Calmodulin Regulation Research Articles

Resolution of bovine brain calcineurin subunits: stimulatory effect of subunit B on subunit A phosphatase activity

Calcineurin was dissociated into subunits A and B by SDS and the dissociated subunits were separated by Sephadex G-100 column chromatography in SDS. The phosphatase activity was associated with the A subunit and was detected only in the presence of MnCl 2 of the various divalent cations tested. The Mn 2+-dependent phosphatase of A subunit was stimulated (4-5-fold) by calmodulin. The subunit B increased only modestly Mn 2+ stimulated phosphatase activity of subunit A but markedly increased it when assay also contained calmodulin. These results support the view that subunit B plays an important role in Mn 2+/calmodulin regulation of subunit A phosphatase activity. They also lend further support to our earlier postulate ([1984] FEBS Lett. 169, 251-255) that Mn 2+ is a powerful regulator of calcineurin phosphatase.

Calmodulin regulation of cellular cyclic AMP production in response to arginine vasopressin, prostaglandin E2 and forskolin in rat renal papillary collecting tubule cells in culture.

The effect of calmodulin on the stimulation of cyclic AMP production by arginine vasopressin (AVP), prostaglandin E2 (PGE2) and forskolin was examined in cultured renal papillary collecting tubule cells of the rat. In the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine submaximal concentrations of AVP (1 nmol/l), PGE2 (20 nmol/l) and forskolin (240 nmol/l) significantly increased cellular cyclic AMP accumulation by 2.3-, 6.0- and 8.4-fold respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7), attenuated the AVP-, PGE2- and forskolin-stimulated cellular production of cyclic AMP in a dose-related manner. Cellular production of cyclic AMP was inhibited by 50% (ID50) by doses ranging from 16 to 28 mumol trifluoperazine/l and 35 to 44 mumol W-7/1. Basal accumulation of cellular cyclic AMP was also decreased by treatment with either trifluoperazine or W-7, but the effective dose was higher than that which inhibited cellular cyclic AMP production stimulated by AVP, PGE2 and forskolin. Since forskolin directly activates adenylate cyclase at a site of the catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-guanine nucleotide regulatory-catalytic units, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin-activated adenylate cyclase in the papillary collecting tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cooperative calcium binding and calmodulin regulation in the calcium-dependent adenosine triphosphatase purified from the erythrocyte membrane

Calcium binding to calcium-dependent ATPase purified from erythrocyte membrane was assessed by measurements of the ATPase intrinsic fluorescence. Calcium-binding isotherms obtained by fluorescence titration are identical to curves representing the Ca 2+-concentration dependence of ATPase activity, and demonstrate that cooperativity is in fact a feature of the binding mechanism rather than an apparent effect of enzyme kinetics. Loss of cooperativity and a reduction of the ATPase affinity for calcium is observed at very low enzyme concentrations. This effect of enzyme dilution is prevented by calmodulin at 37°C but not at 25°C. It is suggested that calcium binding by erythrocyte-membrane ATPase is influenced by hydrophobic interactions of binding domains, exhibiting a dissociation constant between 10 −7 and 10 −8 M in the absence of calmodulin, at 37°C and in a specific set of experimental conditions. The dissociation constant is decreased by calmodulin. Erythrocyte membrane Ca 2+-ATPase Calmodulin Purified Ca 2+-ATPase Intrinsic fluorescence Cooperativity

Studies on role of calmodulin in Ca2+ regulation of rabbit ileal Na and Cl transport

To study the role of calmodulin in regulation of rabbit ileal active electrolyte transport by Ca2+, the effects of the Ca2+-calmodulin antagonist naphthalenesulfonamides W12 and W13 and the weak Ca2+-calmodulin antagonist promethazine, a phenothiazine, were studied on basal ileal Na and Cl transport and on secretion stimulated by Ca2+ and by cAMP. The naphthalenesulfonamides and promethazine all stimulated basal ileal Na and Cl absorption. The stimulation of Na absorption was dependent on Cl in the bathing solutions, and the stimulation of Cl absorption was Na dependent. This suggests that the transport process stimulated was the neutral, linked NaCl absorptive process. W13, which is a better Ca2+-calmodulin antagonist but has similar hydrophobic properties to W12, stimulated active ileal absorption at a lower concentration than W12, suggesting that the naphthalenesulfonamide-induced stimulation of active ileal absorption was not due to the hydrophobic properties of these drugs but could be due to their effects on the calcium-binding protein calmodulin. W12, W13, and promethazine did not alter paracellular transport, not affecting dilution potentials or structural features of the paracellular pathway. W12 and W13 did not decrease the changes in active ileal Na and Cl transport caused by increasing ileal cAMP content or intracellular Ca2+. This suggests that calmodulin is not directly involved in the active electrolyte secretion caused by increased intestinal Ca2+ or cAMP.

Protein carboxylmethylation and nervous system function

Enzymatic protein- O-carboxylmethylation transfers methyl groups from S-adenosylmethionine to aspartyl and/or glutamyl residues of various methyl acceptor proteins. The function of this post-translational modification of protein, originally detected as “methanol-forming” activity in pituitary gland, has remained enigmatic in nervous tissue. Theories concerning the function of protein methylation have focused on possible roles in neurotransmitter release, neurophysin carboxylmethylation, regulation of calmodulin and calmodulin-binding proteins, chemotaxis, processing of precursor peptides, and repair/recognition of racemized D-amino acids. However, difficulties in establishing quantitative and temporal relationships between methylation and the biochemical event described have led to controversies. Similarly, the alkaline lability of the carboxylmethyl ester bond has led to difficulties in using the high resolution gel electrophoresis systems so successfully used in characterization of other post-translational events. Recent studies localizing protein- O-carboxylmethyltransferase to neurons in the rat brain suggest that this enzyme may be involved in signal transduction in the CNS. Alternative theories concerning protein methylation will be discussed and future directions for research in this area will be outlined.

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.

Evidence for a role of calmodulin in cellular cAMP production in response to vasopressin, prostaglandin E2 and forskolin in cultured rat renal papillary collecting tubule cells

In the present study, the role of calmodulin in the cellular action of arginine vasopressin (AVP), prostaglandin (PG) E2 and forskolin on adenosine-3', 5'-monophosphate (cAMP) production was examined in cultured rat renal papillary collecting tubule cells. In the presence of the phosphodiesterase inhibitor, submaximal concentrations of 10(-9) M AVP, 2 X 10(-8) M PGE2 and 2.4 X 10(-7) M forskolin significantly increased cellular cAMP accumulation by 2.3, 6.0 and 8.4-fold, respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), attenuated the cellular production of cAMP in a dose-related manner in response to all three stimuli. A dose which inhibited the cellular production of cAMP by 50% (ID50) ranged from 1.6 X 10(-5) to 2.8 X 10(-5) M for trifluoperazine and from 3.5 X 10(-5) to 4.4 X 10(-5) M for W-7. Basal accumulation of cellular cAMP was also decreased by treatment with either trifluoperazine or W-7, but an effective dose was relatively higher than that which inhibited agents-stimulated cellular cAMP production. Since forskolin is recognized as activating adenylate cyclase at one step of the catalytic component and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic component, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin-activated adenylate cyclase in the papillary collecting tubule cells. Further study demonstrated the inhibition of AVP- or PGE2-induced cellular cAMP production by treatment with either a calcium-free medium or verapamil, a blocker of cellular calcium uptake, before and during the experiment. These findings suggest that an increase in cytosolic calcium, which interacts with calmodulin to form an active complex, is, at least in part, due to the increased cellular influx of calcium from the extracellular space.

Reversible effects of phenothiazines on frog gastric stimulation.

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and promethazine (PZ) were tested for effects on stimulus-secretion coupling in in vitro bullfrog gastric mucosa. When added to histamine-stimulated tissues, the drugs caused H+ secretion to decrease and transepithelial resistance to increase over a 2-h time course. The potency sequence was TFP (IC50 = 40 microM) greater than CPZ (IC50 = 72 microM) congruent to PZ (IC50 = 72 microM). Anesthetics and other phenothiazines with weak anticalmodulin activity had no effect on secretory parameters. In the presence of histamine, further addition of isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) plus dibutyryl cAMP (DBcAMP), IBMX alone, or forskolin (a specific activator of adenylate cyclase) to phenothiazine-inhibited tissues caused full resumption of secretory activity. If TFP (50 microM) was added before stimulation with histamine, the normal increases in tissue cAMP content (which occurs primarily in oxyntic cells), oxyntic cell apical membrane elaboration (morphometric analysis of electron micrographs), and H+ secretion were all blocked. Subsequent addition of IBMX or IBMX plus DBcAMP completely reversed the TFP effect. These results indicate that the histamine-sensitive adenylate cyclase may be the site of TFP inhibition and Ca2+-calmodulin regulation; since these drugs inhibited stimulation by DBcAMP plus IBMX, they may also be exerting additional effects distal to cAMP generation.

Activation of plant aspartate kinase by calcium and calmodulin-like factor from plants

Aspartate kinase partially purified from plants is activated by a protein factor prepared from the same plant material. This activation is further stimulated by calcium and inhibited by anticalmodulin compounds such as trifluoperazine and chlorpromazine. The results suggest that the characteristics of regulation of the enzyme activity by the protein factor are similar to calmodulin regulation of plant and animal enzymes.

Regulation of calmodulin- and dopamine-stimulated adenylate cyclase activities by light in bovine retina.

Neural retina from most species contains 3,4- dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca2+-binding protein, calmodulin, and that calmodulin can increase dopamine-sensitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine- and calmodulin-stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low-intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a particulate fraction washed to deplete it of endogenous Ca2+ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, guanosine-5'-(beta,gamma-imido)triphosphate ( GppNHp ), was significantly (60%) greater in particulate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn2+-, and GTP-stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological regulation of calmodulin's activity

Pharmacological regulation of calmodulin's activity

Calmodulin regulation of the ATP-dependent calcium uptake by inverted vesicles prepared from rabbit synaptosomal plasma membranes

Calmodulin has been shown to activate the ATP-dependent Ca 2+ uptake in inside-out vesicles which have been prepared from rabbit synaptosomal plasma membranes by the methodology of Gill et al. (Gill, D.L., Grollman, E.F. and Kohn, L.D. (1981) J. Biol. Chem. 256, 184–192). Following extensive washings of these membranes with EGTA/EDTA solutions, the Ca 2+ uptake activity demonstrated an affinity for calmodulin of 30 nM and an affinity for Ca 2+ of 2 μM. The activity was completely inhibited by the anticalmodulin compound R24571 ( K i ⋍ 8 μ M ). The molecular weight of the ATPase molecule, revealed by a combination of the [ 125I]calmodulin overlay technique and [ 32P]phosphoenzyme electrophoresis, was 145000. The overlay technique also revealed that the mechanism of activation is via a direct binding of calmodulin to the pump molecule.

Early, anti-immunoglobulin induced events prior to Na+-K+ pump activation: an analysis in a monoclonal human B-lymphoma cell population.

Events following F(ab)2 anti-delta immunoglobulin stimulation of monoclonal (leukemic) human B cells prior to Na+-K+ pump activation were investigated in vitro. This pump activation, measured by ouabain-sensitive 86Rb+ uptake, appeared susceptible to the phospholipid-interacting drugs tetracaine and quinacrine, to the antioxydant nordihydroguaiaretic acid (NDGA), and to the calmodulin antagonist trifluoperazine, while much less susceptible to the methylation inhibitor-3-deazaadenosine. The Ca++ ionophore A 23187 appeared to induce pump activation in a way similar to anti-delta, as it was susceptible to the same drugs and as anti-delta had no additional stimulating effect on A 23187-stimulated cells. However, whereas the anti-delta-induced activations appeared independent of the extracellular Ca++ activity, [Ca++]e, the activation by A 23187 was potentiated by addition of the Ca++ chelator ethyleneglycol-bis (beta-aminoethyl ether) N, N'-tetracetic acid (EGTA). Estimations by fluorescent chelator method (quin 2) showed anti-delta to increase the intracellular Ca++ activity, [Ca++]i both in the absence and presence of EGTA. A 23187 increased [Ca++]i strongly in Ca++ medium, but was weaker, more similar to the anti-delta response, in EGTA medium. It is suggested that Na+-K+ pump activation after anti-Ig stimulation in B cells may follow Ca++ mobilization from internal stores. The trifluoperazine susceptibility suggests that calmodulin regulation is involved.

Calcium/calmodulin inhibition of coupled NaCl transport in membrane vesicles from rabbit ileal brush border.

The role of Ca2+ and calmodulin in regulating coupled NaCl transport has been investigated in membrane vesicles from rabbit ileal brush border. Uptake of 22Na+ and 36Cl- was determined by a rapid filtration technique in vesicles isolated with a sucrose density gradient ultracentrifugation method. Ca2+ on the inside of the vesicle inhibited Na+ uptake when Cl- was the anion and Cl- uptake when Na+ was the cation by approximately equal to 30%. Ca2+ on the outside had no effect. When gluconate was the anion or when choline was the cation, Na+ or Cl- uptake was reduced by only 9-12%. A similar inhibition of D-[3H]mannitol uptake (10-17%) suggests this was due to a nonspecific decrease in the membrane permeability. Other cations such as Ba2+ and Mg2+ had no effect, but La3+ inhibited Na+ and Cl- uptake to the same degree as Ca2+. Calmodulin (2 microM) in combination with Ca2+ (1 microM, free concentration) significantly inhibited Na+ uptake when Cl- was the anion by 21-32% and Cl- uptake when Na+ was the cation by 20-27%. This effect was completely reversed by 10 microM trifluoperazine. When gluconate was the anion or when choline was the cation, Na+ or Cl- uptake was unaffected by Ca2+/calmodulin and trifluoperazine. The Ki for Ca2+ inhibition of Cl- -coupled Na+ uptake was reduced from 200 microM to 0.2 microM by incubation with 20 microM calmodulin. The Ki for exogenously added calmodulin studied at 1 microM Ca2+ was 0.2 microM. The Ki for trifluoperazine inhibition of the Ca2+/calmodulin response was 3 microM. These results represent compelling evidence for intracellular Ca2+/calmodulin regulation of coupled NaCl transport across the intestinal microvillus membrane. The exact mechanism of this regulation remains to be delineated.

Cations and calmodulin in normal and neoplastic cell growth regulation.

The purpose of this presentation is to review pertinent literature pertaining to the role of divalent cations and calmodulin in regulating growth of nonneoplastic and neoplastic cells and to examine the anticancer efficacy of some calmodulin inhibitors. Although normal eukaryotic cell replication and proliferation is closely controlled by a complex system of endogenous substances, it is likely that the coordination of purposeful interactions between these substances is the ultimate responsibility of two groups of cellular components, namely the divalent cations Ca2+ and Mg2+ and the versatile intracellular Ca2+-binding protein calmodulin (CaM). When free Ca2+ enters normal cells, it acts as a positive signal for proliferation; this action appears to be specifically associated with the late G1 phase, just prior to DNA synthesis. This period is designated G1/S and is considered to contain Pardee's "restriction point." Reduction of extracellular Ca2+ concentrations between physiological levels (1-0.1 mM) results in gradually reduced rates of cell proliferation; at Ca2+ concentrations of 0.1 mM or less, normal cell proliferation is reversibly inhibited. Since an extracellular concentration of about 0.7 mM Mg2+ is required for Ca2+ to initiate cell replication, it has been proposed that Ca2+ and Mg2+ act in concert via a common mechanism, however, in contrast to Ca2+, Mg2+ appears to be required throughout the entire cell cycle. Intracellular Ca2+ can activate CaM which, in turn, can modulate various cellular processes that affect cell proliferation, including cyclic nucleotide metabolism, protein phosphorylation, polyamine metabolism, prostaglandin metabolism, Ca2+ transport, DNA synthesis, and microtubular function including mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of calmodulin and myosin light chain kinase

Myosin light chain kinase is one of the calmodulin-activatable enzymes. It was first discovered in skeletal muscle and subsequently in smooth and cardiac muscles and in non-muscle tissues. It is suggested that the enzyme is involved in contraction-relaxation cycle of smooth muscle. The myometrium plays a role in the maintenance of pregnancy and parturition and is under influence of steroid hormones such as estrogen and progesterone. In the present study, we attempted to examine the alteration by the hormones of levels in calmodulin and myosin light chain kinase in the myometrium. Injections of estrogen and progesterone to castrated rabbits produced significant increases of 2.8- and 2.1-fold in the enzyme activity per mg protein and of 1.4- and 1.2-fold in the calmodulin content per mg protein, respectively. The increases caused by estrogen treatment were inhibited by the administration of cycloheximide. Human chorionic gonadotropin increased the level of myosin light chain kinase activity by inducing ovulation and stimulating secretion of progesterone from corpus luteum.

Calmodulin regulation of phospholipid and fatty acid methylation by rat liver microsomes.

The addition of micromolar Ca2+ in the presence of ATP (10 microM) to rat liver microsomes stimulates phospholipid methylation and the formation of fatty acid methyl esters. Our data indicate that calmodulin mediates the effect of Ca2+ on both phospholipid and fatty acid methylation. This evidence includes: 1) chlorpromazine, an inhibitor of Ca2+ effects mediated through calmodulin, prevented the activation by Ca2+ plus ATP of the methylation reactions; 2) isolation of the microsomes in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (5 mM), to remove endogenous calmodulin also prevented Ca2+ plus ATP stimulation of both phospholipid and fatty acid methylation; 3) the addition of exogenous calmodulin to microsomes isolated in the presence of 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored Ca3+ plus ATP stimulation of both classes of lipids: and 4) pre-incubation of liver microsomes with an antiserum against calmodulin prevented the activation of lipid methylation by Ca2+ plus ATP.

GTP is not required for calmodulin stimulation of bovine brain adenylate cyclase.

The importance of guanyl nucleotides for calmodulin stimulation of bovine cerebral cortex adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was examined by using a partially purified calmodulin-sensitive adenylate cyclase that was resolved from calmodulin-insensitive forms of the enzyme. By using 5'-adenylyl imidodiphosphate as a substrate, in the absence of an ATP-regenerating system, it was determined that GTP is not required for calmodulin stimulation of the enzyme. Maximal activation by 5'-guanylyl imidodiphosphate (p[NH]ppG) was 5.3-fold, whereas the combination of p[NH]ppG and calmodulin stimulated the enzyme 27-fold. Although GDP inhibited p[NH]ppG stimulation of the calmodulin-sensitive adenylate cyclase, it did not affect calmodulin stimulation. In addition, calmodulin did not alter the kinetics for activation of the enzyme by p[NH]ppG. It is concluded that GTP is not required for calmodulin stimulation of brain adenylate cyclase and that calmodulin regulation of this enzyme is probably not due to effects of calmodulin on the affinity of the guanyl nucleotide regulatory complex for guanyl nucleotides.

Calcium-controlled peroxidase secretion by sugarbeet cell suspensions in relation to habituation

Sugarbeet cells from normal and habituated callus released peroxidases in liquid cultures, in proportion to their endogenous level. Calcium promoted this release more in the normal than in the habituated line. Treatment of the cells with sodium azide, sodium hydrogenarsenate or phenothiazine inhibited the calcium effect, which indicated a dependence on metabolic energy and on calmodulin regulation. The Ca ionophore Ro (bromolasalocid ethanolate) restricted peroxidase release.

Function and regulation of calmodulin in endocrine cells

Calmodulin is the major intracellular receptor for Ca 2+ in all eukaryotic cells. This protein binds Ca 2+ with high affinity, has no alternative function, has multiple acceptor protein and is ubiquitous and structurally conserved. Calmodulin is constituatively expressed in 13 hormone-dependent systems. However the protein is elevated in both endocrine and non-endocrine tumors compared to non-transformed counterparts. The synthesis of Calmodulin is also regulated during the cell cycle and is restricted to the g 1/s boundary. Anti-calmodulin drugs can selectively and reversibly block cells in early S. The data are compatible with a role for Calmodulin in DNA synthesis. One of the major calmodulin acceptor proteins present in all cells is the myosin light chain kinase (MLCK). Both CaM and MLCK are localized on stress fibers in the cytoplasm of interphase cells and appear to be one primary enzyme system responsible for the Ca 2+-dependent regulation of secretion from endocrine cells. Calmodulin and MLCK are also found in the nucleoli of interphase cells by light and electron-microscopic as well as by biochemical techniques. These molecules are restricted to the fibrillar component of the nucleolus and the localization is markedly altered by actinomycin D. Calcium is known to regulate the structure of nucleoli by a mechanism involving contractility. Our data suggest that this mechanism may be dependent upon CaM and MLCK.