Calmodulin effect on purified rat cortical plasma membrane Ca(2+)-ATPase in different phosphorylation states.

The plasma membrane Ca(2+)-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca(2+) concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca(2+)-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca(2+)-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca(2+)-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca(2+)-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca(2+)-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca(2+)-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca(2+)-ATPase.

Plasma membrane Ca2+-ATPase in excitable and nonexcitable cells.

There is a significant number of data confirming that the maintenance of calcium homeostasis in a living cell is a complex, multiregulated process. Calcium efflux from excitable cells (i.e., neurons) occurs through two main systems--an electrochemically driven Na+/Ca2+ exchanger with a low Ca2+ affinity (K0.5 = 10-15 microM), and a plasmalemmal, specific Ca2+-ATPase, with a high Ca2+ affinity (K0.5 < 0.5-1 microM), whereas in nonexcitable cells (i.e., erythrocytes) the calcium pump is the sole system responsible for the extrusion of calcium ions. The plasma membrane Ca2+-ATPase (PMCA) is a ubiquitously expressed protein, and more than 26 transcripts of four PMCA genes are distributed in a tissue specific manner. Differences in the structure and localization of PMCA variants are thought to correlate with specific regulatory properties and may have consequences for proper cellular Ca2+ signaling. The regulatory mechanisms of calcium pump activity have been studied extensively, resulting in a new view of the functioning of this important molecule in the membranes.

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity.

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17beta-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca2+-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10-11-10-6 M, showed an inverted U-shape potency in the regulation of Ca2+-ATPase activity. At physiologically relevant concentrations (10-8-10-9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10-11-10-6 M) and 17beta-estradiol (10-12-10-9 M) caused a dose-dependent increase in the hydrolytic ability of Ca2+-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.

Characterization of erythrocyte compounds in asphyxiated newborns.

Perinatal hypoxic-ischemic damage remains a major cause of acute mortality in infants. In our study we have shown that ATP-powered calcium pump was degraded in asphyxiated erythrocyte membranes. Moreover, the activity of Ca2+-ATPase, the enzyme that is solely responsible for maintenance of calcium homeostasis in erythrocytes, was reduced by 50% compared to healthy newborns. We have also detected the enhanced lipid peroxidation in asphyxiated erythrocyte ghosts. To elucidate the potential mechanisms of the calcium pump damage, we have examined the effect of peroxynitrite on Ca2+-ATPase purified from adult human erythrocyte membranes. We have concluded that calcium pump is a direct target for peroxynitrite action in vitro. Our results indicate that erythrocyte membrane compounds could be a primary target for asphyxia-induced damage, and the impairment of the plasma membrane Ca2+-ATPase function could be, in part, mediated by reactive oxygen species.

Can magnesium sulfate reduce the risk of cerebral injury after perinatal asphyxia?

Hypoxia-ischemia produces brain damage by processes that continue for many hours after reoxygenation/reperfusion. This provides a window of opportunity for therapy aimed at preventing further loss of brain cells. Sulfate magnesium can prevent posthypoxic brain injury by blocking glutamate receptors within the calcium (Ca++) ion channel. We used sulfate magnesium in nine newborn infant after perinatal hypoxia. We investigated the brain damage, by ultrasound examination, on third day, in first, second and third week, and third, sixth month of life. We have estimated the neurological development in the first week of life and third and twelfth month of life. We did not find deviations in ultrasound examination. We did not observe convulsions. We did not observe any side effect of this therapy. The examination at 1 of year of life in all of children was correct.

Protein kinases A and C phosphorylate purified Ca2+-ATPase from rat cortex, cerebellum and hippocampus.

The plasma membrane Ca2+-ATPase (PMCA), the enzyme responsible for the maintenance of intracellular calcium homeostasis, is regulated by several independent mechanisms. In this paper we report that the protein kinases A and C differentially activate the Ca2+-ATPase purified from synaptosomal membranes of rat cortex, cerebellum and hippocampus. The effect of protein kinases was more pronounced for the cortical enzyme, whereas cerebellar and hippocampal Ca2+-ATPases were activated to a lesser degree. The preparation of Ca2+-ATPase contained the phosphoamino acids, i.e., P-Ser and P-Thr, indicating that the enzyme was purified in phosphorylated state. The phosphorylation of Ca2+-ATPase by PKA and PKC increased the amount of phosphoamino acids, but in a region-dependent manner. Using the specific antibodies against N-terminal portion of four main PMCA isoforms we have characterized the isoforms composition of Ca2+-ATPase purified from the nervous endings of examined brain areas. Our results indicate that the activity of calcium pump is related to its phosphorylated state, and that the phosphorylation is region-dependent. Moreover, the differences observed could be related to the composition of PMCA isoforms in the different brain areas. Phosphorylation of the plasma membrane Ca2+-ATPase appears to be a mechanism to control its activity. The results support also the possible involvement of PKA and PKC.

Neuroactive steroids modulate in vitro the Mg(2+)-dependent Ca(2+)-ATPase activity in cultured rat neurons.

1. The in vitro effect of neuroactive steroids on the Mg(2+)-dependent Ca(2+)-ATPase activity in neuronal membranes isolated from primary cell culture of rat cortex was examined. 2. A 1-hr treatment of neuronal cell culture with 17-beta-estradiol (10 pM) and pregnenolone sulfate (1 microM) resulted in an increase in the enzyme activity of as much as 130% and 160%, respectively. 3. Neuroactive steroids moderately decreased the stimulation of the Mg(2+)-dependent Ca(2+)-ATPase activity by 72 nM calmodulin, by 20-30%. 4. The effects of hormones on the ATPase activity were irreversible after extensive washing of the membranes. 5. These results suggest that 17-beta-estradiol and pregnenolone sulfate at physiological concentrations could participate in the regulation of neuronal calcium homeostasis at a membrane level.

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells.

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from rat cerebellum by calmodulin affinity chromatography. The enzyme was identified as plasma membrane Ca2+-ATPase by its interaction with calmodulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the study was to establish whether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated from excitable cells and whether their effects are comparable to those on the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosphorylation and activation of the SM versus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SM Ca2+-ATPase was approximately 3 fold lower and significantly less stimulated by calmodulin. The initial rate of PKC catalyzed phosphorylation (in the presence of 12-myristate 13-acetate phorbol) was approximately two times slower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approached maximum level at around 5 min, comparable level of phosphorylation of SM Ca2+-ATPase was observed only after 30 min. The PKC-catalyzed phosphorylation resulted in a statistically significant increase in Ca2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase. The differences may be associated with diversities in Ca2+-ATPase function in erythrocytes and neuronal cells and different isoforms composition.

Okadaic acid as a probe for regulation in vitro of Mg(2+), Ca(2+)-ATPase activity in rat cortical and cerebellar synaptosomal membranes.

The in vitro effect of okadaic acid on basal phorbol 12-myristate 13-acetate (PMA)-, and cyclic adenosine monophosphate (cAMP)-stimulated Mg(2+)-dependent Ca(2+)-adenosine triphosphatase (ATPase) activity in synaptosomal membranes isolated from rat brain cortex and cerebellum was investigated. The basal activity was enhanced by okadaic acid in both examined regions. This inhibitor differed in the regulation of Mg2+, Ca(2+)-ATPase activity in PMA- and cAMP-incubated membranes. Stimulation by calmodulin (CaM) of basal Mg2+, Ca(2+)-ATPase activity declined in cortex and cerebellum after treatment with okadaic acid. The presence of PMA or cAMP decreases the stimulatory effect of CaM. These results suggest that Mg2+, Ca(2+)-ATPase activity in the rat-brain synaptosomal membrane may be regulated in vitro by dephosphorylation processes.

Neuroactive steroids modulate in vivo the Mg2+/Ca(2+)-ATPase activity in rat cortical and cerebellar synaptosomal membranes.

Estradiol and pregnenolone sulfate administered in vivo inhibited the Mg2+/Ca(2+)-ATPase activity in synaptosomal membranes from rat cortex by 20% and 30%, respectively. In cerebellum, estradiol decreased the activity up to 43%. The calmodulin-stimulated activity declined in cortex after treatment with estradiol and pregnenolone sulfate, but significantly increased in cerebellar membranes. Dehydroepiandrosterone sulfate had influence neither on enzyme activity, nor on stimulation by calmodulin in both examined rat brain regions.

Serotonin, histamine and somatostatin modulation of PMA-stimulated phosphorylation of 130 kDa Ca2+ pump-like protein from rat cerebellum synaptosomal membranes.

1. Influence of some neurotransmitters and neuromodulators on the PMA-stimulated phosphorylation in vitro of calcium pump-like protein from rat cerebellum synaptosomal membranes was examined. 2. The prolonged time (up to 6 min) of synaptosomal membranes preincubation with 1 and 10 microM serotonin results in the increase of phosphorylation. The decrease of phosphorylation up to 80% of control value was observed for 100 microM serotonin. 3. The most stimulating effect on 130 kDa protein phosphorylation was observed with 1 microM of histamine (160% of control value). 4. 1 and 0.1 microM somatostatin triggered a short-time transient increase of 130 kDa phosphorylation (up to 135% of control value).

Characterization of 130 kDa protein from rat cerebellum synaptosomal membranes phosphorylated by PKC.

1. The effect of endogenous PMA-stimulated phosphorylation of the protein in the molecular weight range of 130 kDa in rat cerebellum synaptosomal membranes was examined. 2. The 50% inhibition of the phosphorylation of 130 kDa protein by 5 microM polymyxin B was observed after 6 min of preincubation. 3. The sensitivity of 130 kDa protein for phosphorylation in the presence of exogenous protein kinase C suggests, that this protein could serve as a physiological substrate of protein kinase C. 4. Partial characterization of 130 kDa protein was performed. Upon incubation with [gamma-32P]ATP the 130 kDa protein formed Ca(2+)-dependent, hydroxylamine-sensitive phosphointermediate, which was inhibited by 50 microM vanadate, but not 0.5 mM vanadyl. 5. One-dimensional peptide mapping by proteolysis of 130 kDa protein with V8 protease was obtained.