Sodium Nitrite Alone Protects the Brain Microsomal Ca2+-ATPase Against Potassium Cyanide-induced Neurotoxicity In Rats

The effect of a short-term oral administration of potassium cyanide (KCN) (200 ppm in diet) with or without sodium nitrite (NaNO₂) pretreatment on rat brain microsomal Ca^2± ATPase was investigated. The specific activity value of the enzyme significantly decreased (p<0.05) by 50% compared with control and by 63% for KCN-treated rats compared with KCN-treated rats pretreated with NaNO₂. There was no significant difference at the h=0.05 level between the values obtained for the control and KCN-treated rats pretreated with NaNO₂. These results show both that feeding lowers brain microsomal Ca²⁺-ATPase activity and that NaNO₂ has a protective role (antidote function) in that respect.     

Increase in calcium content and Ca2+-ATPase activity in the brain of fasted rats: Comparison with different ages

The effect of fasting on calcium content and Ca²⁺-ATPase activity in the brain tissues of 5 weeks and 50 weeks old rats was investigated. Brain calcium content and Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of the brain homogenate from young and elderly rats were significantly increased by overnight–fasting. These increases were appreciably restored by a single oral administration of glucose solution (400 mg/100 g body weight) to fasted rats. In comparison with young and elderly rats, brain calcium content and microsomal Ca²⁺-ATPase activity were significantly elevated by increasing ages. The effect of ageing was not seen in the brain mitochondrial Ca²⁺-ATPase activity. When calcium (50 mg/100 g) was orally administered to young and elderly rats, brain calcium content was significantly elevated. The calcium administration–induced increase in brain calcium content was greater in elderly r crease in Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of brain homogenates from young rats. In aged rats, the microsomal Ca²⁺-ATPase activity was not further enhanced by calcium administration, although the mitochondrial enzyme activity was significantly raised. The present study demonstrates that the fasting–induced increase in brain calcium content is involved in Ca²⁺-ATPase activity raised in the brain microsomes and mitochondria of rats with different ages, supporting a energy–dependent mechanism in brain calcium accumulation.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Role of Ca2+,Mg2+-ATPases in Diabetes-Induced Alterations in Calcium Homeostasis in Input Neurons of the Nociceptive System

In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca²⁺ in input neurons of the nociceptive system increases, Ca²⁺ signals are prolonged, while Ca²⁺ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca²⁺,Mg²⁺-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca²⁺,Mg²⁺-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca²⁺,Mg²⁺-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca²⁺ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Streptozotocin-induced diabetes increases (Ca2+-Mg2+)-ATPase activity in hepatic plasma membranes of rats: Involvement of protein kinase C

The alteration in calcium transport in the liver of rats with streptozocin(STZ)-diabetic state was investigated. STZ (6 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 2 weeks later they were sacrificed by bleeding. STZ administration caused a remarkable elevation of serum glucose concentration. Liver calcium content was significantly increased by STZ administration. Hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was markedly elevated by STZ administration. This increase was completely abolished by the presence of staurosporine (10^-7-10^-5 M), an inhibitor of protein kinase C, in the enzyme reaction mixture, suggesting an involvement of protein kinase C signalling. Moreover, the STZ-induced increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was significantly raised by the presence of okadaic acid (10^-5 and 10^-4 M). Meanwhile, the STZ-increased (Ca²⁺-Mg²⁺)-ATPase activity was not appreciably altered by the presence of anti-regucalcin IgG in the reaction mixture, indicating that the activatory protein regucalcin does not participate in the elevation of the enzyme activity. The present study demonstrates that STZ-induced diabetes causes the increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity of rats.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Acetaminophen at Different Doses Protects Brain Microsomal Ca2+-ATPase and the Antioxidant Redox System in Rats

Acetaminophen, an analgesic and antipyretic drug, rescues neuronal cells from mitochondrial redox impairment and reactive oxygen species (ROS). Excessive administration of acetaminophen above the recommended daily dose range has some negative effects on the brain. We investigated the effects of different doses of acetaminophen on Ca²⁺-ATPase and the antioxidant redox system in rats. Seventy rats were randomly divided into seven equal groups. The first was used for the control. One dose of 5, 10, 20, 100, 200, and 500 mg/kg acetaminophen was intraperitoneally administered to rats constituting the second, third, fourth, fifth, sixth, and seventh groups, respectively. After 24 h, brain cortical samples were taken and brain microsomal samples were obtained by ultracentrifugation. Brain and microsomal lipid peroxidation (LP) and brain calcium levels in the sixth and seventh groups were increased compared to control. LP levels in the second, third, and forth groups; brain vitamin E levels; brain and microsomal glutathione peroxidase (GSH-Px); and Ca²⁺-ATPase activity in the sixth and seventh groups were lower than in control, although brain vitamin E concentrations in the second, third, fourth, and fifth groups and microsomal GSH-Px activity in the third and fourth groups were higher than in control. Brain cortical β-carotene and vitamin A concentrations did not differ in the seven groups. In conclusion, 5–100 mg/kg acetaminophen seems to have protective effects on oxidative stress-induced brain toxicity by inhibiting free radicals and supporting the antioxidant redox system.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Neutral organic solute effects on the activity of the plasma membrane Ca2+-ATPase

We have compared effects of dimethylsulfoxide (Me₂SO) and two polyols on the Ca²⁺-ATPase purified from human erythrocytes. As studied under steady-state conditions over a broad solute concentration range and temperature, Me₂SO, glycerol, and xylitol do not inhibit the Ca²⁺-ATPase activity; this is in contrast to numerous other organic solutes that we have investigated. Under specific experimental conditions, Me₂SO (but not glycerol) substantially increases Ca²⁺-ATPase activity, suggesting a possible facilitation of enzyme oligomerization. The activation is more pronounced at low Ca²⁺ concentrations. In contrast to glycerol, Me₂SO shows no protective effect on enzyme structure as assessed by determining residual Ca²⁺-ATPase activity after exposing the enzyme to thermal denaturation at 45°C. Under these conditions several other organic solutes strongly enhance the denaturating effect of temperature. Because of the temperature dependence of its effect on the Ca²⁺-ATPase activity we believe that Me₂SO activates the Ca²⁺-ATPase by indirect water-mediated interactions.     

Consequence of aflatoxin B1 ingestion on the membrane-bound liver endoplasmic reticulum Ca2+-ATPase of protein-undernourished Fischer F344 rats

Ingestion of the mycotoxin Aflatoxin B₁ (AFB₁) by protein-undernourished Fischer F344 rats for twelve weeks resulted in significant (p<0.05) increases in the proliferation of liver cells, reduced body weight and increased microsomal Ca²⁺-ATPase activity. Cyt. P₄₅₀ content, -Glutamyl Transferase (GGT) and Glutathione Transferase (GST) activities were unaffected. The ingestion of AFB₁ by normal rats had no effect on all the parameters investigated. It appears that the microsomal Ca²⁺-pumping ATPase of Protein-Undernourished (PU) Fischer F344 rats is more sensitive to the mycotoxin AFB₁ than its counterpart in well-nourished rats. The use of PU rat as animal model for studies on AFB₁ toxicity, particularly the effect of AFB₁ on the regulation of intracellular calcium ion concentration ([Ca²⁺]_i), is suggested.     

亲和层析纯化肌质网Ca2+－ATP酶

建立了一种亲和层析纯化肌质网Ca²⁺-ATP酶的方法．用非离子型去污剂C₁₂E₈ 溶解肌质网,再通过反应红－120琼脂糖亲和层析柱使肌质网Ca²⁺-ATP酶纯度从粗品中的65％提高到99％,并具有较高ATP水解活性．经SDS－聚丙烯酰胺凝胶电泳检测,为电泳纯．     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Effects of selenium deficiency on Ca transport function of sarcoplasmic reticulum and lipid peroxidation in rat myocardium

Two groups of weanling Sprague-Dawley rats were fed a low-selenium basal diet (Se 0.009 mg/kg) and the same diet supplemented with sodium selenite (Se 0.25 mg/kg), respectively, for 1, 2, and 3 months. At each feeding time, the Ca²⁺-ATPase activity, Ca²⁺ uptake rate and the capacity of Ca²⁺ uptake in isolated cardiac sacroplasmic reticulum from the Se-deficient rats were decreased significantly compared to those from the Se-supplemented rats, the contents of lipid peroxide in postmitochondrial supernatant and isolated sarcoplasmic reticulum from the Se-deficient rats were significantly higher than that from Se-supplemented rats. Compared to the Se-supplemented rats, the cytosolic glutathione peroxidase activity in Se-deficient rats decreased significantly. In addition, significant linear negative correlations of lipid peroxide in postmitochondrial supernatant to sarcoplasmic reticular Ca²⁺-ATPase activity, Ca²⁺ uptake rate and to whole blood selenium concentration were observed. The results suggest that the enhancement of lipid peroxidation via the depressed glutathione peroxidase activity might be responsible for the decrease of Ca²⁺-ATPase and Ca²⁺ uptake activities in sarcoplasmic reticulum in Se-deficient animals.     

Phosphorylation by protein kinase C and the responsiveness of Mg2+-ATPase to Ca2+ of myofibrils isolated from stunned and non-stunned porcine myocardium

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca²⁺ sensitizer without phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in non-stunned myocardium. This finding was consistent with the observed leftward shifts of the pCa²⁺/Mg²⁺-ATPase curves of isolated myofibrils induced by [+]EMD 60263. The aim of the present investigation was to study the possible involvement of protein kinase C in the mechanism of reduced Ca²⁺ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca²⁺-stimulated Mg²⁺-ATPase and in the pCa₅₀ of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-³²P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the³² P incorporation counted by the Molecular Imager. Ca²⁺/phosphatidylserine/sn-1,2 diolein-dependent³² P incorporation catalyzed by excess of purified rat brain protein kinase C in C-protein, TnT and TnI subunits did not show any differences between myofibrils from non-stunned and stunned myocardium. However, protein kinase C-induced phosphorylation of myofibrils isolated from ventricular myocardium of sham-operated pigs resulted in a marked leftward shift of the pCa₅₀ from 6.03 ± 0.04 to 6.44 ± 0.06 (p < 0.05), while porcine heart cyclic AMP-dependent protein kinase-induced phosphorylation resulted in an expected small rightward shift to 5.97, although statistical significance was not reached. Protein kinase C-induced phosphorylation also stimulated (80%) the maximal myofibrillar Mg²⁺-ATPase activity. [+]EMD 60263 (3 µM) produced a leftward shift of the myofibrillar pCa²⁺/Mg²⁺-ATPase curve which was unaffected by prior protein kinase C-induced phosphorylation. In conclusion, the findings with isolated myofibrils from myocardium of anaesthetized open-chest pigs indicate that protein kinase C might be involved in the mechanism of reduced Ca²⁺ responsiveness of myofilaments in stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca²⁺-stimulated Mg²⁺-ATPase, the pCa₅₀ and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

Antimicrotubular drugs binding to vinca domain of tubulin

The effect of oxidative stress on the Ca²⁺-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO₄/EDTA (0.2 mol Fe²⁺ per mg of protein) at 37°C for 1 h in the presence or absence of antioxidants. FeSO₄/EDTA decreased the maximum velocity of Ca²⁺-ATPase reaction without a change of affinity for Ca²⁺ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca²⁺-ATPase and decrease in tryptophan fluorescence, while the loss of –SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca²⁺-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca²⁺-ATPase activity.     

Topiramate modulates hippocampus NMDA receptors via brain Ca2+ homeostasis in pentylentetrazol-induced epilepsy of rats

Background: Increase in neuronal Ca²⁺, activation of hippocampus N-methyl-D-aspartate receptor (NMDAR) and defects in enzymes such as brain cortex microsomal membrane Ca²⁺-ATPase (MMCA) are thought to play a role in epilepsy. Topiramate (TOP) is a novel drug with broad antiepileptic effect, and its effect on brain cortex MMCA is not known. We investigated effects of TOP on pentylentetrazol (PTZ)-induced MMCA activity and NMDAR subunits in rat brain.

Materials and methods: Thirty-two rats were randomly divided into four equal groups. The first group and second groups were used for the control and PTZ groups, respectively. 50 and 100?mg TOP were administered to rats constituting the third (TOP50) and fourth (TOP100) groups for 7 days, respectively. At the end of 7 days, all groups except the first received a single dose PTZ. Brain and hippocampus samples were taken at 3?hrs after PTZ administration.

Results: The microsomal MMCA activity was lower in the PTZ group than in control although the MMCA activities were higher in the treatment group than in PTZ group. Brain cortex total calcium levels, the hippocampus NMDAR 2A and 2B subunit concentrations were higher in the PTZ group than in control although their concentrations were decreased by TOP50 and TOP100 administration. Total brain cortex calcium and hippocampus NMDAR 2A and 2B subunit concentrations were higher in TOP100 group than in TOP50 group.

Conclusion: The two doses of TOP modulated MMCA activity, total brain cortex calcium and hippocampus NMDAR 2A and 2B subunit concentrations in the epileptic rats.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.