CaM-dependent Phosphodiesterase Research Articles

Phosphodiesterase 1 inhibition underlies long QT phenotype caused by the human N98S-calmodulin mutant.

We previously demonstrated that mice heterozygous for the p.N98S mutation in the calmodulin (CaM)-encoding Calm1 gene (Calm1N98S/+) exhibit β-adrenergic hyperresponsiveness of cardiac L-type current (ICa.L). We hypothesized that N98S-CaM-mediated inhibition of Ca2+/CaM-dependent phosphodiesterase (PDE1) increases cAMP-signaling at the L-type channel, amplifying the effect of β-adrenergic stimulation on ICa.L. Peak ICa.L densities were significantly larger (−13.9±0.6 pA/pF vs. −10.3±0.3 pA/pF;mean±SEM at 0 mV) and ICa.L activated at more negative potentials (Vhalf:-16.2±0.8 mV vs. -12.9±0.8 mV) in Calm1N98S/+vs. Calm1+/+ ventricular myocytes (VMs) treated with the β-adrenergic agonist isoproterenol (ISO; 5 nM;P = 0.005; 21 cells/genotype). Substitution of external Ca2+ with Ba2+ plus dialysis of VMs with 5 μM ryanodine, 1 μM thapsigargin and 10 mM BAPTA abrogated ICa.L potentiation, indicative of its Ca2+-dependence. PDE1-mediated, i.e., Ca2+-sensitive, cAMP hydrolysis was absent in Calm1N98S/+ VM lysates, whereas it contributed 31±3% to the total PDE catalytic activity in Calm1+/+ lysates (n = 6/genotype). The PDE1 inhibitor (PDE1i) Lu-AF50827 (200 nM) eliminated PDE1 activity from and replicated ISO-induced ICa.L potentiation in Calm1+/+ myocytes but had no additional effect on peak ICa.L of ISO-stimulated Calm1N98S/+ myocytes. The protein kinase A (PKA) inhibitor Rp-cAMPS abrogated ISO-evoked ICa.L potentiation of Calm1N98S/+ VMs, indicating that PKA overactivity is both necessary and sufficient for ICa.L potentiation. ISO did not prolong action potential duration at 90% repolarization (APD90) in Calm1+/+ VMs (1 Hz; n = 6), but its combination with PDE1i increased APD90 by 8±2 ms (n = 6; P = 0.005). Despite the larger peak ICa.L density of ISO-stimulated Calm1N98S/+ myocytes, magnitudes of action potential-evoked peak [Ca2+]i elevations with ISO were indistinguishable between the two genotypes. We conclude that N98S-CaM-induced PDE1 inhibition amplifies cAMP signaling at the L-type channel, causing PKA-mediated ICa.L potentiation, but does not impact sarcoplasmic reticulum Ca2+ cycling, compromising feedback inhibition of L-type channels and prolonging APD.

Multifunctional Cationic Peptide Fractions from Flaxseed Protein Hydrolysates

The aim of this work was to determine the multifunctional properties of flaxseed protein-derived cationic peptide fractions. Alcalase hydrolysis of flaxseed protein fractions liberated cationic peptides, which were separated into two major fractions (FI and FII) by chromatography using a cation-exchange column. Due to their cationic property, the peptide fractions bound and inactivated calmodulin (CaM, a negatively charged enzyme activator protein) with concomitant inhibition of CaM-dependent phosphodiesterase (CaMPDE); this activity was substantially reduced as CaM concentration increased. Enzyme kinetics studies showed competitive inhibition of CaMPDE by FI and FII with enzyme-inhibitor dissociation constants of 0.0202 and 0.0511mg/ml, respectively. Only the FII peptides showed multifunctional activities by inhibiting CaMPDE, angiotensin converting enzyme (ACE) and renin. Separation of FII peptides by reverse phase HPLC resulted in eight fractions (FII-2 to FII-9) that inhibited the activities of CaMPDE, ACE, and renin but this multifunctional activity was more pronounced in FII-6. From LC-MS analysis, identified peptides present in FII fraction had molecular size range of 330-735Da, which suggests potential for increased absorption. Potential peptide sequences were identified for each of the HPLC fractions and shown to contain either lysine or arginine as the positively charged amino acid residue. The multifunctional properties of the cationic peptide fractions can potentially enhance their use in targeting multiple symptoms of cardiovascular disease, considering that the excessive levels of CaM, CaMPDE, renin and ACE play important roles in enhancing progression and intensity of chronic human diseases.

Prediction and validation of a mechanism to control the threshold for inhibitory synaptic plasticity

Synaptic plasticity, neuronal activity-dependent sustained alteration of the efficacy of synaptic transmission, underlies learning and memory. Activation of positive-feedback signaling pathways by an increase in intracellular Ca2+ concentration ([Ca2+]i) has been implicated in synaptic plasticity. However, the mechanism that determines the [Ca2+]i threshold for inducing synaptic plasticity is elusive. Here, we developed a kinetic simulation model of inhibitory synaptic plasticity in the cerebellum, and systematically analyzed the behavior of intricate molecular networks composed of protein kinases, phosphatases, etc. The simulation showed that Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is essential for the induction of synaptic plasticity, was persistently activated or suppressed in response to different combinations of stimuli. The sustained CaMKII activation depended on synergistic actions of two positive-feedback reactions, CaMKII autophosphorylation and CaMKII-mediated inhibition of a CaM-dependent phosphodiesterase, PDE1. The simulation predicted that PDE1-mediated feedforward inhibition of CaMKII predominantly controls the Ca2+ threshold, which was confirmed by electrophysiological experiments in primary cerebellar cultures. Thus, combined application of simulation and experiments revealed that the Ca2+ threshold for the cerebellar inhibitory synaptic plasticity is primarily determined by PDE1.

Mechanism of Neurotrophic Action of Nobiletin in PC12D Cells

Nobiletin is a nonpeptide compound with a low molecular weight from a citrus fruit and has the activity to rescue bulbectomy-induced memory impairment. Here we describe that nobiletin itself induces neurite outgrowth in PC12D cells, a rat pheochromocytoma cell line, like NGF, and the molecular mechanism of its neurotrophic action. As cultured in the presence of nobiletin or NGF for 48 h and then assayed using a scanning electron microscope, PC12D cells treated with nobiletin showed morphology with flatter and larger cell bodies than the cells cultured with NGF. Nobiletin-induced neurite outgrowth was inhibited by PD98059 and U0126 but not K252a. Consistently, nobiletin caused a concentration-dependent enhancement of Erk/MAP kinase phosphorylation and a sustained increment of phosphorylation of MEK and Erk/MAP kinase, resulting in a stimulation of CREB phosphorylation and CRE-mediated transcription. This compound also increased intracellular cAMP and CRE-mediated transcription in the presence of forskolin and enhanced PKA activity to stimulate phosphorylation of multiple PKA substrates in PC12D cells. Furthermore, nobiletin preferentially inhibited Ca2+/CaM-dependent phosphodiesterase in vitro. This compound failed to stimulate phosphorylation of Erk5, which is known to be induced by NGF/TrkA signaling. These results suggest that nobiletin induces neurite outgrowth by activating a cAMP/PKA/MEK/Erk/MAP kinase-dependent but not TrkA-dependent signaling pathway coupling with CRE-mediated gene transcription and may thus become a novel type of biochemical probe for elucidation of the molecular mechanism of neuronal differentiation.

Cyclic AMP-mediated regulation of striatal glutamate release: interactions of presynaptic ligand- and voltage-gated ion channels and G-protein-coupled receptors

The presynaptic regulation of striatal glutamate transmission was investigated using d-[3H]aspartate and mouse striatal slices. Functional changes in voltage-dependent and glutamate receptor-gated ion channels were elicited by pharmacologically modifying intracellular cyclic AMP formation via G-protein-coupled receptor stimulation. The kainate (KA)-evoked release was potentiated by the stimulatory G-protein (Gs)-coupled β-adrenoceptor agonist isoproterenol (ISO) in a concentration-dependent manner. This effect was mimicked by the specific calmodulin (CaM) antagonists trifluoperazine and calmidazolium. Tetrodotoxin (TTX), a blocker of Na+ channels, did not affect the basal release but inhibited to the same degree the releases evoked by kainate alone and by kainate and isoproterenol together. Vinpocetine, a blocker of voltage-dependent Na+ channels, did not alter the basal or the evoked release. The Na+ channel activator veratridine enhanced the basal release in a concentration-dependent manner and isoproterenol attenuated this effect. The opposite effects of isoproterenol on the kainate- and veratridine-evoked releases may reflect prevention of the cyclic AMP-protein kinase A (PKA) phosphorylation cascade in striatal glutamatergic signal transduction. In addition, the calmidazolium-induced potentiation of kainate-evoked release was thwarted by LY354740 and l-2-amino-4-phosphonobutanoate, agonists of the inhibitory G-protein (Gi)-coupled metabotropic group II and III glutamate receptors (mGluRs). Vinpocetine, which inhibits the CaM-dependent phosphodiesterase (PDE1), was likewise inhibitory. In turn, selective agonists and antagonists of the Gq-protein-coupled group I mGluRs and (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylate (AIDA), which modulate the intracellular Ca2+ levels, did not alter the kainate-evoked release.The β-adrenoceptor-mediated cyclic AMP accumulation seems to downregulate Na+ channels but to enhance glutamate release by means of upregulation of kainate receptors. This regulation of presynaptic ligand- and voltage-gated ion channels is affected by the cAMP-protein kinase A-dependent phosphorylation cascade and controlled by Gi-protein-coupled mGluRs.

Calmodulin antagonists and cAMP inhibit ionizing-radiation-enhancement of double-strand-break repair in human cells

Ionizing radiation (IR) enhances double-strand-break (DSB)-repair fidelity in plasmids processed in normal lymphoblasts but not in lymphoblasts from ataxia telangiectasia (A–T) patients. Putatively, signal-transduction pathways mediate this DNA-repair induction. Because IR inhibition of DNA synthesis is defective in A–T cells and is mediated by a calmodulin (caM)-dependent pathway, we evaluated the involvement of caM-dependent pathways in DSB-repair induction. Human lymphoblasts were γ-irradiated with or without treatment with caM antagonists and the cells' abilities to repair shuttle pZ189 carrying a single DSB (linDNA) were assessed. In untreated controls, IR enhanced DSB-rejoining fidelity if transfection occurred promptly but diminished fidelity if transfection was delayed. Treatment with two caM antagonists, W-7 and W-13, prior to irradiation blocked this IR-enhancement of DSB-rejoining fidelity. Vinpocetine, a caM-dependent phosphodiesterase inhibitor, and 8-bromo-cAMP also inhibited IR enhancement of repair fidelity, but caM-dependent protein kinase II inhibitor KN62 had no effect. Other protein kinase inhibitors, staurosporine and genistein, also did not inhibit IR enhancement of DSB repair fidelity. However, staurosporine blocked the twofold reduction in DSB-repair fidelity seen if linDNA transfection was delayed 2 h after irradiation. These findings point to the involvement of caM/cAMP-dependent pathway(s) in mediating IR-enhancement of DSB-rejoining fidelity in mammalian cells.

Ganglioside GD1a enhances immunoglobulin production by human peripheral blood mononuclear cells

Objective We previously reported that ganglioside GD1a greatly enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC) in vitro. We herein examined the mechanism for the stimulatory effect of GD1a. Materials and Methods PBMC from healthy volunteers were cultured with GD1a. The amounts of IgG, IgM, and IgA and cytokine activity in the culture supernatants were measured by enzyme-linked immunosorbent assays. Proliferation was determined by [ 3H] thymidine uptake. Results GD1a at 10 −6 M increased IgG, IgM, and IgA production by PBMC 2.10-fold, 2.10-fold, and 2.23-fold above the control values, respectively. GD1a did not affect the proliferation and viability of PBMC. GD1a did not alter Ig production of B cells alone. Anti-interleukin-6 (IL-6) or anti-IL-10 antibody each partially blocked the GD1a-induced enhancement of Ig production by PBMC, and the addition of both antibodies completely blocked the enhancement. GD1a increased IL-6 and IL-10 production of monocytes without altering those of T cells or B cells. The supernatant from GD1a-treated monocytes enhanced B cell Ig production to a greater extent than that from medium-treated monocytes. The supernatant-mediated effect of GD1a was partially blocked by anti-IL-6 or anti-IL-10 antibody, and the addition of both antibodies completely blocked the GD1a effect. GD1a-induced increases of IL-6 and IL-10 production in monocytes were both blocked by Ca 2+/calmodulin (CaM)-dependent phosphodiesterase (PDE) inhibitors, 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetin, but not by other signal-transducing enzyme inhibitors. The culture with GD1a enhanced Ca 2+/CaM-dependent PDE activity in monocytes. Conclusion These results suggest that GD1a may indirectly enhance B cell Ig production in whole PBMC by increasing IL-6 and IL-10 production of monocytes via promoting Ca 2+/CaM-dependent PDE activity.

Intracellular Application of Calmidazolium Increases Ca2+ Current through Activation of Protein Kinase A in Cultured Vascular Smooth Muscle Cells

In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca²⁺ currents (I_Ca(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak I_Ba (Ca²⁺ channel using 5 mM Ba²⁺ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of –60 mV. To test the effect of CaM on I_Ba, 1 µM calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 nM [Ca]_i). The amplitude of maximally activated I_Ba was –4.3 ± 0.5 pA/pF (n = 13) for control and –8.1 ± 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of I_Ba was not abolished when 5 µM KN-62, a specific inhibitor of CaMK-II, was included in the pipette (–9.5 ± 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal I_Ba. On the other hand, the CMZ stimulation of I_Ba was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca²⁺/CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated I_Ba: control, –4.6 ± 0.4 pA/pF (n = 10); MIBMX, –9.6 ± 1.2 (n = 8), and CMZ, –7.9 ± 0.9 (n = 15). 0.1 mM 8Br-cAMP, a membrane permeable cAMP analogue, stimulated I_Ba by +42%: before, –3.7 ± 0.7 pA/pF; after, –5.2 ± 1.0 (n = 6). In conclusion, Ca²⁺ channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of I_Ba might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.

Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein.

A 19-amino acid residue peptide, Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys- Ala, corresponding to the basic amphiphilic alpha-helix (BAA) motif at the C-terminus of a recombinant tobacco calmodulin-binding protein, TCB60, was synthesized. The interaction of the synthetic binding domain with calmodulin (CaM) was analyzed by gel mobility shift assays, phosphodiesterase competition assays, and fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy. Mobility shift assays showed an apparent 2 kDa increase in CaM Mr in presence of synthetic peptide and CaCl2 in 4 M urea polyacrylamide gel electrophoresis. HPLC measurements of hydrolysis of cyclic AMP by CaM-dependent phosphodiesterase indicated the synthetic peptide competitively inhibits (Ki = 15-20 nM) stimulation of phosphodiesterase activity by CaM. Upon binding CaM, the fluorescence emission maximum of the synthetic peptide, which contained two tryptophanyl residues, shifted toward blue and increased in intensity. The circular dichroism spectra indicated the ellipticity of CaM increased at 208 and 222 nm upon complex formation with the synthetic peptide. 1H NMR studies showed that the peptide interacts with the aromatic residues in domains I and III of CaM. Taken together, these data provide direct evidence that the structurally conserved basic amphiphilic alpha-helix CaM-binding domain of the recombinant tobacco CaM-binding protein interacts with CaM at physiologically significant nanomolar concentrations and the microenvironments of both CaM and the synthetic binding domain are modified upon complex formation.

Characterization of novel calmodulin-binding peptides with distinct inhibitory effects on calmodulin-dependent enzymes

We describe the isolation and interaction with calmodulin (CaM) of two 10-amino-acid peptides (termed peptides 1 and 2; AWDTVRISFG and AWPSLQAIRG respectively) derived from a phage random peptide display library. Both peptides are shorter than previously described CaM-binding peptides and lack certain features found in the sequences of CaM-binding domains present in CaM-activated enzymes. However, 1H NMR spectroscopy and fluorimetry indicate that both peptides interact with CaM in the presence of Ca2+. The two peptides differentially inhibited CaM-dependent kinases I and II (CaM kinases I and II) but did not affect CaM-dependent phosphodiesterase. Peptide 1 inhibited CaM kinase I but not CaM kinase II, whereas peptide 2 inhibited CaM kinase II, but only partially inhibited CaM kinase I at a more than 10-fold higher concentration. Peptide 1 also inhibited a plant calcium-dependent protein kinase, whereas peptide 2 did not. The ability of peptides 1 and 2 to differentially inhibit CaM-dependent kinases and CaM-dependent phosphodiesterase suggests that they may bind to distinct regions of CaM that are specifically responsible for activation of different CaM-dependent enzymes.

Signal transduction: regulation of cAMP concentration in cardiac muscle by calmodulin-dependent cyclic nucleotide phosphodiesterase.

The bovine heart calmodulin-dependent phosphodiesterase can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for calmodulin. The phosphorylation of calmodulin-dependent phosphodiesterase is blocked by Ca2+ and calmodulin and reversed by the calmodulin-dependent phosphatase. The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for calmodulin. The CaM-dependent phosphodiesterase isozymes of heart and brain are regulated by calmodulin, but the affinity for calmodulin are different. Furthermore, the bovine heart CaM-dependent phosphodiesterase isozyme in stimulated at much lower Ca2+ concentration than the bovine brain isozymes. Results from this study suggest that the activity of this phosphodiesterase is precisely regulated by cross-talk between Ca2+ and cAMP signalling pathways.

Expression of a calmodulin-dependent phosphodiesterase isoform (PDE1B1) correlates with brain regions having extensive dopaminergic innervation

Cyclic nucleotide-dependent protein phosphorylation plays a central role in neuronal signal transduction. Neurotransmitter-elicited increases in cAMP/cGMP brought about by activation of adenylyl and guanylyl cyclases are downregulated by multiple phosphodiesterase (PDE) enzymes. In brain, the calmodulin (CaM)-dependent isozymes are the major degradative activities and represent a unique point of intersection between the cyclic nucleotide- and calcium (Ca2+)-mediated second messenger systems. Here we describe the distribution of the PDE1B1 (63 kDa) CaM-dependent PDE in mouse brain. An anti-peptide antiserum to this isoform immunoprecipitated approximately 30-40% of cytosolic PDE activity, whereas antiserum to PDE1A2 (61 kDa isoform) removed 60-70%, demonstrating that these isoforms are the major CaM-dependent PDEs in brain. Quantification of PDE1B1 immunoreactivity on immunoblots indicated that striatum contains 3-17-fold higher levels of PDE1B1 than other brain regions, with lowest immunoreactivity in cerebellum. In situ hybridization demonstrated high levels of PDE1B1 mRNA in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Moderate mRNA levels were observed in dentate gyrus, cerebral cortex, medial thalamic nuclei, and brainstem, whereas negligible mRNA was detectable in the globus pallidus, islands of Calleja, substantia nigra, and ventral tegmental area. Immunocytochemistry confirmed that the majority of PDE1B1 protein was localized to the caudate-putamen, nucleus accumbens, and olfactory tubercle. Within the caudate-putamen, PDE1B1 immunoreactivity was ubiquitous, while PDE1A2 immunostaining was restricted to a minor subset of striatal neurons. The expression of PDE1B1 protein and mRNA correlate strongly with areas of the brain that are richest in dopaminergic innervation; indeed, there are strikingly similar distributions for PDE1B1 and D1 dopamine receptor mRNAs. Since D1 receptor binding activates adenylyl cyclase, and striatal neurons lack CaM-sensitive forms of cyclase, the high amount of this PDE implies an important physiological role for Ca(2+)-regulated attenuation of cAMP-dependent signaling pathways following dopaminergic stimulation.

Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2.

Human cytosolic phospholipase A2 (cPLA2, 85 kDa) appears to be pharmacologically distinct from human secretory phospholipase A2 (sPLA2, 14 kDa). Marine natural products and PLA2 substrate and product analogs were potent inhibitors of human recombinant sPLA2 (r-sPLA2), whereas these compounds stimulated, weakly inhibited, or had no effect on cPLA2 activity from the human monocytic cell line U937. In contrast, within a series of seven reported calmodulin (CaM) antagonists tested, significant correlations among the rank order of potencies of these compounds as inhibitors of cPLA2, r-sPLA2, and a CaM-dependent phosphodiesterase were observed. The correlated inhibitory effects of the hydrophobic CaM antagonists on cPLA2 and sPLA2 may reflect a common feature (possibly a hydrophobic domain) shared by these two types of enzymes.

In vitro effects of cadmium, zinc and lead on calmodulin-dependent actions inOncorhynchus mykiss, Mytilus sp., andChlamydomonas reinhardtii

The potential of cadmium, zinc, and lead to interact with calmodulin (CaM) was investigated by examiningin vitro CaM-dependent protein phosphorylation in tissues from rainbow trout (Oncorhynchus mykiss) and sea mussel (Mytilus sp.) and CaM-dependent phosphodiesterase (PDE) activation by algal (Chlamydomonas reinhardtii) extracts. Cadmium, zinc, and lead proved effective in sustaining CaM-dependent protein phosphorylation in systems containing calcium, whereas only lead was capable of CaM activation in systems depleted of calcium. Cadmium lead to a small activation of CaM-dependent PDE activity by algal extracts, corresponding to ∼25% of that induced by calcium. Cadmium-induced PDE-activation could be attributed to the residual calcium present in the extract. The results indicate that metal-induced CaM activation is primarily mediated in the case of cadmium and zinc by resulting calcium/CaM complexes and in the case of lead by lead/CaM complexes.

Interaction of cadmium, lead and zinc with calmodulin from rainbow trout, sea mussels and a green alga

The effect of cadmium, zinc and lead on calcium/Camodulin (CaM)-dependent reactions was determined in vitro. In the presence of calcium, all were effective in sustaining CaM-dependent protein phosphorylations in cytosolic fractions from rainbow trout ( Oncorhynchus mykiss) and sea mussel ( Mytilus sp.) tissues. In systems depleted of calcium only lead was able to activate CaM. Stimulation of CaM-dependent phosphodiesterase (PDE) activity by algal ( Chlamydomonas reinhardtii) extracts in the presence of cadmium corresponded to ∼ 25% of that induced by calcium. On the basis of speciation calculations the cadmium-induced PDE activation could be attributed to residual calcium in the extract. Results of this comparative study indicate the general ability of the metals to induce CaM-dependent reactions. The stimulation of CaM activity is due in the case of Pb 2+ to a mechanism involving mainly its direct interaction with CaM. Cd 2+ and Zn 2+ are effective indirectly through displacement of Ca 2+from cytosolic binding sites, the resulting increase in free calcium ion concentration leading to CaM activation.

High abundance calmodulin from Blattella germanica eggs binds to vitellin subunits but disappears during vitellin utilization

A Ca 2+-binding protein has been purified and characterized from Blatella germanica eggs. This protein has biochemical features in common with calmodulin (CaM). These common features include a relative low molecular weight of ∼ 19 kDa, thermal stability, an acidic pI of 4.0, a low specific absorbance ( E 277nm 1% = 2.9), an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of 1 mM Ca 2+, and calcium-dependent binding to the CaM antagonist W-7. These features, considered together with activation of CaM-dependent phosphodiesterase in a Ca 2+-dependent manner and cross-reactivity with anti-bovine brain CaM antibody, are sufficient to define this protein as bone fide CaM. CaM comprises 1–1.5% of soluble protein of newly ovulated eggs depending on the mode of assay. A rabbit antibody specific for the B. germanica CaM cross-reacts with bovine brain CaM. 14C-Labeled cockroach CaM in a gel-overlay technique binds to vitellin, the major yolk protein of B. germanica egg. The CaM-binding site of vitellin is located on the 95 kDa subunit before degradation and 53 kDa fragment after 95 kDa subunit breakdown. There is sufficient CaM to bind stoichiometrically (1:1) with the vitellin trimer. CaM is present at uniformly high levels until vitellin utilization starts then it is undetectable until the pharate larval stage at the end of embryogenesis.

Interaction of metals with brain calmodulin purified from normal and cadmium exposed rats.

Chronic exposure of cadmium (Cd) to rats (6 mg/kg body weight/day) led to a significant accumulation of Cd in brain and other organs. Calmodulin (CaM) isolated from brains of Cd exposed rats showed a decreased ability to stimulate CaM-dependent phosphodiesterase (PDE) as compared to that purified from unexposed animals. There was a dose dependent inhibition of CaM activity when CaM (from normal and Cd exposed rats) was incubated with different molar ratios of aluminium (Al3+), lead (Pb2+), manganese (Mn2+) and vanadium (V5+). Regression analysis of rat brain CaM activity versus varying metal ion concentration demonstrated negative slopes. However, CaM from the brains of Cd exposed rats was less sensitive to these metals in comparison to the normal rat brain CaM. These data suggest that CaM inhibition may be used as a biological marker of neurotoxicity and for elucidating the possible mechanism by which neurotoxic metals manifest toxic effects.

Synergistic activation of brain adenylate cyclase by calmodulin, and either GTP or catecholamines including dopamine

The effects of calmodulin (CaM), guanosine triphosphate (GTP) and dopaminergic or β-adrenergci agonists on the activities of adenylate cyclase were studied in EGTA-washed lysed synaptosomal membranes from rat striatum and cerebral cortex. Based on the free calcium ion concentration-dependence of the enzymic activity, it was found that the stimulatory effect of CaM and GTP on adenylate cyclase was synergistic with maximum activation atpCa 6.2 for both striatal and cortical membranes. This was not due to the effect of CaM-dependent phosphodiesterase because exogenous CaM did not affect particulate phosphodiesterase activity. Added CaM not only enhanced the adenylate cyclase activity but acted cooperatively with dopaminergic or β-adrenergic agonists in the presence of GTP. Most marked was enhancement found for the striatal SKF 38393- (a specific D 1 agonist) and the cortical isoproterenol-dependent activities. A synergism was also found for CaM and forskolin. These findings strongly suggest that CaM is involved in the striatal dopaminergic as well as in the cortical β-adrenergic systems.

Interaction of the dihydropyridine calcium antagonist, CD-349, with calmodulin

The characteristics of the binding of the 1,4-dihydropyridine Ca 2+ antagonist, 2-nitratopropyl 3-nitratopropyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine 3,5-dicarboxylate (CD-349), to calmodulin (CaM) and the effect of CD-349 on the Ca 2+/CaM-dependent enzyme, cyclic GMP (cGMP) phosphodiesterase (PDE), were investigated. CD-349 showed a Ca 2+-dependent binding to CaM, in equilibrium column binding studies. CD-349 inhibited the [ 3H]CD-349 binding to CaM, at a concentration producing a 50% inhibition ( ic 50) of 2.4 μM, whereas the CaM antagonist, trifluoperazine hydrochloride (TFP), stimulated the [ 3H]CD-349 binding to CaM. Scatchard plot analysis of the binding of CD-349 to CaM revealed that the apparent dissociation constant ( K app) of CD-349 was 2.1 μM and the maximal number of binding sites ( B max) of CD-349 was 1.0 nmol/nmol CaM. In the presence of TFP, the K app and B max values of CD-349 binding to CaM were changed to 1.1 μM and 1.5 nmol/nmol CaM respectively. Although the CaM antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and TFP, decreased and increased, respectively, the fluorescence intensity caused by 2- p-toluidinylnaphthalene -6-sulfonic acid (TNS)-CaM binding, CD-349 only slightly decreased the fluorescence of TNS bound CaM. CD-349 inhibited both basal and Ca 2+/CaM-activated cGMP PDE activity. However, CaM did not competitively antagonize the CD-349-induced inhibition of the Ca 2+/CaM-activated PDE activity. In addition, the kinetic study showed that CD-349 inhibited both basal and Ca 2+/CaM-activated cGMP PDE activity, competitively with cGMP, with almost the same inhibition constant ( K i). These results suggest that CD-349 binds to CaM, with Ca 2+ dependency, at sites differing from those which bind to the CaM antagonist. The inhibitory activity of CD-349 on Ca 2+/CaM-dependent PDE does not seem to be due to a CaM antagonistic action.

Characterization and expression of the unique calmodulin gene of Aspergillus nidulans.

Complete cDNA and genomic clones for the unique calmodulin (CaM) gene of the filamentous fungus Aspergillus nidulans have been isolated and characterized. The gene contains five introns, of which three are at unique positions relative to other CaM genes. The A. nidulans CaM gene is transcribed as a single, 0.85-kilobase mRNA species that encodes a predicted protein 84% identical (93% similar if conservative changes are considered) to vertebrate CaM. The complete cDNA was ligated into a lambda PL promoter-regulated bacterial expression vector to allow expression of A. nidulans CaM in Escherichia coli. The expressed protein was purified from bacterial lysates by phenyl-Sepharose chromatography and migrated as a single species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of Ca2+, A. nidulans CaM exhibited a shift in apparent Mr identical to vertebrate CaM. The bacterially synthesized protein activated vertebrate CaM-dependent phosphodiesterase, CaM-dependent protein kinase II, and myosin light chain kinase with kinetics similar to vertebrate CaM. Isolated conidia (G0 spores) were germinated to induce synchronous cell cycle re-entry and the levels of CaM mRNA and protein determined. Both CaM and its mRNA were regulated during cell cycle re-entry. Calmodulin mRNA levels increased 20-fold as germlings progressed through the G1 phase, while CaM levels increased 2-fold prior to the initiation of DNA synthesis. Messenger RNA levels decreased during S-phase while protein levels increased an additional 2-fold, peaking at the onset of mitosis followed by a subsequent decrease as cells completed mitosis. Disruption of the CaM gene by site-specific homologous recombination was lethal, indicating that CaM is essential for cell cycle progression.