Ultrashort wave alleviates oxygen -glucose deprivation/reoxygenation injury via up -regulation of SPCA1 expression in N2a cells / 中南大学学报(医学版)

OBJECTIVES@#Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca2+-ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level.@*METHODS@#N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting.@*RESULTS@#Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all P<0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences (P<0.01 or P<0.001).@*CONCLUSIONS@#USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

Obesity induces upregulation of genes involved in myocardial Ca2+ handling

Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+) handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c), sarcolemmal Na+/Ca2+ exchanger (NCX), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), ryanodine receptor (RyR2), and phospholamban (PLB) mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control) or high-fat diet (obese) for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05) but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

Thyroid status alters gill ionic metabolism and chloride cell morphology as evidenced by scanning electron microscopy in a teleost Anabas testudineus (Bloch): short and long term in vivo study.

Gill is the main organ of osmotic regulation in teleosts and chloride cells are the sites of ion transport across gill epithelium. Thyroid hormones are implicated in the regulation of osmotic balance in teleosts also. Treatment with 6-propyl thiouracil (6-PTU) inhibited the membrane bound enzyme Na+K+ ATPase in the gill while triiodothyronine (T3) injection stimulated it in a short-term in vivo study in the teleost Anabas testudineus. Na+, K+ and Ca2+ ions were also decreased in the 6-PTU treated fish and the T3 treatment increased their concentrations in the gill lamellae. The gill morphology also changed according to the thyroid status in the long term study. 6-PTU treatment altered the typical serrated morphology of the gill lamellae, while the T3 treatment reversed it. T3 injection increased the density of pavement and chloride cells as evidenced by scanning electron microscopy. The results demonstrate that physiological status of the thyroid influences gill Na+ pump activity and chloride cell morphological changes. Further, the study suggests a regulatory role of T3 on gill ions (Na+, K+ and Ca2+), Na+K+ and Ca2+ ATPase activity and the different gill cell types in A. testudineus.

Effect of methacrylonitrile on membrane bound enzymes of rat brain.

Methacrylonitrile (MeAN) is a plastic monomer. Its effect on membrane bound enzymes like Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, NADH dehydrogenase, alkaline phosphatase (ALP) and various elements like sodium (Na+), potassium (K+), and calcium (Ca2+) in rat brain were studied. Administration of 50 mg/kg body weight/day (0.25 LD50) and 100 mg/kg body weight/day (0.5 LD50) by gavage to rats for 7 days resulted in a significant decrease in activities of Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, and NADH dehydrogenase. A significant reduction in calcium content, potassium content and a significant increase in sodium content and alkaline phosphatase activity in MeAN treated animals were observed. Inhibition of membrane bound enzymes occurred due to either direct effect of MeAN or indirect effect of changes in ionic homeostasis in MeAN treated animals.

Efeito de íons Cu2+ e Zn2+ em atividade Ca-ATPásica isolada de larvas de Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) / Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae

As ATPases, um importante alvo de inseticidas, são enzimas que hidrolisam o ATP e utilizam a energia liberada no processo para realizar algum tipo de trabalho celular. A larva de Pachymerus nucleorum (Fabricius) possui uma ATPase que apresenta alta atividade Ca-ATPásica, mas não expressa atividade Mg-ATPásica. Nesse trabalho, foi testado o efeito de íons zinco e cobre na atividade Ca-ATPásica dessa enzima. Mais de 90 por cento da atividade Ca-ATPásica foi inibida em 0,5 mM de íons cobre ou 0,25 mM de íons zinco. Na presença de EDTA, mas não na sua ausência, a inibição por zinco foi revertida pelo aumento da concentração de cálcio. A inibição por íons cobre, não foi revertida nem na presença e nem na ausência de EDTA. O tratamento da fração ATPase com cobre, previamente ao ensaio de atividade ATPásica, não inibiu a atividade Ca-ATPásica sugerindo que o íon cobre não liga diretamente a enzima. Os resultados sugerem que íons zinco e cobre formam complexo com o ATP e se ligam à enzima inibindo sua atividade Ca-ATPásica.

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90 percent of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.

Calcium Uptake and Release through Sarcoplasmic Reticulum in the Inferior Oblique Muscles of Patients with Inferior Oblique Overaction

We characterized and compared the characteristics of Ca2+ movements through the sarcoplasmic reticulum of inferior oblique muscles in the various conditions including primary inferior oblique overaction (IOOA), secondary IOOA, and controls, so as to further understand the pathogenesis of primary IOOA. Of 15 specimens obtained through inferior oblique myectomy, six were from primary IOOA, 6 from secondary IOOA, and the remaining 3 were controls from enucleated eyes. Ryanodine binding assays were performed, and Ca2+ uptake rates, calsequestrins and SERCA levels were determined. Ryanodine bindings and sarcoplasmic reticulum Ca2+ uptake rates were significantly decreased in primary IOOA (p < 0.05). Western blot analysis conducted to quantify calsequestrins and SERCA, found no significant difference between primary IOOA, secondary IOOA, and the controls. Increased intracellular Ca2+ concentration due to reduced sarcoplasmic reticulum Ca2+ uptake may play a role in primary IOOA.

Insulin regulates ionic metabolism in a fresh water teleost, anabas testudineus (bloch).

Short term effects of insulin on total brain and branchial Na+K+ ATPase, Ca2+ ATPase and Na+, K+ and Ca2+ ions were investigated in A. testudineus. The increase in brain Ca2+ ATPase after alloxan treatment may account for an increased amount of intracellular calcium required for biochemical events taking place inside the cells. Branchial Na+K+ATPase was significantly stimulated while Ca2+ ATPase significantly inhibited after alloxan treatment. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca2+ transport via; its action on the Ca2+ pump protein rather than the membrane permeability to Ca2+. The increased activity of brain Na+K+ ATPase at 3 and 24 hr by insulin to alloxan pretreated fish may account for the stimulated co-transport of glucose and its utilization for energy requirements and the excitatory action on neurons in the brain. The elevated brain Ca2+ ATPase may be due to the role of calcium as a second messenger in hormone action. At 24 hr, the activity of branchial Na+K+ ATPase and Ca2+ ATPase in alloxan pretreated specimens was significantly stimulated by insulin. This may be due to increased synthesis of these enzyme units. Administration of insulin (lU/fish) in normal fish significantly inhibited the activity of brain and branchial Na+K+ ATPase while brain Ca2+ ATPase showed a stimulatory effect at 3 and 24 hr compared to control. Inhibition of total branchial Ca2+ ATPase activity by insulin may be due to increased Ca2+ concentration. Higher plasma glucose level in alloxan treated groups confirms the diabetic effect of alloxan. Insulin reverses this effect. The possible mechanism by which insulin controls Na+K+ ATPase activity appears to be tissue specific. The results seem to be the first report on the effect of insulin on ATPase activity in a teleost. These data are consistent with the hypothesis that insulin performs a role in hydro mineral regulation in freshwater teleosts.

Phostoxin-induced biochemical and pathomorphological changes in rabbits.

The effect of chronic phostoxin administration on some tissue ATPases, hematology and tissue histopathology was investigated using a combination of gravimetric, enzymatic, colorimetric and histological procedures in New Zealand White rabbits after 2 weeks administration of 0.8mg phostoxin/kg body weight/day, po. The phostoxin treatment led to significant decreases in Na(+)-K+ ATPase activities in renal, hepatic and cardiac tissues. Similar decreases were obtained in the activities of Ca(2+)-ATPase and Mg(2+)-ATPase in liver. In addition, the phostoxin-toxified rabbits manifested significant decreases in hematocrit, red blood cell count, hemoglobin and platelets. Histological examination of the tissues revealed pronounced degenerative changes in liver, heart and kidney.

Role of matrix metalloprotease-2 in oxidant activation of Ca2+ ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane.

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2 (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca2+-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1 : 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca2+-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca2+ATPase activity, H2O2 also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade 14C-gelatin. The protease activity and the Ca2+ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H2O2 was due to reactive oxidant species(es). Both basal and H2O2 stimulated MMP-2 activity and Ca2+ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1 : 10-phenanthroline, a2-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H2O2 increased MMP-2 activity and that subsequently stimulated Ca2+ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H2O2 to the smooth muscle membrane suspension caused submaximal increase in Ca2+ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H2O2 augments further the Ca2+ATPase activity caused by the respective low doses of either H2O2 or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity in the membrane caused by the combined treatment of MMP-2 and H2O2.

Restoration on tissue antioxidants by fenugreek seeds (Trigonella Foenum Graecum) in alloxan-diabetic rats.

The influence of fenugreek seed powder supplementation in the diet on lipid peroxidation and antioxidant status was studied in normal and alloxan-diabetic rats. The protective effect of the aqueous extract of the seeds on the activity of calcium-dependent adenosinetriphosphatase (Ca2+ ATPase) in liver homogenate in the presence of Fe2+/ascorbate in vitro was also investigated. Normal and diabetic rats were provided with a diet supplemented with fenugreek seed powder for 30 days at a dosage of 2 g/kg body weight. The diabetic rats exhibited enhanced lipid peroxidation and increased susceptibility to oxidative stress associated with depletion of antioxidants in liver, kidney and pancreas. However, treatment with fenugreek seed powder normalised the alterations. In normal rats supplementation resulted in increased antioxidant status with reduction in peroxidation. Ca2+ ATPase activity in liver was protected by the aqueous extract to nearly 80% of the initial activity. The findings suggest that the soluble portion of the seeds could be responsible for the antioxidant property.

Energy interconversion by the sarcoplasmic reticulum Ca2+-ATPase: ATP hydrolysis, Ca2+ transport, ATP synthesis and heat production

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca2+-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca2+ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 Kcal/mol depending on whether or not a transmembrane Ca2+ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca2+ transport and the fraction which is dissipated in the surrounding medium as heat.

Interaction of chlorpromazine with low molecular mass ion-transporting ATPase modulator proteins from rat brain cytosol.

Two low molecular mass proteins (13 kDa which inhibits Na+,K(+)-ATPase and 12 kDa which modulates Ca2+, Mg(2+)- and Ca(2+)-ATPases), purified from rat brain cytosol form complexes with chlorpromazine (CPZ) on incubation. The conformational characteristics of the proteins and their complex have been studied by comparing the fluorescence and CD spectra. The tryptophan fluorescence data show that the inhibitor-CPZ complex does not quench the fluorescence of NA+,K(+)-ATPase significantly. CD spectra indicate that the structure of the inhibitor is changed on formation of the complex. The inhibitor-CPZ complex significantly changes the conformation of Na+,K(+)-ATPase. The regulator protein-CPZ complex does not have any appreciable effect on Ca2+, Mg(2+)- and Ca(2+)-ATPase activities. The Trp-fluorescence of Ca2+,Mg(2+)- and Ca(2+)-ATPase are not significantly affected in presence of the complex. CD spectra indicate that the structure of the regulator is abruptly affected on formation of the complex. The conformations of Ca2+,Mg(2+)- and Ca(2+)-ATPases are found to be altered in presence of the complex.

Involvement of a protease in tert-butylhydroperoxide-mediated activation of Ca2+ ATPase in microsomes of pulmonary smooth muscle.

Microsomes isolated from bovine pulmonary artery smooth muscle tissue treated with the oxidant t-buOOH stimulated Ca2+ ATPase activity dose-dependently as also protease activity when tested with a synthetic substrate N-benzoyl-DL-arginine p-nitroanilide. At 300 microM, t-buOOH optimally stimulated these activities. Treatment of the microsomes with t-buOOH stimulated ATP dependent Ca2+ uptake while Na+ dependent Ca2+ uptake was inhibited by t-buOOH. Pretreatment of the microsomes with vitamin E (1 mM) and aprotinin (1 mg/ml) prevented t-buOOH caused stimulation of protease activity and Ca2+ ATPase activity, and also stimulation of ATP dependent Ca2+ uptake while t-buOOH caused inhibition of Na+ dependent Ca2+ uptake was reversed by vitamin E and aprotinin. Treatment of the microsomes with trypsin (1 microgram/ml) stimulated Ca2+ ATPase and ATP dependent Ca2+ uptake while Na+ dependent Ca2+ uptake was inhibited. Pretreatment of the microsomes with aprotinin prevented trypsin caused stimulation of Ca2+ ATPase and ATP dependent Ca2+ uptake, while trypsin caused inhibition of Na+ dependent Ca2+ uptake was reversed by aprotinin.

Localización citoquímica ultraestructural de la actividad de ATPasa de Ca²+ en membranas de eritrocitos humanos invadidos por Plasmodium falciparum / Ultrastructural cytochemical localization of the Ca²+ ATPase activity in the membrane of human erythrocytes invaded by Plasmodium falciparum

Durante el proceso de invasión del eritrocito humano por Plasmodium falciparum el calcio juega un papel importante y su concentración es aumentada notablemnte con relación al eritrocito normal. En este trabajo se propuso determinar la presencia de bombas de calcio como la ATPasa de ca²+ en membranas de eritrocitos invadidos, con el fin de explicar el papel mediador que dicha bomba puede tener en el trasporte de calcio durante la invasión. Para detectar dicha actividad de la ATPasa de Ca²+ se estandarizó un método directo de precipitación de metales pesados en donde el fosfato es el producto de hidrólisis catalizada del ATP. Este método utiliza un medio de incubación que contiene todos los elementos necesarios para que la enzima pueda desarrollar su actividad de ATPasa. El producto de reacción de esta actividad se comprobó por la formación de un precipitado electrodenso que se deposita a lo largo de la superficie de membranas tanto de eritrocitos normales como de los invadidos. Para determinar la actividad de ATPasa en la célula completa mediante este método citoquímico, fué necesario modificar la concentración intracelular de calcio y permeabilizar dicha célula invadida. La actividad de ATPasa fué caracterizada mediante el uso de inhibidores como vanadato, ouabaína y EGTA demostrándose una actividad específica para ATPasa de Ca²+. La especificidad de ésta fué ratificada mediante el uso de inhibidores de calmodulina como trifluoperazina, W7, calmidazolium y un sustituyente de calcio como Lantano. Durante el ciclo asexual eritrocítico, la actividad en la membrana del eritrocito permaneció constante duratnte todo el ciclo y la membrana de la vacuola parasitófora mostró una mayor actividad en parásitos de 18-20 horas de crecimiento y posteriormente una disminución y pérdida de actividad durante la maduración del parásito. Se encontró actividad de ATPasa de Ca²+ en otras estructuras membranales como los clefts distribuídos en el citosol de la célula roja invadida

Specific activities of seminal vesicular phosphomonoesterases and Mg2+-,Ca2+- and Na+/K(+)-adenosine triphosphatases in hypo- and hyperthyroid albino rats.

Hypothyroidism (surgical thyroidectomy) inhibited the activities of acid phosphatase and Mg(2+)-ATPase in seminal vesicular tissue and fluid and that of Ca(2+)- and Na+/K(+)-ATPases in fluid alone, and T4 supplementation restored normalcy in all, except acid phosphatase. Hyperthyroidism (T4 25 micrograms/100g body weight/day for 60 days, im) enhanced the activities of alkaline phosphatase and ATPases in seminal vesicular tissue and fluid, and decreased acid phosphatase activity in tissue alone. Withdrawal of T4 treatment from hyperthyroid rats (after 30 days) augmented the activity of ATPases in tissue and impaired the same in fluid, while phosphomonoesterases remained at hyperthyroid level. The results suggest specific responses of various seminal vesicular phosphatases to altered thyroid hormone status. Modification in the specific threshold of androgen/estrogen action on different phosphatases in seminal vesicles appears to be the plausible mechanism underlying these changes in hypo- and hyperthyroid conditions.

Interactions of the regulatory ligands Mg2+ and MgATP2- with the renal plasma membrane Ca(2+)-ATPase: effects of osmolytes that stabilize or destabilize protein structure

In this report we analyze the kinetics of activation of the plasma membrane Ca(2+)-ATPase from kidney proximal tubules by the regulatory ligands Mg2+ and MgATP2-, and we examine modifications in the effects of these ligands that are promoted by organic solutes of natural occurrence that stabilize or destabilize protein structure and function. The solutes tested were trimethylamine-N-oxide (TMA-O), sucrose and urea. TMA-O and sucrose were chosen as representative of the different methylamines and polyols, respectively, that accumulate in living organisms. The results lead to the conclusion that free Mg2+ and the MgATP2- complex both activate the rate-determining E2-->E1 transition during the catalytic cycle of the enzyme, by binding to nonidentical and independent regulatory sites. They also indicate that TMA-O, sucrose and urea not only promote global modifications in the enzyme structure, but also modify specific interactions of the ligands Mg2+ and MgATP2- at their regulatory sites

Intracellular calcium homeostasis in Leishmania mexicana. Identification and characterization of a plasma membrane calmodulin-dependent Ca(2+)-ATPase

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin...

Methyl parathion induced regional alterations in the regulatory proteins during critical stage of central nervous system development in albino rat pups.

Sublethal doses of methyl parathion (O,O-dimethyl-O-nitrophenyl- thiophosphate) injected intraperitoneally to 15 and 21 day old rat pups induced regional alterations in the central nervous system (CNS) in the levels of total RNA, total proteins, modulatory protein Calmodulin (CaM), in the activity levels of membrane bound enzyme Ca(2+)-ATPase and phospholipids. Levels of RNA and total proteins increased considerably in 15 days old methyl parathion treated (MPT) rat pups. Contrary to this the RNA and total protein content exhibited remarkable decrease in 21 day old methyl parathion treated animals. Calmodulin level showed an increase in cerebral cortex and brain stem and decrease in cerebellum and spinal cord in 15 day old methyl parathion treated rat pups. Whereas the level of Calmodulin decreased in cerebral cortex and cerebellum and increased in brain stem and spinal cord in 21 day old methyl parathion treated rat pups. Activity levels of calcium dependent ATPase showed significant inhibition in all the regions of Central Nervous System (CNS) of 15 and 21 day old methyl parathion treated rat pups. Phospholipids showed a general increase in all the regions of Central Nervous System on methyl parathion exposure. In the light of these observations, it has been suggested that the molecular regulatory mechanisms involving Ca2+/CaM are rendered inefficient due to toxic impact of methyl parathion.

Effect of H2-receptor antagonists, cimetidine and ranitidine on reproductive functions in male mice.

Na+,K(+)-ATPase and Ca(2+)-ATPase in testis were inhibited with an oral administration of cimetidine and ranitidine. Cimetidine at dose level of 100 and 30 mg while ranitidine at 70 and 10 mg per kg body wt inhibited the enzyme activities, 24 hr after single administration or daily administration for 15-days. Mg(2+)-ATPase activity was increased with cimetidine while ranitidine inhibited the enzyme. Michaelis-Menten kinetic characteristics revealed mixed type of inhibition for Na+,K(+)-ATPase with cimetidine, whereas it was noncompetitive for Ca(2+)-ATPase with cimetidine as well as ranitidine administration. Inhibition of Na+,K(+)-ATPase with ranitidine was also of noncompetitive type. Mg(2+)-ATPase behaved differently with administration of ranitidine at both the time points used i.e. noncompetitive type of inhibition after 24 hr and mixed type after 15-days. Histologically, signs of degeneration of testicular elements appeared after administration of cimetidine with a significant decrease in tubular diameter and germinal epithelial cell height. Ranitidine administration did not produce any change in the seminiferous tubules of testis. Scanning electron microscopy of spermatozoa from cimetidine-treated mice exhibited distinct departure from the normal morphology such as, (i) breaks at various places along distal portion of the tail, (ii) roughening, wrinkling and disorganization of plasma membrane of the head region, (iii) decapitation of the head and (iv) changes in shape of cytoplasmic droplet. Ranitidine administration showed normal morphology of the spermatozoa.