Biological Activity Of Calmodulin Research Articles

Involvement of altered cytoskeletal protein phosphorylation in aluminum-induced CNS dysfunction.

In the present investigation, we have studied the effects of aluminum (10 mg/kg of body weight per day, i.p.) and desferrioxamine (6 mg/kg of body weight per day, i.p.) alone and in combination for 4 weeks on the regulation of phosphorylation of neuronal proteins. A marked decrease in the biological activity of calmodulin was observed after aluminum treatment; however, in combination with desferrioxamine, a reversal in the levels of calmodulin, in terms of nmol cAMP hydrolyzed/min/mg protein, was observed. Exogenous addition of calmodulin had an inhibitory effect on calmodulin-mediated synaptosomal protein phosphorylation in aluminum-exposed animals. An almost complete reversal of this inhibition was observed following coexposure to aluminum and desferrioxamine. Cyclic AMP-dependent synaptosomal protein phosphorylation was, however, stimulated following aluminum exposure. Coadministration of desferrioxamine along with aluminum was found to mitigate the neurotoxic effect of aluminum.

Effect of Lead on the Biological Activity of Calmodulin in Rat Brain

In the present investigation, we observed that lead in vitro activates calmodulin at lower concentrations, and the maximum activation was observed at 30 μM concentration. In vivo lead exposure (50 mg/kg body weight, intragastrically) for a period of 8 weeks also stimulated the activity of calmodulin by 45%. The addition of trifluoperazine resulted in partially inhibiting the lead-stimulated calmodulin activity, whereas the calcium-stimulated calmodulin activity was completely inhibited by trifluoperazine. Studies with purified calmodulin from the brain of control and lead-treated animals indicate that approximately 4 mole of calcium was present bound/mole of calmodulin in control animals and this fraction was reduced in lead-treated animals to approximately 3 mole of calcium/mole of calmodulin. Lead distribution revealed that approximately 68% of the total lead present was bound to calmodulin and the remaining 32% present was bound to non-calmodulin binding sites following lead exposure. These results indicate that in vivo lead exposure is able to displace and mimic the action of calcium and this may constitute a molecular mechanism of lead neurotoxicity.

In vitro effects of organophosphorus compounds on calmodulin activity

In vitro effects of organophosphorus compounds (OP), such as malathion (M), methyl parathion (MP) and ethyl parathion (EP), on calmodulin (CaM) activity and its active conformation were studied to understand the mechanism(s) of neurotoxicity, since CaM is known to regulate Ca2+ transport and the enzymes involved in signal transduction and nucleotide metabolism. The biological activity of CaM was assessed as a measure of phosphodiesterase (PDE) stimulation. The effect of OP compounds on the active conformation of CaM was determined by studying the binding of fluorescence probes, namely N-phenyl-1-naphthylamine (NPN), and changes in dansyl-calmodulin fluorescence. Dansylated calmodulin was also used to study the effect of OP compounds on complex formation between CaM and PDE. All three OP compounds inhibited the CaM activity and its active conformation in a concentration-dependent manner. Malathion was less effective in comparison to EP and MP, with IC50 values of 37 microM, 34.5 microM and 32 microM, respectively, for CaM activity. EP and MP significantly altered NPN and dansyl-calmodulin fluorescence (50 microM concentrations of OP compounds), whereas M did not show any significant effect on NPN fluorescence. All these compounds significantly affected complex formation between the dansylated CaM and PDE. These results suggest that OP compounds may be interacting with CaM, altering its active conformation, and thus may be inhibiting its biological activity.

Kinetic studies on the biological activity of calmodulin in canine heart and liver during endotoxin shock.

Effects of endotoxin administration on the biological activity of calmodulin isolated from canine heart and liver were studied. Calmodulin was isolated and purified to homogeneity. The biological activity of calmodulin was determined by its ability to activate Ca(2+)-dependent phosphodiesterase. Results obtained 4 h after endotoxin administration show that the Vmax and A0.5 for calmodulin, the Vmax and Km for cAMP, and the Vmax and the Hill coefficient for Ca2+ were unchanged, while the S0.5 for Ca2+ for the activation of phosphodiesterase were significantly increased in the heart. The kinetic parameters as described above were not significantly altered in the liver. These data indicate that the biological activity of calmodulin is inhibited in the heart during endotoxin shock and that the nature of inhibition is associated with a mechanism involving a decrease in the affinity (1/S0.5) towards Ca2+ binding. Since calmodulin plays an important role in the regulation of cardiac function through calmodulin-dependent calcium transport systems, our findings may have a pathophysiological significance in contributing to the understanding of myocardial dysfunction in endotoxin shock.

Modulation of Calmodulin Properties by Amiodarone and its Major Metabolite Desethylamiodarone*

Long-term amiodarone therapy is invariably associated with some side effects. Although its mechanism of action, as an antiarrhythmic drug is well understood, the side effect profile of amiodarone is not yet established. To determine possible mechanisms, the interaction of amiodarone and its major metabolite desethylamiodarone with calmodulin was investigated, since calmodulin is known to regulate Ca2+ transport, cell proliferation and the enzymes involved in signal transduction and nucleotide metabolism. The interaction between the drugs and calmodulin was studied by monitoring intrinsic tyrosine fluorescence of calmodulin and by using a fluorescent probe, N-phenyl-1-naphthylamine (NPN). 14C-Chlorpromazine displacement studies were conducted to differentiate the specific binding sites. The effect on the biological activity of calmodulin was determined with calmodulin dependent phosphodiesterase and Ca2(+)-ATPase. The dansyl calmodulin was used as fluorescent probe to study the effect of these drugs on complex formation between calmodulin and phosphodiesterase. Both amiodarone and desethylamiodarone decreased tyrosine fluorescence of calmodulin with IC50 of 4.9 and 4.4 microM respectively and these interactions were Ca2(+)-dependent. NPN fluorescence was also affected in a concentration dependent manner. These drugs also displaced bound 14C-chlorpromazine from calmodulin and the effect was biphasic. However, desethylamiodarone was more potent than amiodarone. The binding of 3H-amiodarone to calmodulin was modified by a variety of compounds, one class of compounds decreased and the other increased 3H-amiodarone binding to calmodulin. Only, desethylamiodarone inhibited the phosphodiesterase activation by calmodulin with IC50 of 13.2 microM without changing the basal enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Cadmium inhibits brain calmodulin:In vitro andin vivo studies

The toxic effects of divalent cations especially Cd{sup 2+}, are related to their chemical and physical characteristics viz. ion polarizability, electronic structure and the hard and soft characteristics. Cadmium ion, a hazardous environmental pollutant in areas in which cadmium nickel batteries are manufactured, accumulates in tissues such as kidney, liver, brain or bone with a very slow turnover and can cause pathophysiological disorders. Calmodulin (CaM), a Ca{sup 2+} binding protein is found in many if not most eukaryotic cells. The protein mediates the control of a large number of enzymes by Ca{sup 2+} fluxes and alterations in these fluxes have been postulated to be involved in steps leading to irreversible cell damage. The present study demonstrates an interaction of Cd{sup 2+} with CaM resulting in an impairment of CaM-dependent phosphodiesterase (PDE) stimulation in vitro. Biological activity of CaM is also affected in brain (cerebral cortex) of Cd-exposed rats in vivo.

Altered calmodulin activity in buccal epithelial cells from cystic fribrosis patients

Boiled extracts of buccal epithelial cells from control subjects and cystic fibrosis patients were shown to possess calmodulin like activity, as assessed by their ability to activate calmodulin-deficient cyclic AMP phosphodiesterase. Estimation of calmodulin content, using pure calmodulin as standard revealed that control extracts contained 3.08 ± 0.71 sem ( n = 7) μg calmodulin/mg protein and cystic fibrosis extracts 0.88 ± 0.30 sem ( n = 12) μg calmodulin/mg protein ( p < 0.02 for difference from control). The results indicate that the biological activity of calmodulin is altered in buccal epithelial cells from cystic fibrosis individuals.

Compartmentalization of calmodulin and tubulin in the male C57BL/6J mouse brain: Heterogeneity of age changes in calmodulin compartments

Calmodulin (CaM) and tubulin were analyzed by radioimmunoassay in subcellular fractions prepared from cerebral cortex and striatum of aging male C57BL/6J mice. Three fractions were prepared by a new procedure: (1) cytosol (soluble); (2) EGTA-releasable, membrane-bound; and (3) detergent-extractable (Triton X-100), membrane-bound fractions. CaM concentration in all three fractions prepared from striatum showed small (10–15%, p <0.05) decreases with age (3–31 months). Cortical CaM concentrations were less affected by age, and only the EGTA-releasable fraction decreased. To compare functional activity and immunoreactivity of CaM, soluble CaM was also assayed by the activation of cyclic nucleotide phosphodiesterase (PDE). The radioimmunoassay and PDE activation assays gave equivalent results, suggesting that no alteration occurred with age in biological activity of CaM, via post-translational modification or other mechanisms. Soluble and particulate tubulin concentration did not change significantly with age in either brain region. The changes observed in striatal CaM, particularly in membrane-bound compartments, could contribute to the age-related decline in mammalian basal ganglial function.