Calcium-calmodulin Protein Kinase Research Articles

Modulation of the alveolar macrophage superoxide production by protein phosphorylation.

Stimulation of alveolar macrophages (AM) with adenosine-5-diphosphate (ADP) results in transient production of superoxide anion radical (O2.-; superoxide) and H2O2 in a metabolic event known as the respiratory burst. Initiation of the respiratory burst appears to depend on activation of protein kinase activity, whereas protein phosphatases might involved in termination of the burst. The involvement of protein kinase C was suggested by inhibition by bisindolylmaleimide I (GF 109203X), a relatively specific inhibitor. KN-62, an inhibitor of calcium-calmodulin protein kinase II, also partly inhibited the respiratory burst stimulated by ADP and phorbol esters. The role of protein phosphatases in termination of the ADP-stimulated respiratory burst of AM was examined with calyculin A (CA) (25-75 nM) or okadaic acid (OA) (1-5 microM), two inhibitors of protein phosphatase 1 and 2a (PP1;PP2a). A dose-dependent prolongation of the respiratory burst was observed in the presence of these inhibitors. CA and OA also markedly enhanced the rate of superoxide production stimulated by ADP, consistent with involvement of PP1/PP2a in regulating both the rate of activation and timing of termination. Treatment of AM with cyclosporin A (CsA) (1-50 microM), an inhibitor of the calcium-dependent protein phosphatase 2b (PP2b), stimulated superoxide production by itself and significantly prolonged the duration of ADP-stimulated superoxide production. CsA, however, did not increase the ADP-stimulated rate of superoxide production. Thus, PP1/PP2a appear to be the primary phosphatases for controlling the intensity of the respiratory burst during receptor-elicited superoxide production in AM, whereas PP1/PP2a and PP2b play a role in turning off the respiratory burst.

Memory Formation: The Sequence of Biochemical Events in the Hippocampus and Its Connection to Activity in Other Brain Structures

Recent data have demonstrated a biochemical sequence of events in the rat hippocampus that is necessary for memory formation of inhibitory avoidance behavior. The sequence initially involves the activation of three different types of glutamate receptors followed by changes in second messengers and biochemical cascades led by enhanced activity of protein kinases A, C, and G and calcium–calmodulin protein kinase II, followed by changes in glutamate receptor subunits and binding properties and increased expression of constitutive and inducible transcription factors. The biochemical events are regulated early after training by hormonal and neurohumoral mechanisms related to alertness, anxiety, and stress, and 3–6 h after training by pathways related to mood and affect. The early modulation is mediated locally by GABAergic, cholinergic, and noradrenergic synapses and by putative retrograde synaptic messengers, and extrinsically by the amygdala and possibly the medial septum, which handle emotional components of memories and are direct or indirect sites of action for several hormones and neurotransmitters. The late modulation relies on dopamine D1, β-noradrenergic, and 5HT1A receptors in the hippocampus and dopaminergic, noradrenergic, and serotoninergic pathways. Evidence indicates that hippocampal activity mediated by glutamate AMPA receptors must persist during at least 3 h after training in order for memories to be consolidated. Probably, this activity is transmitted to other areas, including the source of the dopaminergic, noradrenergic, and serotoninergic pathways, and the entorhinal and posterior parietal cortex. The entorhinal and posterior parietal cortex participate in memory consolidation minutes after the hippocampal chain of events starts, in both cases through glutamate NMDA receptor-mediated processes, and their intervention is necessary in order to complete memory consolidation. The hippocampus, amygdala, entorhinal cortex, and parietal cortex are involved in retrieval in the first few days after training; at 30 days from training only the entorhinal and parietal cortex are involved, and at 60 days only the parietal cortex is necessary for retrieval. Based on observations on other forms of hippocampal plasticity and on memory formation in the chick brain, it is suggested that the hippocampal chain of events that underlies memory formation is linked to long-term storage elsewhere through activity-dependent changes in cell connectivity.

In vivo and in vitro interaction of flunarizine with d-fenfluramine serotonergic effects

Flunarizine (35 mg/kg), but not haloperidol and trifluperazine, counteracted the initial indole depletion induced by d-fenfluramine (dF) in vivo (5 mg/kg), without affecting ex vivo [ 3H]-serotonin (5-HT) uptake by synaptosomes or changing the brain concentrations of the parent drug and its main active metabolite, d-norfenfluramine (dNF). The long-term indole depletion induced by repeated doses of dF (5 mg/kg b.i.d. for 4 days) was also reversed by flunarizine pretreatment. Flunarizine, methiothepin, and trifluperazine, but not haloperidol, reduced in vitro the Ca 2+-dependent [ 3H]5-HT release stimulated by 0.5 μM dF and dNF from superfused synaptosomes. At the concentrations used in release experiments the drugs were not active on [ 3H]5-HT uptake nor on the calcium-calmodulin protein kinase activity, thus excluding an effect on the uptake carrier or on phosphorylation of synaptic proteins involved in exocytosis, respectively. The drugs did not consistently affect [ 3H]5-HT release induced by depolarization, or dNF-induced [ 3H]dopamine release in vitro. The fact that flunarizine, as methiothepin and 5-HT uptake inhibitors, counteract dF-induced indole depletion in vivo suggests a relation between the reduction of the Ca 2+-dependent release of [ 3H]5-HT induced by dF in vitro and the protective effect on the short- and long-lasting depletion of indoles induced in vivo by high doses of dF.

Neocortex and hippocampus contain distinct distributions of calcium‐calmodulin protein kinase II and GAP43 mRNA

Calcium-calmodulin protein kinase II and GAP43 are two molecules which have been linked to synaptic plasticity. Localization of mRNA for these molecules identifies the neuronal populations which have the potential to utilize these mechanisms. General descriptions for calcium-calmodulin protein kinase II or GAP43 mRNA have been previously reported. In light of recent evidence that suggests that at some sites these two molecules may interact, we sought to determine the cortical distribution in detail, and to examine the extent of overlap between neuronal populations containing each mRNA. To this end we have used in situ hybridization techniques to study the distribution of calcium-calmodulin protein kinase II and GAP43 mRNA in adjacent sections of adult rat forebrain. Overall, the distribution patterns were distinct but partially overlapping. For both calcium-calmodulin protein kinase II and GAP43, mRNA levels were highest in hippocampus, allo- and neocortex, compared to moderate to low levels in striatum and thalamic nuclei. Within the heavily labeled regions certain populations expressed both calcium-calmodulin protein kinase II and GAP43 mRNA at high levels, while other populations were selective for calcium-calmodulin protein kinase II. In the hippocampus, the stratum pyramidale of CA1-3 expressed high levels of both calcium-calmodulin protein kinase II and GAP43 mRNA. Granule cells of the fascia dentata and the stratum radiatum of CA3 both contained moderate to high levels of calcium-calmodulin protein kinase II mRNA, but near background levels of GAP43 mRNA label. Within the neocortex, deep layers were distinguished from superficial layers by their lack of calcium-calmodulin protein kinase II mRNA expression within the neuropil, and the presence of GAP43 mRNA in neurons located in layer V and the deepest part of layer VI. Thus, layer V and deep layer VI neurons showed high levels of label for both GAP43 and calcium-calmodulin protein kinase II mRNA, while neurons of superficial layers contained only calcium-calmodulin protein kinase II mRNA. These markers differentiate neuronal populations which can also be distinguished on the basis of their ability to undergo specific forms of synaptic plasticity. These different forms of plasticity may be due in part to the laminar-specific patterns of GAP43 and calcium-calmodulin protein kinase II mRNA that we have described.

Mechanisms of aluminium fluoride- and insulin-stimulated p33 mRNA accumulation in rat hepatoma cells: Involvement of a G protein and kinase action and demonstration of effects on mRNA turnover

The insulin signalling pathway to control nuclear p33 gene expression was examined. An AlF 4-stimulated pertussin toxin-insensitive G protein was shown to be involved. The action of AlF 4 − was completely blocked by deferoxamine. Insulin action was markedly stimulated in the presence of AlF 4 −. cAMP and diacylglycerol concentrations were examined as possible regulators but no increases were detected. The effects of AlF 4 − and of insulin were completely inhibited by the general kinase inhibitor H-7. A second calcium calmodulin protein kinase inhibitor, W-7, had no detectable effect. Insulin and AlF 4 − were shown to stabilize p33 mRNA.

Defective sarcolemmal phosphorylation associated with noninsulin-dependent diabetes

Noninsulin-dependent diabetes is associated with a decrease in the activity of sarcolemmal phosphatase 1, but no change in the activities of phosphatase 2A, 2B, or 2C. Also unaffected by diabetes were the activities of protein kinase C, cAMP-dependent protein kinase and calcium-calmodulin protein kinase. Because of the decrease in phosphatase 1 activity, 32P incorporation into sarcolemmal phosphoproteins catalyzed by either intrinsic protein kinases or extrinsic cAMP-dependent protein kinase was elevated in the diabetic. Among the proteins whose phosphorylation was elevated in diabetes was the phospholamban-like protein, which has been implicated in the regulation of ATP-dependent calcium transport. The phosphate-linked increase could be prevented by exposing the membranes to a phosphatase inhibitor and either extrinsic cAMP-dependent protein kinase or alamethicin. In addition to the phosphatase-linked effects, analysis of individual sarcolemmal phosphoproteins by SDS-polyacrylamide gel electrophoresis indicated that diabetes caused a specific elevation in membrane phosphorylation of some proteins (43 kDa and 78 kDa), but a decrease in the phosphorylation state of other phosphoproteins (31 kDa and 49 kDa). The data indicate that membrane phosphorylation is dramatically altered by diabetes. The possibility that this contributes to altered myocardial function is discussed.

The phosphorylation of choline acetyltransferase

Human placental Choline Acetyltransferase (ChAT) has been shown to be phosphorylated in vitro by kinases present in rat brain. Phosphorylation occurs at a single site with the exclusive phosphoamino acid being serine. ChAT phosphorylation was shown to be calcium, and not cyclic nucleotide, dependent and was inhibited by inhibitors of calcium/calmodulin protein kinases including anti-calmodulin anti-sera. ChAT phosphorylation was stimulated by calmodulin (9 fold) and, to a lesser extent, by phosphatidylserine (4 fold). These results indicate the involvement of a calcium/calmodulin and possibly also a calcium/phospholipid kinase. This finding was confirmed by demonstrating ChAT phosphorylation using both purified multifunctional calcium/calmodulin protein kinase (CaMK) and calcium/phospholipid protein kinase C (PKC) from rat brain. A stoichiometric incorporation of 0.9 mol phosphate/mol ChAT was achieved by CaMK. Phosphorylated ChAT could be isolated from freshly prepared rat brain synaptosomes. The results obtained with this model system support the hypothesis that in vivo a fraction of ChAT exists phosphorylated.