ATPase Activity In Microsomes Research Articles

SERCA Inhibition as a Potential Therapeutic Target for Breast Cancer

Chemotherapy is both a palliative treatment for many cancers and adjuvant treatment before, during, or after local treatment (surgery and/or radiation therapy) in single or combination regimens. Despite current treatments, approximately 1 in 39 women (3%) still die from breast cancer. A major complication with systemic therapy is drug resistance which limits the efficacy of clinical chemotherapy. Hence, current research aims to uncover novel targets for breast cancer therapy. Various studies have shown altered calcium signaling in breast cancer cells, therefore, calcium modulators may be potential therapeutic targets. Studies have also shown that Thapsigargin (TG) and cyclopiazonic acid (CPZ), two potent and specific inhibitors of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), can impair cancer cell growth. However, TG and CPZ also impair non-malignant cells, which results in significant toxicity, thus preventing use in chemotherapy. Using spectrometry UV-visible measurements to assess calcium loading and ATPase activity in SR microsomes, our lab has found that various drugs which display cell protective effects in ischemia also inhibit SERCA. They include novel benzothiazepines (BZTs; K201, CGP37157, PH00095) and have shown to inhibit SERCA in a calcium dependent mechanism. They decreased SR uptake when cytosolic Ca2+ levels ([Ca2+]c) are low, similar to competitive inhibition of Ca2+ uptake, which produces mild store depletion. Contrarily, TG and CPZ block Ca2+ uptake at all [Ca2+]c and largely deplete SR stores. Using various SERCA inhibitors, we sought out to determine their effects on breast cancer cell lines of different subtypes. MTS assay studies show that TG and CPZ significantly reduce cell viability in MCF7 and MCF10A cell lines while having no significant effects on triple negative breast cancer (TNBC) cell line MDAMB231. Preliminary studies also show no significant effect by TG on BT20 cells, another TNBC cell line. BZT K201 also significantly reduced cell viability in MCF7 cells but not MDAMB231. Immunoblot shows MCF7 cells have significantly higher SERCA2 expression than MDAMB231. We observe no significant differences in plasma membrane calcium channel ATPase (PMCA) protein expression. Preliminary RT-PCR studies estimate MCF7 cells have a higher fold change in SERCA genes. Thus, targeting SERCA may serve as a potential therapeutic target for breast cancer. Here, we find that SERCA inhibitors TG, CPZ, and BZT K201 reduce cell viability in MCF7 cells. Still, these studies indicate that SERCA inhibition may only be efficacious in certain breast cancer cell types which do not express a TNBC phenotype. TNBC cell lines (MDAMB231, BT20) are not significantly affected by SERCA inhibition, which may be due to higher dependence for signaling on plasmalemma calcium channels and transporters. Additional studies are required to confirm the expression profiles of calcium transporters among cell lines, including those in the plasmalemma (PMCA, calcium channels), which may help to understand if alternative sources of Ca2+ for intracellular signaling make TNBC cell lines more resistant to SERCA inhibitors.

Plasma membrane H+‐ATPase activity and graft success of breadfruit (Artocarpus altilis) onto interspecific rootstocks of marang (A. odoratissimus) and pedalai (A. sericicarpus)

Breadfruit (Artocarpus altilis) is primarily grown as a staple tree crop for food security in Oceania. Significant wind damage has driven interest in developing its dwarfing rootstocks. Due to the predominantly vegetative propagation of the species, grafting onto interspecific seedlings is an approach to identifying dwarfing rootstocks. However, grafting of breadfruit onto unrelated Artocarpus species has not been investigated. Here we first report the success of breadfruit grafting onto interspecific rootstocks, marang (A. odoratissimus) and pedalai (A. sericicarpus). To address the low graft survival, we investigated the relationship of plasma membrane (PM) H+ -ATPase activity to graft success. We provide the first evidence for a positive correlation between PM H+ -ATPase activity and graft survival. The graft unions of successful grafts had higher PM H+ -ATPase activity compared to those of failed grafts. Rootstocks with low PM H+ -ATPase activity in leaf microsomes before grafting had lower graft survival than those with high enzyme activity, with graft success of 10% versus 60% and 0% versus 30% for marang and pedalai rootstocks, respectively. There was a positive correlation between graft success and the PM H+ -ATPase activity measured from the rootstock stem microsomes 2months after grafting [marang, r(7)=0.9203, P=0.0004; pedalai (r(7)=0. 8820, P=0.0017]. Removal of scion's own roots decreased the leaf PM H+ -ATPase activity of grafted plants regardless of the final graft outcome. Recovery of the enzyme activity was only found in the successful grafts. The function of PM H+ -ATPase in graft union development and graft success improvement is discussed.

Pharmacological Targeting of Serca May Have Potential for Cellular Protection

We previously reported that CGP-37157 and K201, two benzothiazepines (BZT) with cardioprotective action, inhibit the Ca2+ ATPase of sarcoplasmic reticulum (SR) intracellular Ca2+ stores (SERCA). We tested if SERCA block could be part of the mechanism by which drugs protect cells from ischemic damage. We also screened structural characteristics in BZT that could affect their drug potency as SERCA inhibitors. SR microsomes isolated from rabbit skeletal muscle (SkM) and pig heart ventricle were utilized to measure modulation of SR Ca2+ loading and SERCA-mediated ATPase activity by drugs. Seven out of twenty cell-protective drugs tested (including pimozide, EGCG, KN-93 and carvedilol) inhibited, at least partially, SR Ca2+ loading and Ca2+-stimulated ATPase activity in SkM and heart SR microsomes. We also screened ten novel BZT derivatives and seven FDA-approved benzodiazepines (BZD; including bromazepam and clonazepam), which have close homology to CGP37157. Ca2+-dependent block of SERCA was found in four BZT's, which displayed higher (PH000995, PH000902) and similar potency (PH000902, PH006796) compared to CGP. BZD were all ineffective, which suggest that the sulfur atom in the BZT ring (substituted by nitrogen in BZD) is crucial for their SERCA blocking ability. All compounds above, were tested on ryanodine receptor (RyR) activity (planar bilayers, SR leak, [3H] ryanodine binding). None of these agents directly inhibited RyR function in heart and muscle. In contrast, some agents (BZT and BZD) had mild agonistic action on channel function. We think that SERCA block by these drugs, which persists at pH ∼6.5, may benefit ischemic cells by preventing SR Ca2+ overload, known to trigger, upon reperfusion, abnormal RyR-mediated Ca2+ leak associated with cell death and tissue injury. BZT have potential as templates for therapeutic targeting of SERCA (Supported by AHA and Eskridge Foundation).

Effect of feeding lipids recovered from fish processing waste by lactic acid fermentation and enzymatic hydrolysis on antioxidant and membrane bound enzymes in rats.

Fish oil recovered from fresh water fish visceral waste (FVW-FO) through lactic acid fermentation (FO-LAF) and enzymatic hydrolysis (FO-EH) were fed to rats to study their influence on lipid peroxidation and activities of antioxidant and membrane bound enzyme in liver, heart and brain. Feeding of FO-LAF and FO-EH resulted in increase (P < 0.05) in lipid peroxides level in serum, liver, brain and heart tissues compared to ground nut oil (control). Activity of catalase (40-235%) and superoxide dismutase (17-143%) also increased (P < 0.05) with incremental level of EPA + DHA in diet. The increase was similar to cod liver oil fed rats at same concentration of EPA + DHA. FO-LAF and FO-EH increased (P < 0.05) the Na(+)K(+) ATPase activity in liver and brain microsomes, Ca(+)Mg(+) ATPase in heart microsome and acetylcholine esterase in brain microsomes when fed with 5% EPA + DHA. There was also significant change in fatty acid composition and cholesterol/phospholipid ratio in microsomes of rat fed with FVW-FO. Feeding FVW-FO recovered by biotechnological approaches enhanced the activity of antioxidant enzymes in tissues, modulates the activities of membrane bound enzymes and improved the fatty acid composition in microsomes of tissues similar to CLO. Utilization of these processing wastes for the production of valuable biofunctional products can reduce the mounting economic values of fish oil and minimize the environmental pollution problems.

Cannabinoid receptors in submandibular acinar cells: functional coupling between saliva fluid and electrolytes secretion and Ca2+ signalling

Cannabinoid receptors (CBRs) belong to the G protein-coupled receptor superfamily, and activation of CBRs in salivary cells inhibits agonist-stimulated salivation and modifies saliva content. However, the role of different CBR subtypes in acinar cell physiology and in intracellular signalling remains unclear. Here, we uncover functional CB(1)Rs and CB(2)Rs in acinar cells of rat submandibular gland and their essential role in saliva secretion. Pharmacological activation of CB(1)Rs and CB(2)Rs in the submandibular gland suppressed saliva outflow and modified saliva content produced by the submandibular gland in vivo. Using Na(+)-selective microelectrodes to record secretory Na(+) responses in the lumen of acini, we observed a reduction in Na(+) transport following the activation of CBRs, which was counteracted by the selective CB(1)R antagonist AM251. In addition, activation of CB(1)Rs or CB Rs caused inhibition of Na(+)-K(+) 2 -ATPase activity in microsomes derived from the gland tissue as well as in isolated acinar cells. Using a Ca(2+) imaging technique, we showed that activation of CB(1)Rs and CB(2)Rs alters [Ca(2+)](cyt) signalling in acinar cells by distinct pathways, involving Ca(2+) release from the endoplasmic reticulum (ER) and store-operated Ca(2+) entry (SOCE), respectively. Our data demonstrate the expression of CB(1)Rs and CB(2)Rs in acinar cells, and their involvement in the regulation of salivary gland functioning.

1-[5-(2-Fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine Monofumarate (TAK-438), a Novel and Potent Potassium-Competitive Acid Blocker for the Treatment of Acid-Related Diseases

Proton pump inhibitors (PPIs) are widely used in the treatment of acid-related diseases. However, several unmet medical needs, such as suppression of night-time acid secretion and rapid symptom relief, remain. In this study, we investigated the pharmacological effects of 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine monofumarate (TAK-438), a novel potassium-competitive acid blocker (P-CAB), on gastric acid secretion in comparison with lansoprazole, a typical PPI, and SCH28080 [3-(cyanomethyl)-2-methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine], a prototype of P-CAB. TAK-438, SCH28080, and lansoprazole inhibited H(+),K(+)-ATPase activity in porcine gastric microsomes with IC(50) values of 0.019, 0.14, and 7.6 μM, respectively, at pH 6.5. The inhibitory activity of TAK-438 was unaffected by ambient pH, whereas the inhibitory activities of SCH28080 and lansoprazole were weaker at pH 7.5. The inhibition by TAK-438 and SCH28080 was reversible and achieved in a K(+)-competitive manner, quite different from that by lansoprazole. TAK-438, at a dose of 4 mg/kg (as the free base) orally, completely inhibited basal and 2-deoxy-d-glucose-stimulated gastric acid secretion in rats, and its effect on both was stronger than that of lansoprazole. TAK-438 increased the pH of gastric perfusate to a higher value than did lansoprazole or SCH28080, and the effect of TAK-438 was sustained longer than that of lansoprazole or SCH28080. These results indicate that TAK-438 exerts a more potent and longer-lasting inhibitory action on gastric acid secretion than either lansoprazole or SCH28080. TAK-438 is a novel antisecretory drug that may provide a new option for the patients with acid-related disease that is refractory to, or inadequately controlled by, treatment with PPIs.

Modulation of P-glycoprotein activity by the substituted quinoxalinone compound QA3 in adriamycin-resistant K562/A02 cells

QA3 is a derivative of the substituted 1,3-dimethyl-1H-quinoxalin-2-ones, which are compounds that may selectively antagonize P-glycoprotein (P-gp) in multidrug resistance (MDR) cancer cells. Our previous work identified QA3 as a candidate compound for reversing MDR in cancer cells. In the present study, we found that QA3 significantly decreases the intracellular level of ATP, stimulates ATPase activity in membrane microsomes and decreases protein kinase C (PKC) activity. These results indicated that QA3 inhibits P-gp activity by blocking ATP hydrolysis and ATP regeneration. Furthermore, QA3 triggered and increased adriamycin-induced K562/A02 cell apoptosis as evidenced by Annexin V-FITC plus PI staining. Western blot analysis showed that the levels of cleaved caspase-9 and cleaved caspase-3 proteins increased, and similarly, the levels of procaspase-9 and procaspase-3 decreased after QA3 treatment. Consequently, poly ADP-ribose polymerase (PARP) activity increased as evidenced by the presence of the PARP cleavage product in K562/A02 cells. QA3 also enhanced the potency of adriamycin against K562/A02 cells as demonstrated by increased apoptosis and activation of caspase-9,-3 and PARP. These data support the observation that P-gp activity is inhibited after QA3 treatment. Moreover, these results indicate that QA3 is a novel MDR reversal agent with potent inhibitory action against P-gp MDR cancer cells.

The long-lasting effect of TU-199, a novel H+, K(+)-ATPase inhibitor, on gastric acid secretion in dogs.

We have used Heidenhain-pouch dogs to investigate the effects of (+/-)-5-methoxy-2-{[(4-methoxy-3,5-dimethylpyrid-2-yl)methyl]sulph inyl}-1H-imidazo[4,5-b]pyridine (TU-199), an imidazopyridine derivative, on gastric acid secretion stimulated by histamine, carbachol and tetragastrin. We have also investigated the duration of the antisecretory effect of TU-199 using a measurement of intragastric pH for 24 h in gastric fistula dogs whose gastric acid secretion was stimulated by histamine. Single oral administration of TU-199 (0.1, 0.2 and 0.4mgkg(-1)) dose-dependently suppressed gastric acid secretion stimulated by histamine infusion. Oral treatment with TU-199 (0.2, 0.4 and 0.8 mg kg(-1)) also dose-dependently inhibited acid secretion induced by carbachol and tetragastrin. The inhibitory effect of TU-199 on stimulated gastric acid secretion was more potent than that of omeprazole, a well-known H+,K(+)-ATPase inhibitor in dogs. Repeated oral treatment with TU-199 at a dose of 0.2 mg kg(-1) once a day for seven days markedly suppressed histamine-stimulated gastric acid secretion in dogs. This inhibitory effect of TU-199 reached a maximum level after three or four doses and was more pronounced than that of omeprazole or lansoprazole. In gastric fistula dogs, the duration of intragastric pH-elevation by administration of TU-199 (0.3 mg kg(-1)) was much longer than that of omeprazole (0.6mgkg(-1)) or lansoprazole (0.9mgkg(-1)). The IC50 values (doses resulting in 50% inhibition) of TU-199, omeprazole and lansoprazole with regard to H+,K(+)-ATPase activity in dog gastric mucosal microsomes were 8.6, 8.8 and 9.9 microM, respectively. These results indicate that TU-199 inhibits gastric acid secretion via suppression of a H+,K(+)-ATPase activity. Our findings also suggest that TU-199 might have potent and long-lasting effects on gastric acid secretion.

Involvement of Microsomal Na+/K+ ATPase Activity in the Mechanism of Action of Dopaminergic D2 Receptors

Problem statement: Our previous research has shown that mitochondrial ATPase may have a role in the action of dopaminergic D2 receptors. It is therefore, thought of interest to investigate the effect of bromocriptine, sulpiride or their combination on the mitochondrial and microsomal ouabain sensitive and ouabain insensitive Na+/K+-ATPase in the liver of rats. Approach: In vivo treatment was carried out to study the effects of bromocriptine mesylate 10 mg kg-1, sulpiride 10 mg kg-1; or bromocriptine mesylate 10 mg kg-1 + sulpiride 10 mg kg-1, compared to a control group, in order to measure liver Na+/K+-ATPase activity. The drugs were injected intraperitoneally daily for 14 days. Mitochondrial and microsomal fractions were obtained by the methods of Schwartz. Results: Pretreatment of rats with both bromocriptine and sulpiride given simultaneously resulted in complete abolition of stimulation caused by bromocriptine and inhibition that resulted from sulpiride on mitochondrial as well as microsomal ATPase activities. Conclusion: This study indicated that the in vivo administration of dopaminergic agonist and antagonist drugs result in a significant alteration not only in the mitochondrial ATPase enzyme activity but also in the microsomal ATPase enzyme activity. Therefore, it is quite possible that ATPase of the tow fractions play important physiological as well as pathological roles in the mechanism of action of dopaminergic receptors.

Na +, K +-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

This investigation provides an extensive characterization of the modulation by ATP, Mg 2+, Na +, K + and NH 4 + of a gill microsomal (Na +,K +)-ATPase from Callinectes danae acclimated to 15‰ salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K + modulated by NH 4 +, discussed regarding NH 4 + excretion in benthic marine crabs. The (Na +,K +)-ATPase hydrolyzed ATP at a maximum rate of 298.7 ± 16.7 nmol Pi min − 1 mg − 1 and K 0.5 = 174.2 ± 9.8 mmol L − 1 , obeying cooperative kinetics ( n H = 1.2). Stimulation by sodium ( V = 308.9 ± 15.7 nmol Pi min − 1 mg − 1 , K 0.5 = 7.8 ± 0.4 mmol L − 1 ), magnesium (299.2 ± 14.1 nmol Pi min − 1 mg − 1 , K 0.5 = 767.3 ± 36.1 mmol L − 1 ), potassium (300.6 ± 15.3 nmol Pi min − 1 mg − 1 , K 0.5 = 1.6 ± 0.08 mmol L − 1 ) and ammonium ( V = 345.1 ± 19.0 nmol Pi min − 1 mg − 1 , K 0.5 = 6.0 ± 0.3 mmol L − 1 ) ions showed site–site interactions. Ouabain inhibited (Na +,K +)-ATPase activity with K I = 45.1 ± 2.5 μmol L − 1 , although affinity for the inhibitor increased ( K I = 22.7 ± 1.1 μmol L − 1 ) in 50 mmol L − 1 NH 4 +. Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F 0F 1- and K +-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other.

Mechanism of Reversal of Phospholamban Inhibition of the Cardiac Ca2+-ATPase by Protein Kinase A and by Anti-phospholamban Monoclonal Antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.

Characterization of Ca 2+-ATPase activity in gill microsomes of freshwater mussel, Lamellidens marginalis (Lamarck) and heavy metal modulations

Freshwater mussel Lamellidens marginalis showed a Ca 2+-stimulated ATPase activity in the microsomal fraction of gill. Mg 2+ was also found to be an activating cation for this ATPase enzyme. Ca 2+ ATPase showed maximal activity at 40 °C and between pH 7.5–8.0. Substrate specificity of Ca 2+ ATPase was highest with ATP, followed by GTP and other trinucleotides such as UTP, CTP and ADP also showed some amount of hydrolysis. Ca 2+ ATPase showed slight inhibition with ruthenium red and sodium vanadate and is insensitive to sodium azide, ouabain, oligomycin B and phenylalanine. Heavy metals like Hg 2+, Cu 2+ and Zn 2+ showed 50% inhibition of Ca 2+ ATPase activity at concentrations of 0.25 mM, 0.5 mM and 1 mM, respectively whereas Cd 2+ and Ni 2+ showed 39% and 28% inhibition of enzyme activity at 1 mM concentrations.

Estímulo no crescimento e na hidrólise de ATP em raízes de alface tratadas com humatos de vermicomposto: I - efeito da concentração

O vermicomposto contém uma concentração elevada de substâncias húmicas e já é bem conhecido o efeito do seu uso sobre as propriedades do solo. No entanto, a ação direta das substâncias húmicas sobre o metabolismo das plantas é menos conhecida. O objetivo deste trabalho foi avaliar o uso de humatos extraídos de vermicomposto de esterco de curral com KOH 0,1 mol L-1 sobre o desenvolvimento e metabolismo de ATP em plântulas de alface. Após a germinação, plântulas de alface foram tratadas com os humatos em concentrações que variaram de 0 a 100 mg L-1 de C, durante quinze dias. Foram avaliados o crescimento da raiz e a atividade das bombas de H+ isoladas da fração microssomal do sistema radicular. Foi observado aumento na matéria fresca e seca do sistema radicular, bem como no número de sítios de mitose, raízes emergidas do eixo principal, na área e no comprimento radiculares, com o uso do humato na concentração de 25 mg L-1 de C. Também foi observado, nessa concentração, aumento significativo na hidrólise de ATP pelas bombas de H+, responsáveis pela geração de energia necessária à absorção de íons e pelo crescimento celular.

Atividade ATPásica e pirofosfatásica em microssomos de raízes de milho colonizadas com fungos micorrízicos arbusculares

No presente trabalho, foi avaliada a influência de duas espécies de fungos micorrízicos arbusculares (FMAs) Glomus clarum e Gigaspora margarita sobre as atividades ATPásica e pirofosfatásica de microssomos obtidos por meio de fracionamento celular de raízes de milho colonizadas aos 20, 30, 40 e 60 dias do plantio. Ambos os fungos proporcionaram aumentos significativos nas atividades das ATPases e pirofosfatases; entretanto, as maiores atividades foram observadas nas raízes colonizadas pelo fungo G. clarum. Os dados cinéticos indicaram a presença de uma ativação diferencial das H+-ATPases e H+-pirofosfatases presentes nas membranas das células radiculares, dependendo da espécie fúngica e do estádio da colonização das raízes pelos FMAs. Como indicadores da eficiência da micorrização, foram avaliados a altura e o conteúdo de nutrientes na parte aérea das plantas. Os dados obtidos mostraram a primeira evidência cinética de estimulação de atividade pirofosfatásica em membranas microssomais de raízes colonizadas e descreveram um padrão inédito de ativação diferencial para a hidrólise de ATP, dependendo da espécie (G. clarum ou G. margarita) e do estádio de colonização.

Diabetes-induced decrease in rat brain microsomal Ca2+-ATPase activity.

The Ca(2+)-ATPase activity of rat brain microsomes was studied in streptozotocin (STZ)-induced diabetes. Male rats, 200-250 g, were rendered diabetic by injection of STZ (45 mg kg(-1) body weight) via the teil vein. Brain tissues were collected at 1, 4 and 10 weeks after diabetes was induced for determination of Ca(2+)-ATPase activity, lipid peroxidation and tissue calcium levels. Diabetic rats had significantly elevated blood glucose levels compared to controls. Blood glucose levels were 92.92 +/- 1.22 mg dl(-1) (mean +/- SEM) for the control group, 362.50 +/- 9.61 mg dl(-1) at 1 week and >500 mg dl(-1) at 4, 8 and 10 weeks for the diabetics. Enzyme activities were significantly decreased at 1, 4, 8 and 10 weeks of diabetes relative to the control group (p < 0.001). Ca(2+)-ATPase activity was 0.084 +/- 0.008 U l(-1), 0.029 +/- 0.005 U l(-1), 0.029 +/- 0.006 U l(-1), 0.033 +/- 0.003 U l(-1) and 0.058 +/- 0.006 U l(-1) (mean +/- SEM) at control, 1, 4, 8 and 10 week of diabetes respectively. The change in calcium levels in diabetic rat brain at 8 and 10 weeks of diabetes was significantly higher than that of the control group (p < 0.05). On the other hand lipid peroxidation measured as TBARS (thiobarbituric acid reactive substances) was significantly higher at 8 and 10 weeks of diabetes (p < 0.05). The increase in lipid peroxidation observed in diabetic rat brain may be partly responsible for the decrease in calcium ATPase activity.

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.

Chemical nature of soil humified fractions and their bioactivity

The aim of this work was to evaluate the humus composition from an Ultisol from Campos dos Goytacazes, RJ, Brazil. Soil samples of four depths (0-0.05, 0.05-0.10, 0.10-0.20 and 0.20-0.40 m) and its chemical nature were analysed by elemental composition, E4/E6 ratios and Fourier transformed infrared spectroscopy. The bioactivity of these humified substances was evaluated through their action on maize root growth and H+-ATPase activity of roots microsomes. In topsoil, the content of high condensed alkaline soluble humic substances is greater than that found in the subsuperficial layers. The chemical nature of humic and fulvic acids also varied with the soil depth. The humic acids isolated from the soil samples exhibited higher bioactivity compared with the fulvic acids. Moreover, the results suggest that more condensed humic substances can promote highest stimulation of the microsomal H+-ATPases from maize roots. These data reinforce the concept that the activity of the H+ pumps can be used as a biochemical marker for evaluation of humic substances bioactivity.

Juglone disrupts root plasma membrane H+-ATPase activity and impairs water uptake, root respiration, and growth in soybean (Glycine max) and corn (Zea mays).

Juglone is phytotoxic, but the mechanisms of growth inhibition have not been fully explained. Previous studies have proposed that disruption of electron transport functions in mitochondria and chloroplasts contribute to observed growth reduction in species exposed to juglone. In studies reported here, corn and soybean seedlings grown in nutrient solution amended with 10, 50, or 100 microM juglone showed significant decreases in root and shoot dry weights and lengths with increasing concentrations. However, no significant differences in leaf chlorophyll fluorescence or CO2-dependent leaf oxygen evolution were observed, even in seedlings that were visibly affected. Disruption of root oxygen uptake was positively correlated with increasing concentrations of juglone, suggesting that juglone may reach mitochondria in root cells. Water uptake and acid efflux also decreased for corn and soybean seedlings treated with juglone, suggesting that juglone may affect metabolism of root cells by disrupting root plasma membrane function. Therefore, the effect of juglone on H+-ATPase activity in corn and soybean root microsomes was tested. Juglone treatments from 10 to 1000 microM significantly reduced H+-ATPase activity compared to controls. This inhibition of H+-ATPase activity and observed reduction of water uptake offers a logical explanation for previously documented phytotoxicity of juglone. Impairment of this enzyme's activity could affect plant growth in a number of ways because proton-pumping in root cells drives essential plant processes such as solute uptake and, hence, water uptake.

양액내 염류농도 증가에 의한 상추뿌리의 마이크로솜 ATPase 활성증가

염류장애에 의한 작물의 생육저해 원인을 밝혀내기 위하여 상추를 대조양액과 대조양액에 30 mM 및 50 mM <TEX>$KNO_3$</TEX>를 첨가하여 염류농도를 높인 양액 등 3가지 조건에서 재배하였으며, 이때 이들 양액의 EC는 각각 1.0, 4.5, 6.5 dS/m 이었다. 뿌리세포의 원형질막 및 액포막에 위치한 <TEX>$H^+$</TEX>-ATPase 활성은 각각에 특이적 저해제인 vanadate와 <TEX>$NO_3^-$</TEX>를 이용하여 측정하였다. 대조양액에서 재배한 상추 뿌리의 ATPase 활성은 <TEX>$356\pm1.5$</TEX> nmol/min/mg protein이었으며, 30 mM과 50 mM <TEX>$KNO_3$</TEX>를 첨가한 양액에서는 활성이 대조활성에 비하여 각각 1.6배, 1.9배 증가하였다. 이것은 상추가 염류장애시 뿌리조직의 ATPase 활성증가를 통하여 적응함을 보여주는 것이며, 활성증가는 주로 액포막 <TEX>$H^+$</TEX>-ATPase 활성증가에 의해 이루어짐을 확인하였다. 마이크로솜 ATPase 활성에 미치는 여러 가지 중금속 이온들의 효과를 측정하였으며, 중금속 이온은 100 <TEX>${\mu}M$</TEX> 농도에서 콩류에 따라 활성을 10<TEX>$\sim$</TEX>25% 저해하였다. 특히, <TEX>$Cu^{2+}$</TEX>는 주로 액포막 <TEX>$H^+$</TEX>-ATPase 활성을 저해함을 확인하였다. 본 연구의 결과로부터 상추는 염류집적 환경에서 뿌리의 ATPase 활성, 특히 액포막에 위치한 <TEX>$H^+$</TEX>-ATPase 활성을 증가시켜 적응하며, <TEX>$Cu^{2+}$</TEX>는 주로 액포막 <TEX>$H^+$</TEX>-ATPase 활성을 저해하는 성분으로 뿌리의 ATPase 활성변화 연구에 유용하게 이용될 수 있음을 확인하였다. In order to investigate the mechanism of growth inhibition by salt stress, lettuces were grown hydroponically in three different nutrient solutions, normal and 30 mM or 50 mM <TEX>$KNO_3$</TEX>-added nutrient solutions, and the electrical conductivities of these solutions were 1.0, 4.5, and 6.5 dS/m, respectively. The activities of plasma and vacuolar <TEX>$H^+$</TEX>-ATPases in the root tissue of lettuce were measured by specific inhibitors, 100 <TEX>${\mu}M$</TEX> vanadate and 50 mM <TEX>$NO_3^-$</TEX>, respectively. Microsomal ATPase activity of lettuce grown in the normal nutrient solution was <TEX>$356\pm1.5$</TEX> nmol/min/mg protein. When lettuces were grown in 30 mM and 50 mM <TEX>$KNO_3$</TEX>-added nutrient solutions, total activities of microsomal ATPases were increased by 1.6 and 1.9 times, respectively, and the increases were mainly mediated by vacuolar <TEX>$H^+$</TEX>-ATPase. These results show that lettuces adapt themselves to salt-stressed condition by increasing the activities of <TEX>$H^+$</TEX>-ATPases. Effects of various heavy metal ions were investigated on the microsomal ATPases and various metal ions at 100 <TEX>$\mu M$</TEX> inhibited the activities by 10<TEX>$\sim$</TEX>25%. <TEX>$Cu^{2+}$</TEX> showed the highest inhibitory effect on the vacuolar <TEX>$H^+$</TEX>-ATPase. These results suggest that lettuce increases the activities of root ATPases, specially that of vacuolar <TEX>$H^+$</TEX>-ATPase, in salt-stressed growth conditions and <TEX>$Cu^{2+}$</TEX> could be a useful tool to control the activity of vacuolar <TEX>$H^+$</TEX>-ATPase.

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity.

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.