Adenosine Triphosphatase System Research Articles

Study of Antioxidant and Membrane Resistant Peculiarities of a New Cyan Containing Lactone in Membranes of Hepatocytes with Sarcoma-45

The antioxidant and membrane resistant peculiarities of a new derivative (2-cyan-3,4,4-trymethil-2-buten-4-olyd - CTBO) of cyan containing unsaturated lactones have been studied in membranes of hepatocytes with Sarcoma-45 1. The results of our previous research 1, 2, 3 showed significant changes of phospholipid (PL) exchange in hepatocytes of microsomal membranes at experimental animals vaccinated with Sarcoma-45 tumor strain. It is manifested in significant changes of quantitative and qualitative contents of membrane phospholipids separate fractions, increase of cytotoxic lysophospholipids (LPCs), phosphatidylinositol (PI) and phosphatidic acid (PA) levels, significant decrease of phosphatitylcholines (PC) and sphingomyeline (SP) contents, statistically significant changes of PL/PL ratio, peroxidation ratio intensity, dramatic increase of phospholipase A2 (PLA2)activity, quantitative and qualitative changes of adenyl nucleotides, as well as disorders of adenosine triphosphatase (ATPase) system activity 3, 4, 5, 6, 7.

Proton pump inhibitors: use, action and prescribing rationale

Proton pump inhibitors (PPIs) are a group of drugs that work by inhibiting the K+/H+- adenosine triphosphatase (ATPase) system, also known as the proton pump, in the gastric parietal cell wall. They therefore efficiently reduce hydrochloric (HCl) acid secretion in the stomach. PPIs are used as the treatment of choice for gastro-oesophageal reflux disease, peptic ulcer disease, non-steroidal anti-inflammatory drugassociated ulcers, Helicobacter pylori eradication and excess acid secretion in Zollinger-Ellison syndrome. They are also used as a short term therapy to treat intermittent gastric acid reflux when antacids and H2 receptor antagonists have proven to be ineffective. Their use, however, must be monitored to prevent secondary disease such as Clostridium difficile-associated diarrhoea and rebound excess gastric acid secretion (irritable bowel syndrome).

Influence of Hypoxia and Sex on Hyponatremic Encephalopathy

Over the past 20 years it has become increasingly apparent that hyponatremic encephalopathy is a major cause of inhospital morbidity and mortality, particularly in postoperative patients. The factors that may lead to death or permanent brain damage and the susceptible patient groups have been gradually elucidated. Hyponatremic encephalopathy most commonly leads to brain damage in young women and in prepubescent children. The causes of brain damage include brain edema, cerebral hypoxemia, decreased brain blood flow, increased intracranial pressure, and improper therapy. Cerebral hypoxia occurs through a combination of impaired brain adaptation and cerebral vasoconstriction. Brain adaptation consists largely of brain cell loss of sodium and potassium by means of the Na-K adenosine triphosphatase (ATPase) system. There is also loss of organic osmolytes. The brain Na-K ATPase system is impaired by a combination of vasopressin plus estrogen and is stimulated by testosterone. Similarly, vasopressin plus estrogen leads to cerebral vasoconstriction, resulting in a decrement of brain oxygen utilization and cerebral blood flow. Vasopressin also directly decreases brain production of ATP. The combination leads to hypoxic brain damage, which appears to be the major cause of brain damage associated with hyponatremic encephalopathy. Measurement of arterial P o 2 in patients with symptomatic hyponatremia usually demonstrates a P o 2 <50 mm Hg. Improper therapy is another possible cause of brain damage in patients with hyponatremic encephalopathy. The type and distribution of such lesions are similar to those found in patients with hyponatremic encephalopathy who have severe hypoxia. Current scientific knowledge indicates that patient survival can be improved through aggressive treatment of hypoxia associated with hyponatremic encephalopathy, particularly in young women.

Antagonistic effects of simultaneous exposure of ergot alkaloids on kidney adenosine triphosphatase system.

Much of the research on fescue toxicosis has concentrated on evaluating animal response to grazing endophyte-infected (E+) versus endophyte-free tall fescue or the effects of single toxins such as ergonovine (EN), ergovaline (EV), or ergotamine (ET) on animal performance. Such approaches have eliminated the opportunity to test the possible additive, synergistic, or antagonistic interactions of one or more ergot alkaloids with the other ergot alkaloids found in E+ tall fescue. This study was conducted to determine the effects of simultaneous exposure of pairs of EN, EV, and ET on the kidney adenosine triphosphatase (ATPase) system in vitro. Tests were performed using three separate rat kidney homogenates and were repeated four times at concentrations of 0, 75, and 200 microM. Individually, EN, EV, and ET induced dose-dependent inhibitions of kidney Na(+)/K(+) ATPase, with EN being most potent, followed by purified EV, and then by ET. The ergot alkaloids inhibited Mg(2+) ATPase to a lesser degree than Na(+)/K(+) ATPase, with EN again being the most potent toxin. Simultaneous exposure to any combination of the ergot alkaloid pairs tested (EV + ET, EV + EN, and ET + EN) resulted in significant interactions (P < 0.05), indicating antagonistic effects on the inhibition of Na(+)/K(+) ATPase and Mg(2+) ATPase for most concentration combinations. These interactions suggest that in studies of the effects of any ergot alkaloid on animal performance, effects of other ergot alkaloids may also be present. Effects may not be additive, as was the case in this study, and the presence of one toxin may enhance or hinder the effectiveness of others.

Contact Tolerance

Contact tolerance describes an immunological state which is caused by ordinary contact allergens painted in doses too low to sensitize, either once or repeatedly, onto healthy intact skin. The tolerance state accomplished by this means in BALB/c and C57BI/6 mice was found to be mediated by hapten-specific T cells that adoptively transferred tolerance to naive recipients. Furthermore, these cells were shown to be sensitive to cyclophosphamide, to express the Lyt2+ (CD8) phenotype, and to produce, upon restimulation in vitro, predominantly anti-inflammatory cytokines such as IL-4, IL-5 and IL-10. These data indicate that contact tolerance of the low zone type is actively mediated by Th2-like CD8 T cells rather than arising as a consequence of clonal anergy. The induction of contact tolerance appeared to be strictly dose-dependent. As opposed to sensitizing doses of allergen, subsensitizing doses did not involve epidermal Langerhans cells discernibly. This was suggested by their normal ultrastructure, their unaffected adenosine triphosphatase system, the inefficacy of functional blocking and excision experiments. Both radiolabeled and fluorescent contact sensitizers were observed to readily enter the bloodstream, thereby being dispersed throughout the body. Presumably, contact tolerance is induced systemically rather than locally. The presence of hapten-specific tolerance can only be uncovered through a subsequent attempt to sensitize. If the attained sensitization turns out to be significantly lower than that of immunologically naive controls, and if sensitization to chemically unrelated sensitizers is not impaired, hapten-specific tolerance does exist. Thus, contact tolerance is a result obtained from experimental sensitization in animals. Nonetheless, it is assumed to occur also in humans, although it is not demonstrable unless different proofs of existence become available.

Induction of Low Zone Tolerance to Contact Allergens in Mice Does Not Require Functional Langerhans Cells

Epidermal Langerhans cells are known to be the major controlling element in the development of contact hypersensitivity. Haptenic molecules permeating the skin are taken up locally by Langerhans cells and then presented to T lymphocytes in the regional lymph nodes. Despite the presence of functional Langerhans cells, however, subsensitizing doses of hapten applied epicutaneously induce tolerance. We examined epidermal Langerhans cells at the site of contact with picryl chloride or oxazolone in BALB/c and C57B1/6 mice with regard to their responding to either subsensitizing or sensitizing doses of allergen. Subsensitizing doses did not interfere with the membranous adenosine triphosphatase system on Langerhans cells, known to relate to functional readiness of the cell. Accordingly, on electron microscopy the ultrastructure of Langerhans cells was found to be like that in untreated skin. In contrast, sensitizing doses caused a significant depletion of adenosine triphosphatase-positive Langerhans cells, and electron microscopy revealed marked cellular activation of Langerhans cells, with enlarged nuclei and increased numbers of mitochondria and Birbeck granules. Furthermore, subsensitizing doses induced tolerance regardless of whether Langerhans cells were functionally intact or had their function blocked arbitrarily. Blocking was achieved either by preceding ultraviolet B irradiation at the site of application or by painting of a sensitizer before painting another sensitizer on the same site. Moreover, not even surgical removal of the site within minutes after painting could prevent the induction of tolerance. The data suggest that subsensitizing doses of contact allergens painted on normal murine skin bypass involvement of epidermal Langerhans cells.

The effects of cadmium in vitro on adenosine triphosphatase system and protection by thiol reagents in rat brain microsomes.

Cadmium (Cd) inhibited the activities of Na(+)-K+ ATPase (IC50 = 5.0 x 10(-5) M), K(+)-p-nitrophenyl phosphatase (PNPPase) (IC50 = 4.0 x 10(-5) M) and 3H-ouabain binding (IC50 = 7.5 x 10(-5) M) in rat brain microsomes. Monothiols (cysteine but not glutathione and D-penicillamine) and dithiols (dimercaprol, dimercaptosuccinic acid and dithiothreitol) offered varied levels of protection against Cd-inhibition of Na(+)-K+ ATPase. Protection of Na(+)-K+ ATPase by these sulfhydryl (SH) agents was higher at 7.5 as compared to 8.5 pH. The present data suggest that Cd-inhibited Na(+)-K+ ATPase, by interfering with phosphorylation of enzyme molecule and dephosphorylation of the enzyme-phosphoryl complex and exerts a similar effect to that of SH-blocking agents.

Effect of mercuric chloride on the kinetics of cationic and substrate activation of the rat brain microsomal ATPase system

Mercuric chloride (HgCl 2), a neurotoxic compound, inhibited the adenosine triphosphatase (ATPase) system in a concentration-dependent manner. Hydrolysis of ATP was linear with time with or without HgCl 2 in the reaction mixtures. Higher inhibition of (Na +−K +)ATPase activity by HgCl 2 was observed in alkaline (8.0 to 9.0) pH and at lower temperatures (17 to 32°). Activation energy values were increased slightly in the presence of HgCl 2. Activation of (Na +−K +)ATPase by ATP in the presence of HgCl 2 showed a decrease in V max from 15.29 to 5.0 μmol of inorganic phosphate (P i)/mg protein/hr with no change in K m . Similarly, activation of K +-stimulated p-nitrophenyl phosphatase (K +-PNPPase) in the presence of HgCl 2 showed a decrease in V max from 3.26 to 1.35 μmol of p-nitrophenol (PNP)/mg protein/hr with no change in K m . K +-activation kinetic studies indicated that HgCl 2 decreased V max from 14.01 to 4.30 μmol P i/mg protein/hr in the case of (Na +-K +)ATPase and from 3.45 to 2.40 μmol PNP/mg protein/hr in the case of K +-PNPPase with no changes in K m . Na +-activation of (Na +-K +)ATPase in the presence of HgCl 2 showed a decrease in V max from 11.06 to 3.23 μmol P i/mg protein/hr and an increase in K m from 1.06 to 2.08 mM. Preincubation of microsomes with sulfhydryl (SH) agents dithiothreitol, cysteine and glutathione protected HgCl 2-inhibition of (Na +−K +)ATPase. The data suggest that HgCl 2 inhibited (Na +-K +)ATPase by interfering with the dephosphorylation of the enzymephosphoryl complex.

Ultracytochemical localization of membrane-bound enzymes in the node of Ranvier

The localization of ouabain-sensitive, potassium dependent p-nitrophenylphosphatase, a component of the transport adenosine triphosphatase (Na-K-ATPase) system, and the activity of its associated enzymes, magnesium-dependent ATPase, calcium-dependent ATPase and thiamine pyrophosphatase were ultracytochemically investigated in the node of Ranvier. In the nodal region, the axolemma, which lies above the dense cytoplasmic undercoating, showed remarkably strong activity. The results suggested that the enzymes observed are associated structurally and functionally with the dense cytoplasmic undercoating and the saltatory conduction.

Cytochemical localization of K+-NPPASE in the heminode of Ranvier in the rat optic nerve.

The localization of ouabain sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase), a component of the transport adenosine triphosphatase (Na+, K+-ATPase) system, was ultracytochemically investigated in the heminode of Ranvier in the rat optic nerve. K+-NPPase activities were noted on the nodal and paranodal axolemma, and on the paranodal myelin lateral loops. In the nodal region, the axolemma, which lies above the dense cytoplasmic undercoating, showed remarkably strong K+-NPPase activity.

Erythrocyte ghost Na+,K+-ATPase and blood pressure.

To examine the relationship between body mass index, blood pressure, and the Na+,K+-adenosine triphosphatase (ATPase) system, we measured the erythrocyte ghost Na+,K+-ATPase and the erythrocyte Na+ concentration in 120 blacks and 127 whites (136 males and 111 females). Blacks showed a 13.9% higher erythrocyte Na+ (7.63 +/- 0.19 vs 6.70 +/- 0.11 [SEM] mEq/L; p = 0.0001) and a 16.1% lower erythrocyte ghost Na+,K+-ATPase activity (140.3 +/- 4.2 vs 167.3 +/- 4.7 nmol inorganic phosphate/mg protein/hr; p = 0.0002) than whites. Male subjects demonstrated a 6.4% higher erythrocyte Na+ (7.35 +/- 0.17 vs 6.91 +/- 0.14 mEq/L; p = 0.043) and an 11.5% lower Na+,K+-ATPase activity (145.7 +/- 3.7 vs 164.7 +/- 5.5 nmol inorganic phosphate/mg protein/hr; p = 0.0015) than female subjects. Significant (p less than 0.001) negative correlations were identified for the systolic, diastolic, and mean blood pressure levels and the erythrocyte ghost Na+,K+-ATPase. These findings were complemented by positive correlations for the blood pressure levels and erythrocyte Na+ concentrations. The body mass index was negatively correlated with erythrocyte ghost Na+,K+-ATPase and it accounted for 6.7%, 5.6%, and 6.1% of the variabilities in the systolic, diastolic, and mean blood pressure levels, respectively. Variabilities of 1.4% systolic, 12.3% diastolic, and 11.1% in mean arterial pressure were attributable to the erythrocyte ghost Na+,K+-ATPase activity. Provided that findings in erythrocytes also reflect the relative status of the vascular smooth muscle cell Na+,K+-ATPase, the predisposition of black, male, and obese persons to hypertension may relate, among other factors, to a lower activity of this enzyme system, which results in an increased vascular tone.

Effects of tricyclohexylhydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents.

Tricyclohexylhydroxytin, commonly known as Plictran, inhibited Na+, K+-ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 microM. Both K+-stimulated para-nitrophenylphosphatase and [3-H]-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 microM, respectively. Altered pH and Na+, K+-ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na+, K+-ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na+, K+-ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K+ activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na+, K+-ATPase, K+-para-nitrophenylphosphatase, and [3-H]-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na+, K+-ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.

Muscle contraction and free energy transduction in biological systems.

Muscle contraction occurs when the actin and myosin filaments in muscle are driven past each other by a cyclic interaction of adenosine triphosphate (ATP) and actin with cross-bridges that extend from myosin. Current biochemical studies suggest that, during each adenosine triphosphatase cycle, the myosin cross-bridge alternates between two main conformations, which differ markedly in their strength of binding to actin and in their overall structure. Binding of ATP to the cross-bridge induces the weak-binding conformation, whereas inorganic phosphate release returns the cross-bridge to the strong-binding conformation. This cross-bridge cycle is similar to the kinetic cycle that drives active transport and illustrates the general principles of free energy transduction by adenosine triphosphatase systems.

Metabolic, ionic, and electrical responses of gerbil brain to ischemia

The effects of short- or long-term complete cerebral ischemia were studied in the gerbil brain using a multi-parameter monitoring system. Metabolic (NADH redox state) and hemodynamic responses were monitored by surface fluorometry-reflectometry. Ionic activities (K+ and pH) were measured by surface macroelectrodes. Electrical activity was evaluated by monitoring the general electrocorticogram (ECoG) as well as local DC steady potential (two sites). Two groups of gerbils were studied to compare the effects of 4-5 min occlusions with those of 30 min complete ischemia. During bilateral carotid artery occlusion the cortex is exposed to complete ischemia resulting in the complete depletion of O2 with attendant maximal reduction of NADH. Extracellular K+ began to increase as soon as energy reserves were decreased with a time course suggesting two different kinetic areas. Surface pH decreased very shortly after the occlusion. During the recovery phase, NADH was reoxidized soon after recirculation, whereas the pH and K+ recovery showed a short delay. ECoG did not recover even when all other parameters reached base-line levels. The recovery of all the measured parameters was correlated to the duration of the ischemic insult; i.e., the recovery from 30 min of ischemia took significantly longer than after 5 min of ischemia. We conclude that pH recovery depends on recirculation and adequate O2 supply to the tissue, whereas K+ recovery required not only an adequate O2 supply but also the integrity of the adenosine triphosphatase system.

Kinetic studies of the Na+-K+-ATPase enzyme system in brain and heart of aging rats.

Kinetic analyses of the Na+-K+-adenosine triphosphatase (ATPase) system were performed in brain and heart preparations from young mature (4 mo old) and healthy aged (25 mo old) rats. The K+-activated p-nitrophenylphosphatase (K+-pNPPase) method was used to assess activity of the enzyme system. Ouabain inhibition of K+-pNPPase activity was also examined. A significant age-related decrease in maximal velocity (Vmax) was found in cardiac K+-pNPPase activity, but no changes were seen in the K+ concentration for half-maximal velocity (K0.5). No age differences in Vmax or K0.5 were seen for brain. No differences in ouabain inhibition were found in either brain or heart. In a second experiment, the major component of the age-related decline in cardiac K+-pNPPase activity was found to occur between 5 and 14 mo of age, a period during which plasma thyroxine had previously been found to decline in the same animals. Since peripheral Na+-K+-ATPase activity is partly thyroid hormone dependent, the age-dependent decrease in cardiac enzyme activity appears to be secondary to neuroendocrine changes.

Mechanism and Regulation of Mitochondrial ATP Synthesis

This chapter presents an experimental work to elucidate how various components of the F 0 F 1 - adenosine triphosphatase (ATPase) system function and interact to carry out and regulate adenosine triphosphate (ATP) synthesis. The chapter discusses the reaction mechanism of F 1 as disclosed by studies with the uncoupler-reversible inhibition of the enzyme by bathophenanthroline-metal chelates. The interaction of F 0 and F 1 based on resolution-reconstitution experiments and the studies of the structure and function of oligomycin sensitivity-conferring protein (OSCP) are presented. The regulation of ATP hydrolysis and ATP synthesis by means of the ATPase-inhibitor (AI) protein, with special reference to the effects of Ca 2 + and Mn 2 + are discussed. The chapter presents attempts to establish which amino acids are destroyed by photooxidation in the presence of Rose Bengal and protected by BPh 3 F 2+ . The derivatives of BPh, which contain a covalent photoaffinity label, such as azide groups attached to the phenyl groups of BPh are prepared and the amino acid residue, which binds the inhibitory chelate, are determined.

Cytochemical localization of ouabain-sensitive, K +-dependent p-nitro-phenylphosphatase (transport ATPase) in the mouse central and peripheral nervous systems

Enzyme activity, representing the sites of K +-dependent p-nitrophenylphosphatase (K +-pNPPase), a component of the transport adenosine triphosphatase (Na +, K +-ATPase) system, has been localized at the ultrastructural level in both cerebral cortex and in sciatic nerve of the mouse. Normal mice and animals with mechanically injured blood-brain barrier (BBB) were used. In the cerebral cortex, positive reaction was found in synapses, plasmalemma of neurites (axons and dendrites), in endothelial cells of microblood vessels and in the plasmalemma of mesothelial cells of the pia mater. In the sciatic nerve, a strong reaction was present in the nodes of Ranvier, with weaker reaction in the internodal areas of the axolemma. In the endothelial cells of normal blood vessels, the reaction product was localized on the luminal and abluminal, or only on the abluminal plasmalemma. After damage of BBB, numerous invaginations, pits and pinocytic vesicles showing positive reaction in their limiting membrane appeared in the endothelial cells.

Quercetin inhibition of the induction and function of cytotoxic T lymphocytes

Quercetin is a naturally occuring flavonoid, chemically related to cromolyn. Quercetin has been shown to inhibit antigen- and mitogen-induced histamine release from rat mast cells and basophils of subjects with hay fever, to increase cyclic adenosine monophosphate (AMP) in Ehrlich ascites tumor cells and to inhibit phosphodiesterase and certain adenosine triphosphatase (ATPase) systems. We have studied the effect of quercetin on mouse T cell responses. When 5 × 10−6 to quercetin is present throughout either allogeneic mixed leukocyte culture (MLC) or cytotoxic T lymphocyte (CTL) assay culture, inhibition of in vitro CTL generation or effector function results, respectively (inhibition is 75–100% at 2 × 10−5 M and 100% at 5 × 10−5 M). Quercetin also inhibits concanavalin A-induced DNA synthesis. Addition of Cu2+ strongly blocks the effects of quercetin in all systems tested, in a concentration dependent fashion, while Mg2+ and Ca2+ have little or no effect and Mn2+ and Co2+ have a significant but slight blocking effect on quercetin-mediated inhibition of both CTL generation and function. In kinetic studies, evidence was obtained for the existence of a major quercetin-sensitive step in CTL induction, between 3 and 24 hr of the MLC.

A serotonin agonist-antagonist reversible effect on Na+-K+-ATPase activity in the developing rat brain.

In brain of adult and developing rats the Na+-K+-adenosine triphosphatase (Na+-K+-ATPase) system seems to react to serotonin (5-HT) changes induced pharmacologically. A 5-HT agonist (quipazine) elicits a response of the enzyme activity in the cerebral cortex in vivo, which is neutralized with a 5-HT antagonist (methysergide). This effect was observed from day 21 to adulthood. Also in a state of 5-HT receptor hypersensitivity (rats treated early with 5,6-dihydroxytryptamine), the response of Na+-K+-ATPase to the 5-HT agonist was higher than without neurotoxic lesion of 5-HT paths. These data suggest an involvement of the Na+-K+-ATPase system in 5-HT receptor sensitivity in the rat brain.

Effects of mycotoxins on mixed-function oxidase and adenosine triphosphatase systems in neonatal rats. I. Aflatoxin B 1/rubratoxin B

The effects of aflatoxin B 1 and/or rubratoxin B on hepatic monooxygenase and hepatic, brain and renal ATPase activities were examined in neonatal rats exposed to a single treatment of one or both toxins. Animals received orally 0·4 mg aflatoxin B 1/kg, 2 mg rubratoxin B/kg or the same doses of aflatoxin B 1 and rubratoxin B in combination, within 24 hr of birth, and were killed 12 days later. Given alone, rubratoxin B caused a statistically significant decrease in cytochrome P-450 content. Hepatic and renal oligomycin-sensitive ATPase activity was depressed by the aflatoxin B 1 and rubratoxin B combination but this effect appeared to be the result of the aflatoxin treatment. The only interaction observed after exposure of pups to the mycotoxin combination was a tendency towards a decrease in NADPH-dependent dehydrogenase.