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PubMed1983

Calcium/calmodulin-regulated guanylate cyclase of the excitable ciliary membrane from Paramecium. Dissociation of calmodulin by La3+: calmodulin specificity and properties of the reconstituted guanylate cyclase.

Ca2+-regulated guanylate cyclase in ciliary membranes from Paramecium contained tightly bound calmodulin. Antisera against calmodulin from Tetrahymena and soybean inhibited enzyme activity. EGTA did not easily release calmodulin; however, La3+ inhibited guanylate cyclase by dissociation of calmodulin. While La could not replace Ca in the activation of guanylate cyclase, it substituted for Ca2+ in the activation of calmodulin-dependent phosphodiesterase from pig brain independently of whether homologous or Paramecium calmodulin was used. After removal of endogenous calmodulin from guanylate cyclase, reconstitution was achieved with calmodulin from Paramecium, Tetrahymena, pig brain, and soybean. Ca2+-binding proteins lacking trimethyllysine like calmodulin from Dictyostelium, parvalbumin, and troponin C failed to restore enzyme activity. The properties of the native and reconstituted guanylate cyclase/calmodulin complex were compared. Reassociation of calmodulin with its target enzyme was weak since all calmodulin remained in the supernatant after a single centrifugation. While most enzyme characteristics remained unchanged in the reconstituted complex, the inhibition by Ca greater than 100 microM was of a mixed-type compared to noncompetitive inhibition in the native enzyme. The regulation of the enzyme by cations was also altered. Whereas Ca was the most potent and specific activator of the native enzyme, in the reconstituted system Sr was far more effective.

Animals↗
PubMed1993

Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation.

When extracts of mouse testis were Western-blotted against a monoclonal antibody which reacts with calmodulin in the presence of Ca2+, all calmodulin was associated with the macromolecules of molecular weight above 50 kDa. Immuno-electron microscopy of testes using this antibody indicated that calmodulin is localized at higher density in the nucleus and cytoplasm of germ cells during the developmental phase between pachytene and round spermatid, showing the highest level just before meiotic divisions. There was no special association of calmodulin to any organelles in these cells. Extremely low levels of calmodulin occurred in spermatogonia and other testicular tissue cells. Calmodulin decreased dramatically as spermatids underwent metamorphosis, becoming detectable only at the perinuclear space of sperm heads. Further relocation to the postacrosomal region occurred during sperm transit to the cauda epididymis. Immunodetection after the calmodulin overlay on ultrathin sections revealed a sharp increase of calmodulin immunogold deposits in the nuclei of spermatids accompanying their condensation. The results indicate that some calmodulin-binding proteins, but not calmodulin itself, accumulate in the nuclei during the final steps of spermiogenesis.

Animals↗
PubMed1984

Comparison of calmodulin-dependent glycogen synthase kinase from skeletal muscle and calmodulin-dependent protein kinase-II from brain.

Calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle and calmodulin-dependent protein kinase-II from rat brain were found to have remarkably similar substrate specificities. Both protein kinases phosphorylated synapsin -I, glycogen synthase, smooth muscle myosin light chains, histone H1 and acetyl-CoA carboxylase at the same relative rates. Site-2 of glycogen synthase was preferentially phosphorylated by both enzymes, followed by a slower phosphorylation of site-1b. Each protein kinase catalysed a 2-fold activation of tryptophan 5-monooxygenase. Calmodulin-dependent protein kinase-II and glycogen synthase kinase exhibited similar immunological cross-reactivity in the presence of Ca2+ and calmodulin, using monoclonal antibody raised against the rat brain enzyme. In the absence of Ca2+ and calmodulin, cross-reactivity of glycogen synthase kinase was decreased, whereas that of calmodulin-dependent protein kinase-II was not. The two enzymes appear to represent different isoenzymes of a multifunctional calmodulin-dependent protein kinase that may mediate many of the actions of Ca2+ in mammalian tissues. The results demonstrate that calmodulin-dependent protein kinase-II is identical to calmodulin-dependent synapsin -I kinase-II, previously shown to be very similar to calmodulin-dependent glycogen synthase kinase [(1983) FEBS Lett. 163, 329-334].

Animals↗
PubMed1985

Cardiac sarcoplasmic-reticulum calmodulin-binding proteins. Modulation of calmodulin binding to phospholamban by phosphorylation.

The gel-overlay technique with 125I-labelled calmodulin allowed the detection of several calmodulin-binding proteins of Mr 280 000, 150 000, 97 000, 56 000, 35 000 and 24 000 in canine cardiac sarcoplasmic reticulum. Only two calmodulin-binding proteins could be identified unambiguously. Among them, the 97 000-Mr protein that undergoes phosphorylation in the presence of Ca2+ and calmodulin, is likely to be glycogen phosphorylase. In contrast, the (Ca2+ + Mg2+)-activated ATPase did not appear to bind calmodulin under our experimental conditions. The second known calmodulin target is dephosphophospholamban, which migrates with an apparent Mr of 24 000. The dimeric as well as the monomeric form of phospholamban was found to bind calmodulin. Phospholamban shifts the apparent Kd of erythrocyte (Ca2+ + Mg2+)-activated ATPase for calmodulin, suggesting thus a tight binding of calmodulin to the proteolipid. Interestingly enough, phospholamban phosphorylation by either the catalytic subunit of cyclic AMP-dependent protein kinase or the Ca2+/calmodulin-dependent phospholamban kinase was found to inhibit calmodulin binding.

Animals↗
PubMed1993

Calmodulin and calmodulin-binding proteins in hair bundles.

Calcium ion plays an important role in the hair cell's mechanoelectrical transduction process; in particular, Ca2+ controls adaptation to protracted mechanical stimuli. Because calmodulin is a ubiquitous intracellular receptor for Ca2+ and has been shown to accumulate at the tips of stereocilia, we determined its concentration and identified the proteins with which it interacts in the hair bundle. By performing quantitative immunoblot analysis on isolated bundles, we ascertained that the average concentration of calmodulin within each stereocilium is approximately 70 microM. Extraction experiments disclosed that, in the presence of 20 microM Ca2+, 50% of the calmodulin is bound to detergent-soluble receptors. To distinguish these receptors, we developed an assay that utilizes calmodulin crosslinked to alkaline phosphatase. This technique is approximately 100-fold more sensitive than calmodulin-binding assays that employ 125I- or biotin-labeled calmodulin. When used with chemiluminescence detection in a blot-overlay assay, the calmodulin-alkaline phosphatase conjugate identified hair-bundle proteins of molecular masses 25, 35, 145, 175, 240, and 350 kDa. We examined the subcellular distribution of these receptors; all but the 240-kDa molecule are soluble in a nonionic detergent. The relatively high concentration of calmodulin and the presence of several calmodulin-binding proteins provide evidence for a role of calmodulin in hair bundles.

Alkaline Phosphatase↗
PubMed1983

On the mechanism of interaction between calmodulin and calmodulin-dependent proteins.

Molecular and kinetic studies of the interaction between calmodulin and calmodulin-dependent proteins have been reviewed. Several calmodulin-dependent proteins have been purified to homogeneity and characterized in terms of subunit structure in recent years. The results indicate that these proteins do not contain a common subunit as the basis of calmodulin binding. A monoclonal antibody capable of interacting with several calmodulin-dependent proteins has been obtained, suggesting that these proteins contain common structure. It seems that hybridoma technology may be used for probing calmodulin-binding domains in the calmodulin-dependent proteins. Using a fluorescent-labelled cyclic nucleotide phosphodiesterase, the interaction between calmodulin and the enzyme in the absence of Ca2+ can be demonstrated, and the equilibrium constant of the reaction can be determined. The study further defines the multiple interactions in the activation of the cyclic nucleotide phosphodiesterase by Ca2+ and calmodulin. Previous kinetic results along with the present results are summarized and used to elucidate the regulatory significance of the multiple Ca2+-binding of calmodulin.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMedDate not supplied

Monovalent cation-insensitive hydrophobic region on calmodulin facilitates the rapid isolation and quantitation of calmodulin free from other Ca2+-dependent hydrophobic proteins.

Calmodulin binds quantitatively to phenyl-Sepharose through its Ca2+-induced hydrophobic binding region. Troponin C and S-100 protein, as well as several other proteins present in rat tissues, also bind to phenyl-Sepharose in a Ca2+-dependent manner. While the Ca2+-dependent binding of calmodulin to phenyl-Sepharose is not altered appreciably by monovalent cations, they do appear to compete for Ca2+ binding to most of the other proteins, including S-100 protein, which exhibit Ca2+-induced interaction with phenyl-Sepharose. The selective elution of these proteins from the phenyl-Sepharose column can be achieved with a 0.5 M concentration of monovalent cations such as K+, Na+, and NH4+ in the presence of a low (100 microM) Ca2+ concentration. Calmodulin-binding proteins associated with calmodulin in crude cell extracts can prevent the interaction of calmodulin with the phenyl-Sepharose, resulting in low recoveries of calmodulin from these tissues. The majority of these interfering proteins are heat labile so that heat treatment (boiling) of the cell extract for a limited time (5 min) negates any binding of these proteins to calmodulin and allows the quantitative recovery of calmodulin by hydrophobic interaction chromatography. This procedure allows the rapid and quantitative recovery of highly purified calmodulin from both cytosolic and Triton X-100-solubilized particulate fractions prepared from various rat tissues. Calmodulin isolated in this manner can be accurately and reliably quantitated by direct protein determination with Coomassie brilliant blue dye or fluorescamine or by the cyclic nucleotide phosphodiesterase stimulation assay.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Alteration of calmodulin-protein interactions by a monoclonal antibody to calmodulin.

The effects of specific anti-calmodulin monoclonal antibodies on the conformation and interaction of calmodulin with two enzymes, the insulin receptor tyrosine kinase and casein kinase II, are examined. Addition of the anti-calmodulin antibody 2D1 in vitro augments phosphorylation of calmodulin by rat hepatocyte insulin receptors 4.9 +/- 0.5-fold (n = 7). Nonimmune immunoglobulin has no effect. Maximal phosphorylation is observed at a molar ratio of calmodulin:antibody of approx. 2:1, with higher concentrations of antibody producing lesser enhancement. Increasing Ca2+ concentrations in the physiological range progressively inhibit phosphorylation both in the absence and presence of antibody 2D1. Phosphate is incorporated predominantly on Tyr-99, which is distant from the antibody binding site. Enhancement of casein kinase II-catalyzed calmodulin phosphorylation is also produced by the antibody 2D1, implying that antibody binding induces a change in calmodulin conformation. In contrast, two other anti-calmodulin monoclonal antibodies, 4F4 and 4G2, decrease phosphorylation of calmodulin by both the insulin receptor kinase and casein kinase II. These data indicate that secondary and tertiary structures are important in enzyme-substrate interactions and suggest that the antibodies may be useful in investigating the mechanism of calmodulin function.

Antibodies, Monoclonal↗
PubMed1985

The effects of chemical modification of calmodulin on Ca2+-induced exposure of a hydrophobic region. Separation of active and inactive forms of calmodulin.

Native calmodulin binds four calcium ions per molecule and exhibits strong Ca2+-dependent binding to phenyl-Sepharose. In contrast, calmodulin inactivated by oxidation of methionine residues or by deamidation binds fewer calcium ions (two per molecule) and shows relatively weak interaction with phenyl-Sepharose. Calmodulin inactivated by modification of lysine residues still is able to bind four calcium ions per molecule and shows strong binding to phenyl-Sepharose similar to native calmodulin. The results suggest that complete exposure of calmodulin's hydrophobic region occurs only after the binding of four ions of calcium to the calmodulin molecule. Thus, phenyl-Sepharose hydrophobic interaction chromatography might be used to separate active calmodulin from inactive forms of calmodulin obtained by oxidation or heat treatment for prolonged periods. As an example, phenyl-Sepharose chromatography can be used to separate free iodide and inactivated species of calmodulin readily from the active, iodinated form of calmodulin following iodination.

Animals↗
PubMed1987

Interaction of calmodulin with iodothyronines: effect of iodothyronines on the calmodulin activation of cyclic AMP phosphodiesterase.

The interaction between calmodulin and iodothyronines and the effect of iodothyronines on the calmodulin activation of cyclic AMP phosphodiesterase were investigated. Binding of [L-125I]triiodothyronine to calmodulin from pig brain, studied by equilibrium dialysis, was dependent on Ca2+, was saturable and reversible, with an apparent Kd of 2.79 microM and binding capacity of 0.5 nmol/20 micrograms of calmodulin L- and D-thyroxine, D-triiodothyronine and tetrac displaced [L-125I]triiodothyronine at concentrations of 8-10 microM; triac, 3,3'-diiodothyronine and reverse-triiodothyronine were weak displacers. In the presence of the antipsychotic drug trifluoperazine, binding decreased in a dose-related manner. Ultraviolet irradiation of calmodulin in the presence of trifluoperazine reduced the binding of [L-125I]triiodothyronine to calmodulin irreversibly. Calmodulin activation of cyclic AMP phosphodiesterase decreased when iodothyronines were bound to calmodulin; the calmodulin-L-triiodothyronine complex was the most active among the stereoisomers of thyroxine and triiodothyronine. These results suggest that, when triiodothyronine was bound to Ca2+-calmodulin, the activation of cyclic AMP phosphodiesterase by the latter is suppressed.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Interaction of calmodulin with the calmodulin binding domain of the plasma membrane Ca2+ pump.

Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump (James et al., 1988) were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with the calmodulin binding domain, supporting the suggestion (Benaim et al., 1984) that the latter, or a portion of it, may act as a natural inhibitor of the pump.

Animals↗
PubMed1994

Phosphorylation of calmodulin on Tyr99 selectively attenuates the action of calmodulin antagonists on type-I cyclic nucleotide phosphodiesterase activity.

Tyr99 phosphorylation of calmodulin appears to induce a distinct conformational change as is evident from the profound attenuation of the Ca(2+)-induced enhancement of calmodulin's mobility seen during SDS/PAGE. The effect of this conformational change appears to be localized, in that both calmodulin and P-Tyr99-calmodulin show identical dose-dependent activation profiles for stimulation of a physiological effector, type-I (Ca2+/calmodulin-stimulated) cyclic nucleotide phosphodiesterase (PDE) activity and their presence engenders similar dose-dependent PDE activation by Ca2+. In marked contrast with this, with P-Tyr99-calmodulin there were 3-4-fold increases in the IC50 values for inhibition of type-I PDE activity by the calmodulin antagonists TFP and W7, together with increased values for Hill coefficients for inhibition. The polybasic compound poly(L-lysine) potently augmented the action of calmodulin as a PDE activator, causing an approx. 7-fold decrease in the EC50 value for activation of PDE. It is suggested (i) that the Tyr99 phosphorylation of calmodulin, which occurs within a high-affinity Ca(2+)-binding domain, induces a localized conformational change in this peptide which can selectively attenuate the action of calmodulin antagonists on type-I PDE activity while leaving unaffected Ca(2+)-dependent activation, and (ii) that polybasic substances on complexing with calmodulin may serve to enhance the sensitivity of type-I PDE to activation by this regulatory peptide.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Regulation of G protein-coupled receptor kinases by calmodulin and localization of the calmodulin binding domain.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 approximately 50 nM) > GRK6 >> GRK2 (IC50 approximately 2 microM) >> GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity.

Amino Acid Sequence↗
PubMed1983

Calmodulin and Ca2+- and calmodulin-dependent protein kinase in rat anterior pituitary gland.

Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.

Animals↗
PubMed1989

The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump.

Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.

Amino Acid Sequence↗
PubMed1987

Identification of calmodulin-like activity in term human amnion: effect of calmodulin inhibitors on prostaglandin biosynthesis.

Human amnion prostaglandin E2 (PGE2) synthesis increases with the onset of labour, and this synthesis is Ca2+-dependent. To understand better the mechanism of Ca2+-stimulated PGE2 biosynthesis, studies were performed to identify the presence of the intracellular Ca2+-mediator, calmodulin, in human amnion and to examine its role in PGE2 synthesis. Calmodulin-like activity was identified by the ability of the microsomal and cytosolic fractions of the 105,000g centrifugation of amnion homogenate to stimulate cyclic AMP-dependent phosphodiesterase activity. Cytosolic fractions consistently stimulated phosphodiesterase activity more than microsomal fractions (P less than 0.001) in paired samples from term human amnions. This activity was calcium-dependent. The cytosolic and microsomal factors increased the Vmax but not the Km of phosphodiesterase. There were no differences in these parameters with the onset of labour. The distribution of calmodulin-like activity between microsomes and cytosol was similar to the distribution of calmodulin mass as determined by radioimmunoassay. Three structurally different inhibitors of calmodulin activity, calmidazolium, trifluoperazine and W7, were tested for their ability to inhibit cytosolic factor-stimulated phosphodiesterase activity and to inhibit PGE2 output from dispersed amnion cells obtained before the onset of labour at term (cesarean section cells) or after spontaneous labour and vaginal delivery (spontaneous labour cells). The 50% inhibitory concentrations of the calmodulin antagonists in the phosphodiesterase assay were: trifluoperazine (6.7 microM), calmidazolium (0.11 microM), and W7 (24 microM). Trifluoperazine inhibited both basal and calcium ionophore (A23187)-stimulated PGE2 output from cesarean section cells and spontaneous labour amnion cells. Calmidazolium inhibited basal PGE2 output in cesarean section cells and spontaneous labour cells, but had no effect on A23187-stimulated output. W7 inhibited only the ionophore-stimulated PGE2 output in cesarean section amnion cells. The rank order of inhibition of both phosphodiesterase activation and basal PGE2 output was: calmidazolium greater than trifluoperazine greater than W7. These results suggest that human amnion contains calmodulin and that its distribution, concentration and activity remain unchanged with the onset of labour. The data suggest, although not conclusively, that calmodulin may, in part, play a role in amnion cell PGE2 production. Further investigation of calmodulin effects upon specific enzymes in the PGE2 synthetic pathway will be necessary to elucidate a role for calmodulin in PGE2 production.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Octopus calmodulin. Structural comparison with bovine brain calmodulin.

A protein previously isolated from octopus optic lobe is shown to have the biochemical characteristics of a calmodulin-like protein. The amino acid composition of the octopus calmodulin is similar to that of another sea invertebrate calmodulin, from Renilla reniformis, in that both contain a single residue of tyrosine which distinguishes them from the vertebrate calmodulins which contain two tyrosines. The 1H NMR spectra of the octopus calmodulin and bovine brain calmodulin are compared in their apo- and calcium-saturated conformations. A comparison of these spectra indicates that the single tyrosine of the octopus calmodulin is in a structurally homologous position to tyrosine-138 of bovine brain calmodulin. 1H NMR and UV difference spectroscopy also demonstrate that the solution conformations of the apo- and calcium-saturated forms of octopus calmodulin are very similar to those of bovine brain calmodulin. It is concluded that both proteins undergo similar calcium-induced changes in tertiary structure, which result in near identical solution conformations.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Differential effects of tamoxifen-like compounds on osteoclastic bone degradation, H(+)-ATPase activity, calmodulin-dependent cyclic nucleotide phosphodiesterase activity, and calmodulin binding.

We studied effects of calmodulin antagonists on osteoclastic activity and calmodulin-dependent HCl transport. The results were compared to effects on the calmodulin-dependent phosphodiesterase and antagonist-calmodulin binding affinity. Avian osteoclast degradation of labeled bone was inhibited approximately 40% by trifluoperazine or tamoxifen with half-maximal effects at 1-3 microM. Four benzopyrans structurally resembling tamoxifen were compared: d-centchroman inhibited resorption 30%, with half-maximal effect at approximately 100 nM, cischroman and CDRI 85/287 gave 15-20% inhibition, and l-centchroman was ineffective. No benzopyran inhibited cell attachment or protein synthesis below 10 microM. However, ATP-dependent membrane vesicle acridine transport showed that H(+)-ATPase activity was abolished by all compounds with 50% effects at 0.25-1 microM. All compounds also inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase at micromolar calcium. Relative potency varied with assay type, but d- and l-centchroman, surprisingly, inhibited both H(+)-ATPase and phosphodiesterase activity at similar concentrations. However, d- and l-centchroman effects in either assay diverged at nanomolar calcium. Of benzopyrans tested, only the d-centchroman effects were calcium-dependent. Interaction of compounds with calmodulin at similar concentrations were confirmed by displacement of labeled calmodulin from immobilized trifluoperazine. Thus, the compounds tested all interact with calmodulin directly to varying degrees, and the observed osteoclast inhibition is consistent with calmodulin-mediated effects. However, calmodulin antagonist activity varies between specific reactions, and free calcium regulates specificity of some interactions. Effects on whole cells probably also reflect other properties, including transport into cells.

Animals↗