Presence Of Exogenous Calmodulin Research Articles

Dantrolene Inhibition of Skeletal Muscle RYR in the Presence of CAM

Dantrolene is a muscle relaxant that has been used clinically as the treatment for malignant hyperthermia (MH). Our group recently reported that dantrolene inhibition of RyR1 and RyR2 required calmodulin (CaM)1. However, nothing more is known about the properties of dantrolene inhibition of RyR1 on single channels. Here, we characterized dantrolene inhibition on RyR1 in the presence of exogenous CaM at various cytosolic [Ca2+] and in the presence of other activators: ATP, halothane and domain peptide, DP4. RyR1 was isolated from rabbit skeletal muscle and incorporated into artificial lipid bilayers and their open probability, Po, was measured using single channel recording. Dantrolene caused inhibition of RyR1 and RyR2, but when CaM was presence in the cytoplasmic bath. In the presence of cytoplasmic [Ca2+] (100 nmol/l) and 2 mmol/l ATP, dantrolene inhibited both RyR1 and RyR2 with identical hyperbolic dose-responses with IC50 of 0.16 ± 0.03 μmol/l and with saturating Po of 52 ± 4 % of control. RyR1 has a bell-shaped cytoplasmic Ca2+ activation curve with half activation at 1.75 ± 0.62 μmol/l and half inhibition at 0.21 ± 0.02 mmol/l and a peak Po = 0.94 ± 0.06. Dantrolene reduced Po in sub-activating and inhibiting [Ca2+] but failed to reduce peak Po. Dantrolene caused 50-60% reduction in Po of RyR1 activated by cytoplasmic Ca2+ alone (100 nmol/l) or in conjunction with halothane (5 mmol/l) or DP4 (10 μmol/l). To conclude, dantrolene inhibits RyR1 activity by 1) decreasing RyR1 sensitivity to Ca2+ activation, 2) increasing sensitivity to Ca2+ inhibition and 3) decreasing channel activation halothane or by 4) reducing the effect of inter-domain disruption in the RyR1 protein.

Distinct Mechanisms of Regulation by Ca2+/Calmodulin of Type 1 and 8 Adenylyl Cyclases Support Their Different Physiological Roles

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.

A new method to precipitate myosin V from rat brain soluble fraction.

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.

The Role of Calmodulin Recruitment in Ca2+ Stimulation of Adenylyl Cyclase Type 8

Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.

Expression and activity of nitric oxide synthase isoforms in rat brain during the development of experimental allergic encephalomyelitis

The activity and expression of nitric oxide synthase (NOS) isoforms and protein nitrotyrosine (NT) residues were investigated in whole encephalic mass (WEM) homogenates during the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. EAE stages (0–III) were daily defined by clinical evaluation, and in the end of each stage, WEMs were removed for analysis of NOS activity, protein NT residues and mRNA for the different NOS isoforms. In the presence of NADPH, WEMs from EAE-III rats showed lower Ca 2+-dependent NOS activity than those from control group. These differences disappeared in the presence of exogenous calmodulin, flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH 4) and NADPH. Of all the cofactors, just the omission of FAD caused comparable decrease of Ca 2+-dependent NOS activity from both groups. Ca 2+-independent NOS activity from EAE-III animals was insensitive to the omission of any of the cofactors, while in control animals this activity was significantly inhibited by the omission of either FAD or BH 4. Increased levels of both iNOS mRNA and protein NT expression were observed in animals with EAE, which also showed lower levels of a thermolabile NOS inhibitor in WEM homogenates and sera than controls. In conclusion, during late EAE stages, constitutive Ca 2+-dependent NOS activity decreases concomitantly with iNOS upregulation, which could be responsible for the high protein NT levels. The differential dependence of iNOS activity on cofactors and the absence of an endogenous thermolabile NOS inhibitor in animals with EAE could reflect additional control mechanisms of NOS activity in this model of multiple sclerosis.

Myosin I from mammalian smooth muscle is regulated by caldesmon-calmodulin.

A single-headed monomeric myosin (myosin I) was isolated from pig urinary bladder smooth muscles and purified to homogeneity. Myosin I from smooth muscle is composed of a 110-kDa heavy chain and three 17-kDa light chains. The heavy chain from smooth muscle myosin I does not cross-react with the antibody against conventional myosin (myosin II) from smooth muscle, but it does show antigenic similarity to adrenal medulla myosin I heavy chain. The light chain from smooth muscle myosin I is similar to calmodulin in molecular weight, amino acid composition, and migration on SDS-polyacrylamide gel electrophoresis in the presence of Ca2+. The high salt ATPase activity of myosin I in the presence of CaCl2 is higher than that in K(+)-EDTA. Smooth muscle actin causes a 5-10-fold activation of the Mg-ATPase activity of myosin I. In the presence of Ca2+, exogenous calmodulin enhances the actin-activated ATPase activity of myosin I, and the increased activity is associated with the binding of exogenous calmodulin to myosin I heavy chain. A maximum of 4 mol of light chains/mol of myosin I heavy chain is observed in the presence of exogenous calmodulin. Caldesmon, a calmodulin/actin-binding protein, inhibits the actin-activated ATPase activity of myosin I. This inhibition is reversed by exogenous calmodulin in the presence of Ca2+. The actin activation of myosin I ATPase exhibits around 50% Ca2+ sensitivity in the presence of exogenous calmodulin. When caldesmon is bound to actin, Ca2+ sensitivity is increased to 80% in the presence of calmodulin. Therefore, smooth muscle caldesmon, which is thought to play a role in the regulation of actin activation of myosin II, also regulates the actin activation of myosin I ATPase in smooth muscle.

Tonoplast localization of a calmodulin-stimulated Ca 2+-pump from maize roots

The subcellular localization of a calmodulin-stimulated calcium (Ca 2+)-ATPase activity from maize roots ( Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca 2+ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure 45Ca 2+ transport in sealed vesicles. Most of the ATP-dependent Ca 2+ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H +/Ca 2+ antiport) and the plasma membrane. When the Ca 2+ transport was measured in the presence of exogenous calmodulin (1 μM), a 3–5-fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, rulling out a direct effect of calmodulin on the H +/Ca 2+ antiport. In conclusion, four different Ca 2+ transporters are present in young maize root cells. A Ca 2+/H + antiport system is present on the tonoplast, whereas, the plasma membrane and the ER posses each a calmodulin-insensitive Ca 2+-ATPase. Finally, a calmodulin-stimulated Ca 2+-ATPase is associated with the tonoplast.

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1–60 μM and 50–370 μM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca 2+-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca 2+-binding affinity was 1.5–2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca 2+-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca 2+-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca 2+-ATPase activity of permeabilized erythrocytes. At Ca 2+ concentrations below 0.7 μM, ouabain (0.5–1 mM) activated whereas at higher Ca 2+ concentrations it inhibited the Ca 2+-ATPase activity. Taking this observation into account the Na +/K +-ATPase was determined as the difference of between the ATPase activities in the presence of Na + and K + and in the presence of K + alone. At physiological concentration of Mg 2+ (370 μM), the addition of 0.3–1 μM Ca 2+ increased Na +/K +-ATPase activity by 1.5–3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg 2+ concentration (e.g., 50 μM) only Na +/K +-ATPase inhibition by Ca 2+ was seen. It was found that at [NaCl] < 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca 2+-free media. This activating effect of furosemide was enhanced with a diminution of [Na +] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na +/K +-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb + influx into intact erythrocytes.

Sensitivity of the Human Myometrial Adenylate Cyclase to Calcium and Calmodulin

The calcium-calmodulin-dependent regulation of adenylate cyclase was studied in membranes from pregnant human myometrium. In the absence or presence of exogenous calmodulin, free calcium concentrations greater than 50 nmol/l inhibited the adenylate cyclase activity. Activation of the enzyme by calmodulin (0.1-1 mumol/l) was calcium-dependent and maximal at 10 nmol/l free calcium. The myometrial adenylate cyclase activity was stimulated by the guanyl nucleotide, Gpp(NH)p. In the presence of the guanyl nucleotide, the activatory effect of the calcium-calmodulin complex disappeared. The activatory effect of exogenous calmodulin was dependent on endogenous calmodulin present in the myometrial membranes. Trifluoroperazine and calmidazolium were able to inhibit the adenylate cyclase activity.

Interactions of cardiac glycosides with cells and membranes. Therapeutic and toxic doses of ouabain acting on sodium and calcium pumps in plasma membranes

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1–60 μM and 50–370 μM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2+-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2+-binding affinity was 1.5–2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2+-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2+-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2+-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 μM, ouabain (0.5–1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2+-ATPase activity. Taking this observation into account the Na+/K+-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 μM), the addition of 0.3–1 μM Ca2+ increased Na+/K+-ATPase activity by 1.5–3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 μM) only Na+/K+-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] < 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2+-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K+-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.

A calmodulin dependent protein kinase activity associated with rabbit heart sarcolemma.

Sarcolemmal vesicles prepared from rabbit heart muscle by differential and discontinuous sucrose density gradient centrifugation, exhibited high marker enzyme activities: Na+ + K+ ATPase 22 microMol Pi x mg-1 x h-1, Adenylate cyclase 500 pmole cAMP x mg-1 x min-1, calcium antagonist receptors 0.7 pmoles x mg-1. Calmodulin in the presence of calcium and gamma-ATP32 stimulated rapidly and specifically the 32P incorporation into two membrane proteins of 54 and 44 kDa. Calmodulin stimulated the phosphorylation of the 44 kDa to a greater extent (17.9 pmol 32P x mg-1 protein) than the 54 kDa protein (1.3 pmoles 32P x mg-1 protein). Removal of endogenous calmodulin from the membrane by EGTA extraction resulted in a 2.5 fold increase in calmodulin dependent 32P incorporation into the two proteins in the presence of exogenous calmodulin. It is suggested that the calmodulin dependent protein kinase activity in heart sarcolemma may mediate the effects of calmodulin in the regulation of Ca2+ transport across the membrane.

Effect of calcium on adenylate cyclase of rat anterior pituitary gland.

The effect of free calcium (Ca2+) on adenylate cyclase (AC) activity of rat anterior pituitary gland have been investigated in order to shed some light on the interrelationships between the two second messengers (cAMP and calcium) which operate in pituitary cells. Anterior pituitary homogenates or crude membranes preparations (obtained using buffers free of divalent cation chelators) were assayed and the concentrations of Ca2+ in the assay mixture containing EGTA were calculated by a computer program for each addition of CaCl2. A wide range of Ca2+ concentrations (from 2 X 10(-9) to 6 X 10(-4)M) was spanned. Ca2+ was found to markedly inhibit pituitary AC and the mathematical analysis of data indicated the presence of two inhibition The two KiS were: 1.78 +/- 0.48 X 10(-7) M and 2.47 +/- 0.52 X 10(-4) M for the homogenates and 1.71 +/- 0.45 X 10(-7) M and 3.15 +/- 0.85 X 10(-4) M for the membrane preparations. No stimulation of the enzyme could be detected at any Ca2+ concentration tested. Furthermore, because of our experimental conditions it is unlikely that there was substantial loss of endogenous calmodulin, or other calcium binding protein(s) required to mediate AC stimulation by calcium. The lack of a calcium-calmodulin stimulation of pituitary AC was confirmed by experiments with anticalmodulin drugs (trifluoperazine and calmidazolium, R24571) and experiments with EGTA-washed membranes in the presence of exogenous calmodulin. At any Ca2+ concentration, the same AC activity was observed in the presence and in the absence of anticalmodulin drugs or added calmodulin. The mechanism of pituitary AC inhibition by Ca2+ was investigated focusing on a range of Ca2+ concentrations near the Ki for the high affinity calcium site and thus similar to the intracellular Ca2+ concentrations. Ca2+ was found to act as a competitive inhibitor of the Mg2+ activation of AC and as a noncompetitive inhibitor with respect to the MgATP2-, the substrate of the enzyme. The effects of Ca2+ on AC were also studied in cell populations and tissues extremely rich in PRL-secreting cells (cell fractions purified from rat anterior pituitaries and human prolactinomas). The pattern of Ca2+ action was found to be nearly superimposable on that observed in total pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)

Antidepressants and protein kinases: Inhibition of Ca 2+-regulated myosin phosphorylation by fluoxetine and iprindole

The effects of several antidepressant and antipsychotic agents on Ca 2+-calmodulin-regulated myosin light chain phosphorylation were evaluated. At a concentration of 100 μM, the antidepressant agents buproprion, mianserin and maprotiline were ineffective; zimelidinem, desipramine and imipramine produced 40–50% inhibition; and iprindole and fluoxetine produced 75–90% inhibition. The efficacies of iprindole and fluoxetine were similar to the phenothiazine antipsychotics chlorpromazine and trifluoperazine. Clozapine, and atypical antipsychotic and the butyrophenone haloperidol were relatively ineffective as myosin light chain phosphorylation inhibitors. IC 50 values of the most effective agents were: trifluoperazine 16 μM, fluoxetine 28 μM, chlorpromazine and iprindole 56 μM. As with trifluoperazine, inhibition of myosin phosphorylation by iprindole was completely attenuated in the presence of exogenous calmodulin. However, a significant component (30%) of the inhibitory effect of fluoxetine was not reversible with calmodulin. These results show that some antidepressant agents, most notably iprindole and fluoxetine, aer capable of antagonizing a calmodulin-regulated protein kinase through calmodulin inhibition; and in the case of fluoxetine, through an additional calmodulin-independent mechanism.