[Signal transduction and cellular responses].
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Animals↗
Biomedical subjects
E Miyamoto
Publications and source records attributed to E Miyamoto.
Activation of Ca2+/calmodulin-dependent protein kinase II by stimulation with bradykinin in neuroblastoma x glioma hybrid NG108-15 cells.
To elucidate the mechanisms of the intracellular signal transduction elicited with bradykinin in NG108-15 neuroblastoma x glioma hybrid cells, we examined the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) by bradykinin stimulation. When the extract of NG108-15 cells was immunoprecipitated with the affinity-purified antibody to brain CaM kinase II, a 50-kDa protein in the immunoprecipitate mainly became autophosphorylated in a Ca2+/calmodulin-dependent manner. The results suggest that the 50-kDa protein is the subunit of CaM kinase II in NG108-15 cells. The Ca2+/calmodulin-independent activity (autonomous activity) of the enzyme increased twice within 10 s by stimulation with 1 microM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases: the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 microM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca2+ with 1 mM EGTA or by the pretreatment with 1 microM nifedipine. Stimulation of 32P-labeled NG108-15 cells with 1 microM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. These results suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells.
Bradykinin↗
Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.
In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.
Animals↗
Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.
Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.
Amino Acid Sequence↗
Increased expression of atrial myosin light chain 1 in the overloaded human left ventricle: possible expression of fetal type myocytes.
We examined the isoforms of myosin light chain 1 in the human left ventricles using pyrophosphate and sodium dodecyl sulfate polyacrylamide gel electrophoresis, peptide mapping, and immunoblotting with monoclonal antibodies against human atrial light chain 1. The relationship between hemodynamic parameters and light chain 1 isoform composition was compared among groups of patients with hypertrophic cardiomyopathy (n = 8), dilated cardiomyopathy (n = 9) and aortic stenosis (n = 5), and controls (n = 6). (1) The light chain 1, which differed from ventricular light chain 1 found in the normal adult ventricle, was highly expressed in the overload left ventricle, and was identical to atrial and fetal ventricular light chain 1 with respect to the physiochemical and immunological properties. (2) The expression of atrial/fetal light chain 1 was augmented in the subendocardial area in comparison with the mid- or subepicardial areas in the hypertrophied left ventricles. (3) The values (%) of the relative expression of atrial/fetal light chain 1 to total light chains 1 determined by densitometric analysis were significantly higher in patients with dilated cardiomyopathy (40.2 +/- 5.8) and those with aortic stenosis (43.1 +/- 6.2) than in the controls (16.9 +/- 2.5) (p less than 0.01), but there was no significant difference between the patients with hypertrophic cardiomyopathy (28.0 +/- 3.7) and the controls. (4) The values of the ratio significantly correlated with those of peak circumferential wall stress (r = 0.53, p less than 0.005). These results suggest that atrial/fetal light chain 1 is expressed in the left ventricles in response to the increased hemodynamic load.
Adult↗
Regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II and calcineurin in the hippocampus of rat brain after transient forebrain ischemia.
We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.
Animals↗
Haemolytic action of N-alkylpolymethylenediamines.
The haemolytic action of various N-alkyl derivatives (lauryl; C12H25-, myristyl; C14H29-, palmityl; C16H33-) of 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentate, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane was examined using rabbit red blood cells. The activities of the various derivatives were compared with those of antiplaque agents commonly used as mouthwashes; cetylpyridinium chloride (CP) and chlorhexidine acetate (CH). The haemolytic activities of these agents were dependent on the length of the N-alkyl chain, whereas the number of methylene groups between the nitrogen atoms had little effect. The order of potency was CP, N-palmityl derivatives, N-myristyl derivatives greater than N-lauryl derivatives greater than CH which was similar to the order of the antiplaque effect evaluated in-vitro.
Animals↗
Increased expression and regional differences of atrial myosin light chain 1 in human ventricles with old myocardial infarction. Analyses using two monoclonal antibodies.
BACKGROUND: This study was designed to examine the expression of atrial/fetal-type myosin light chain 1 (ALC1) in human ventricles with old myocardial infarction and in control hearts. METHODS AND RESULTS: The expression of immunoreactive (ir) ALC1 was examined in the subendocardial and subepicardial myocardium of the infarcted and the noninfarcted regions in the left ventricles with old myocardial infarction (n = 12) and of the control left ventricles (n = 8). For the analysis, we prepared two monoclonal antibodies, KA1 and KB1, that were specific for only ALC1 and for both ALC1 and ventricular myosin light chain 1 (VLC1), respectively. The ir-ALC1 expression ratio [ALC1/(ALC1 + VLC1), %] of the subendocardial myocardium, determined densitometrically by Western blotting with KB1, was significantly higher in the infarcted region (11.4 +/- 7.3%) than in the noninfarcted region (4.7 +/- 2.3%, p < 0.001) and the control ventricle (1.0 +/- 1.5%, p < 0.0001). In the infarcted region, the subendocardial myocardium contained a significantly greater percentage of ir-ALC1 than the subepicardial myocardium (5.8 +/- 6.7%, p < 0.005). The ir-ALC1 expression ratio had a significant negative correlation with the value of tissue protein concentration (milligrams protein per gram wet weight). The immunohistochemical study with KA1 revealed that the surviving myocytes included in the infarcted region, especially in the ventricular aneurysm, expressed ir-ALC1 strongly in comparison with those in the noninfarcted or the control ventricles. CONCLUSIONS: These results demonstrate increased expression of ALC1 and the regional differences in the failing left ventricles with old myocardial infarction. We conclude that the reexpression of ALC1 in infarcted ventricles occurs as one of the regional responses to increased load and may be a useful biochemical marker for the appearance of fetal-type myocytes.
Aged↗
Lysophosphatidylcholine inhibits surface receptor-mediated intracellular signals in endothelial cells by a pathway involving protein kinase C activation.
Lysophosphatidylcholine (lysoPC) transferred from oxidatively modified low density lipoprotein (Ox-LDL) to the endothelial surface membrane has been shown to produce a selective unresponsiveness to cell surface receptor-regulated endothelium-dependent relaxation (EDR) in the rabbit aorta. To determine its mechanism we examined the effects of lysoPC on endothelial surface receptor-mediated transmembrane signals. Incubation for 1 minute with palmitoyl lysoPC (5-10 microM) decreased thrombin (Th, 2 units/ml)- or histamine (His, 0.1 mM)-stimulated inositol 1,4,5-trisphosphate (IP3) production in primary cultures of human umbilical vein endothelial cells (HUVECs). LysoPC also decreased Th- or His-induced intracellular calcium ([Ca2+]i, fura 2) elevation. Pretreatment with protein kinase C (PKC) inhibitors staurosporine (100 nM) or H-7 (50 microM) prevented the inhibitory actions of lysoPC, but HA-1004 had no effect. Incubation for 5 minutes with phorbol 12-myristate 13-acetate (PMA, 100 nM) produced the inhibitory actions on the Th- or His-induced intracellular signals, which closely mimic those exhibited by lysoPC. However, the inhibitory effect of lysoPC was lost in cells that were depleted of PKC by pretreatment for 24 hours with 100 nM PMA. Furthermore, incubation of the cells for 1 minute with lysoPC stimulated PKC activity in the membrane fraction. In organ chamber experiments with porcine coronary artery rings, pretreatment with staurosporine (20 nM) attenuated lysoPC-induced impairment of EDR in response to Th. These results indicate that lysoPC, which accumulates in Ox-LDL and atherosclerotic arterial walls, inhibits the early transmembrane signaling pathway in endothelial cells, and PKC activation could at least partially be involved in the negative regulation by lysoPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
[The activation of Ca2+/calmodulin-dependent protein kinase II in the cell system and its cellular functions].
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Animals↗
Staurosporine prevents the impairment of endothelium-dependent relaxation by oxidized-LDL in pig coronary arteries.
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Alkaloids↗
Protein kinase C and Ca2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles.
A monoclonal antibody was made using the spleen cells of a mouse immunized with chick synaptic membranes and designated as mAb 1D12. It immunoprecipitated 25% of the omega-conotoxin binding protein but no dihydropyridine binding protein solubilized from chick brain membranes. By immunoblotting, a polypeptide of 58-kDa was identified as the antigen of this antibody in chick, rat, rabbit and guinea pig brain. Immunohistochemical observation indicated the immunoreactivity of mAb 1D12 to be localized in the synaptic regions of central and peripheral neurons. In peripheral organs, there was additional staining in the distal portions of nerve fibers. Immunoelectron microscopy showed immunoreactivity to be located in synaptic vesicle and presynaptic plasma membranes. In the subcellular fractionation of rat brain, 58-kDa protein was recovered in the fractions of synaptic vesicles and plasma membranes but not soluble proteins. This protein could be extracted from membranes by Triton X-100 but treatment with EDTA, acid, base or high salt failed to have such effect. Solubilized 58-kDa protein of rat brain was purified by immunoaffinity chromatography using mAb 1D12. Both protein kinase C and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated purified 58-kDa protein, and maxima of 0.47 and 0.94 mol of phosphates, respectively, were incorporated per mol of 58-kDa protein. 58-kDa protein was not phosphorylated by either cAMP-dependent or cGMP-dependent protein kinase. When present in membranes, it was also phosphorylated by protein kinase C and CaM kinase II. Possible involvement of 58-kDa protein in the protein kinase C and CaM kinase II-mediated regulation of synaptic transmission in central and peripheral neurons is discussed.
Animals↗
Antimicrobial and anti-plaque activity of N'-alkyl-N-(2-aminoethyl)piperidine against dental plaque bacteria.
Five N'-alkyl-N-(2-aminoethyl)piperidines were synthesized and their in vitro antimicrobial activities were tested against four micro-organisms related to dental caries (Streptococcus mutans, S. sobrinus, Actinomyces viscosus, and A. naeslundii) which are known to be implicated in dental caries. The tetradecyl and hexadecyl derivatives possessed good bacteriostatic activity. Some derivatives exhibited a rapid bactericidal effect against S. mutans and S. sobrinus in aqueous solution. These compounds also possessed surfactant properties and anti-plaque activity.
Actinomyces↗
Ca2+/calmodulin-dependent protein kinase II immunoreactivity in Lewy bodies.
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the predominant protein kinases in the brain. We found that CaM kinase II immunoreactivity was concentrated in the peripheral halos of Lewy bodies (LBs) in Parkinson's disease and Lewy body-like hyaline inclusions (LBHIs) in amyotrophic lateral sclerosis. An immunoelectron microscopic examination of LBs revealed that the filaments at the periphery of LBs were decorated with immunopositive deposits. Since CaM kinase II has a broad substrate specificity and can phosphorylate neurofilaments and other cytoskeletal proteins, it may play some role in the formation of LBs and LBHIs through the aberrant phosphorylation of the cytoskeletal elements in these inclusions.
Aged↗
Staurosporine: an effective inhibitor for Ca2+/calmodulin-dependent protein kinase II.
We investigated the effect of staurosporine on Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10-100 nM) inhibited the activity of CaM kinase II. The half-maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (beta-casein). (c) Staurosporine suppressed the auto-phosphorylation of alpha- and beta-subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+/calmodulin-independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP-binding site or substrate-binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.
Adenosine Triphosphate↗
[Regulation of Ca2+/calmodulin-dependent protein kinase II in the cell systems in response to cellular stimuli].
Calcium ion (Ca2+) is considered to be involved in the regulation of numerous cellular processes. CaM kinase II is present at the highest concentration in the brain and is considered to be involved in the regulation and coordination of numerous cellular processes. CaM kinase II is activated by Ca2+/calmodulin and simultaneously undergoes autophosphorylation. It has not been determined whether the enzyme is activated in the cell systems in response to the increase in cytoplasmic Ca2+ concentration. We have studied CaM kinase II in several kinds of cells including the primary cultures of cerebellar granule cells and the cell lines of rat embryo fibroblast 3Y1 cells, neuroblastoma cells, PC12 cells and C6 glioma cells. The immunohistochemical analysis demonstrated the presence of CaM kinase II in all of the cells examined. Furthermore, the kinase in cerebellar granule cells was activated by the stimulation of the glutamic acid receptor. Autophosphorylation of CaM kinase II in 3Y1 cells was stimulated by the addition of growth factors. These results suggest that CaM kinase II undergoes activation and autophosphorylation in response to various stimuli to the cells and is regulated in the dynamic state.
Amino Acid Sequence↗
Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II: effects on interaction between enzyme and substrate.
Characteristics of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the cytosol and in the postsynaptic densities (PSD) of rat brain were investigated. Several proteins were surveyed for their abilities to serve as a substrate for non-autophosphorylated and autophosphorylated CaM kinase IIs from the cytosol and PSD. The tested substrates were separated into two groups. Autophosphorylation of the kinase slightly decreased or did not change its activities towards substrates of the first group: myosin light chain of chicken gizzard, synapsin I, tau factor and microtubule-associated protein 2. In contrast, autophosphorylation of the enzyme increased its activities towards substrates of the second group: syntide-2, histone H1, calcineurin and myelin basic protein. The Ca2+/calmodulin-independent kinase activity increased by autophosphorylation with any of substrates tested. Similar results were obtained with the cytosolic and PSD CaM kinase II. Trifluoperazine and mastoparan, calmodulin binding antagonists, inhibited the activity of the non-autophosphorylated CaM kinase II, but had no effect or only a slight inhibitory effect on the activity of the autophosphorylated CaM kinase II, indicating that the autophosphorylated kinase has no requirement for calmodulin for Ca(2+)-dependent activity and/or a higher affinity for calmodulin The results suggest that the autophosphorylation of CaM kinase II is a subtle mechanism for regulating the interaction between the enzyme and substrate.
Animals↗
Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia.
The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.
Animals↗