodulin inhibitor, on TSH-stimulated cyclic AMP levels in cultured thyroid cells. The phenothiazines and W7 were found to inhibit the accumulation of cyclic AMP in response to TSH in a concentration-dependent manner although low concentrations of W7 enhanced TSH-stimulated cyclic AMP accumulation.

The role of calmodulin in human natural killer cell activity.

Calcium is known to play an essential role in the lytic mechanism of natural killer cells (NK), which form a subset of large granular lymphocytes. Many of the intracellular effects of calcium are mediated through the calcium-binding protein calmodulin. In this study we have demonstrated that the specific calmodulin inhibitors (naphthalene-sulphonamides) inhibit NK activity in humans at IC50's of 6.9 microM for W7 and 5.2 microM for W13. Comparison of the potency of these compounds with their less active counterparts suggests that NK activity is calmodulin-dependent.

The mechanism of macrophage activation induced by Ca 2+ ionophore

The mechanism of macrophage activation by Ca 2+ ionophore was studied. Peritoneal exudate macrophages from normal guinea pigs exposed continuously to or pulse treated for 1 hr with the ionophore, A23187, were activated, manifesting increased glucose consumption and inhibition of migration. Highly purified macrophages were also activated as effectively as crude macrophage preparations, and the culture supernatant of spleen lymphocytes treated with A23187 lacked a macrophage activating effect, showing that the macrophage activation resulted from the direct effect of A23187 on macrophages, not via lymphokines produced by lymphocytes. The macrophage activation by A23187 was suppressed in the presence of EGTA, but the suppressive effect was overcome by the addition of Ca 2+, but not of Mg 2+. A dilution experiment with Ca 2+ and Mg 2+ during the pulse treatment of cells with A23187 revealed that the activating effect of A23187 was more dependent on Ca 2+ content than Mg 2+. In addition, the Ca 2+ antagonist, nicardipine, was found to suppress the activating effect of A23187. The Ca 2+ uptake into macrophages was increased by treatment with A23187. These results indicate that Ca 2+ influx into cells is primarily important in the macrophage activating effect of A23187. Trifluoperazine (TFP: a specific inhibitor of calmodulin that is an intracellular Ca 2+ receptor protein) was found to inhibit the activating effect of A23187. Further, the cyclic nucleotides, dibutyryl-cAMP and −cGMP, did not activate macrophages. Therefore, macrophage activation was presumed not to be directly mediated by cyclic nucleotides. All these findings show that macrophage activation with the ionophore proceeds by the following scheme: Ca 2+ influx → activation of Ca 2+ receptor protein, calmodulin → activation of calmodulin-regulated enzymes → metabolic changes, activation. TFP was found to suppress the macrophage activation with highly purified guinea pig macrophage activation factor/macrophage migration inhibitory factor (MIF/MAF) or lipopoly-saccharide (LPS), suggesting that calmodulin also played an important role in macrophage activation with MIF/MAF or LPS.

Follicle-stimulating hormone-regulated granulosa cell steroidogenesis: Involvement of the calcium-calmodulin system

The role of the calcium-calmodulin system in the gonadotropic regulation of ovarian steroidogenesis was studied by investigating the influence of various agents known to alter calcium metabolism or calmodulin activity on basal and follicle-stimulating hormone (FSH)-stimulated production of progesterone by rat granulosa cells in vitro. Lanthanum, a specific calcium antagonist, attenuated FSH-stimulated cyclic adenosine monophosphate (cyclic AMP) and progesterone production. [Ethylene- bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) significantly reduced this steroidogenic response but failed to alter the synthesis of the nucleotide. Although progesterone production was markedly increased by dibutyryl cyclic AMP, this was significantly lowered by an inhibitor of calcium uptake, verapamil. FSH-stimulated production of cyclic AMP and progesterone and dibutyryl cyclic AMP-induced progesterone biosynthesis were all significantly reduced by trifluoperazine, a specific inhibitor of calmodulin. These results indicate the existence of at least two sites in the trophic regulation of granulosa cell steroidogenesis which are calcium- and calmodulin-dependent: one localized at the level of cyclic AMP production, and the second at an unidentified step(s) distal to the formation of this nucleotide.

Effects of calmodulin inhibitor on histamine release from rat peritoneal mast cells induced by concanavalin A and ionophore A23187

1. 1. A specific calmodulin inhibitor, W-7 [ N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], inhibited histamine release from rat mast cells induced by either concanavalin A or A23187 at low concentrations (∼ 30 μM) and at high concentrations (∼ 100 μM) enhanced the release. 2. 2. W-5 [ N-(6-amnohexyl)-1-naphthalenesulfonamide], which has virtually calmodulin-inhibiting activity, did not affect the release.

Purification of cardiac sarcolemmal vesicles: high sodium pump content and ATP-dependent, calmodulin-activated calcium uptake.

Highly purified vesicles of cardiac sarcolemma were prepared from a homogenate of canine ventricular muscle by density gradient centrifugation. The preparation showed an extremely high content of (Na+,K+)-ATPase. The steady state levels of Na+-dependent phosphoenzyme formation in the presence of Triton X-100 and the specific ouabain binding in the absence of Triton X-100 were, respectively, 773 and 907 pmol.mg-1 under the optimum conditions. On the other hand, the amount of Ca2+-dependent phosphoenzyme formed in the absence of Triton X--100 was less than 2 pmol.mg-1. This demonstrates that the preparation was virtually free of contaminant sarcoplasmic reticulum fragments. The preparation showed ATP-dependent Ca2+ uptake. Almost all the Ca2+ accumulated on the addition of ATP was rapidly released by the subsequent addition of NaCl. This finding gives evidence that the ATP-driven Ca2+ pump exists in the cardiac sarcolemma. The Ca2+ uptake was unaffected by 2 microM digitoxin, 1 microM monesin, and 200 microM dinitrophenol. These results exclude the possibility that transmembrane gradients of Na+ and H+ were involved in this Ca2+ uptake. The Ca2+ pump was activated by calmodulin. The concentration of calmodulin giving a half-maximum activation was 0.05 micrograms.ml-1, which is equivalent to 3 nM. This activation was removed by addition of trifluoperazine, a specific inhibitor of calmodulin.

Effect of trifluoperazine on the stimulation by Ca 2+-dependent hormones of gluconeogenesis from glutamine in isolated hepatocytes

In continuation of our previous studies we have investigated some aspects of the hormonal control of glutamine metabolism is isolated rat hepatocytes. (1) Catecholamines, angiotensin II and vasopressin stimulate gluconeogenesis from glutamine more than 2-fold. These effects require the presence of Ca 2+ in the incubation medium. (2) The phenothiazine, trifluoperazine, a purported specific inhibitor of calmodulin, completely blocks the stimulation by catecholamines without affecting the response to the other two hormones. (3) The effectiveness of trifluoperazine in preventing the stimulation of gluconeogenesis by catecholamines was dependent on the concentrations of both the hormone and the inhibitor. (4) Trifluoperazine, at concentrations that prevent stimulation by epinephrine of gluconeogenesis, was as effective as phentolamine in blocking the binding of [ 3H]epinephrine to intact hepatocytes. (5) These studies support the view (Blackmore, P.F., El-Refai, M.F., Dehaye, J.-P., Strickland, W.G., Haghes, B.P. and Exton, J.H. (1981) FEBS Lett. 123, 245–248) that inhibition by trifluoperazine of α-adrenergic stimuli does not necessarily mean that calmodulin is involved in post-receptor events.

Cyclic nucleotide phosphodiesterase of rat pancreatic islets. Effects of Ca2+, calmodulin and trifluoperazine.

Cyclic nucleotide phosphodiesterase activity towards cyclic AMP and cyclic GMP was studied in extracts of rat islets of Langerhans. Biphasic Eadie plots [Eadie (1942) J. Biol. Chem. 146, 85-93] were obtained with either substrate suggesting the presence of both 'high'- and 'low'-Km components. The apparent Km values were 6.2 +/- 0.5 (n = 8) microM and 103.4 +/- 13.5 (6) microM for cyclic AMP and 3.6 +/- 0.3 (12) microM and 61.4 +/- 7.5 (13) microM for cyclic GMP. With cyclic AMP as substrate, phosphodeisterase activity was increased by calmodulin and Ca2+ and decreased by trifluoperazine, a specific inhibitor of calmodulin. With cyclic GMP as substrate, phosphodiesterase activity was decreased by omission of Ca2+ or addition of trifluoperazine. Addition of exogenous calmodulin had no effect on activity. The data suggest that Ca2+ may influence the islet content of cyclic AMP and cyclic GMP via effects on calmodulin-dependent cyclic nucleotide phosphodiesterase(s).