Does melatonin have a time-dependent effect on brain and gill ionic metabolism in a teleost, Anabas testudineus (Bloch)?

Exogenous administration of 0.20, 0.40 and 0.60 microg/g body weight melatonin over a 24 hr cycle caused an inhibition of Na+, K+ ATPase activity in both brain and gills of A. testudineus. However, Ca2+ ATPase activity in the brain was significantly inhibited by the highest dose, and that in the gill at all the doses of melatonin. Evening injection of melatonin had an inhibitory effect on both brain and gill Na+ K+ and Ca2+ ATPase activity. Melatonin treatment in the morning for 12 hrs did not have an effect on brain Na+, K+ ATPase, while Ca2+ ATPase was inhibited. Similar treatment stimulated Na+, K+ and Ca2+ ATPase activity in the gills. Sodium, potassium and calcium ions in the gill were significantly reduced in the evening treated group while no change was observed in the morning melatonin injected group. The results suggest that melatonin elicits a time-dependent effect on the enzymes and ionic content in the brain and gills of A. testudineus.     

Effect of alloxan diabetes on (Na+ + K+)-activated ATPase in brush border membrane of the mucosal cell of rat small intestine]

In order to elucidate a possible relationship between (Na+ + K+)-activated ATPase and intestinal absorption of actively transported monosaccharides enzyme activity was measured in mucosal cells from alloxan diabetic rats. The general effect of increasing capacity of active, Na+-dependent transport processes in diabetes mellitus is associated with a significantly enhanced (Na+ +K+)-activated ATPase activity in mucosal homogenate from diabetic animals. To study the localization of these effects within the cell we isolated purified brush borders and their substructures. To enable a comparison to be made between preparation procedures of diabetic and control animals the fractions were controlled by electronmicroscopy and by measuring the sucrase activity. In the purified brush border fraction of alloxan treated rats there was no significant increase in (Na+ + K+)-activated ATPase activity. Based on these results we conclude that the (Na+ + K+)-activated ATPase in the basolateral membranes was increased in alloxan diabetes, and it seems very likely that this enzyme is involved in the regulation of Na+-dependent transport processes.     

Effects of steroid hormones on total brain Na(+)-k+ ATPase activity in Oreochromis mossambicus

The effects of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen. diethylstilbestrol (DES) on total brain Na(+)-K+- ATPase were investigated in tilapia, O. mossambicus. Exogenous administration of 0.125 and 0.25 microg/g body weight of glucocorticoids and 0.125, 0.25 and 0.5 microg/g body weight of DES for 5 days significantly stimulated Na+(-) K+ ATPase activity by 14-41% in the brain, while 0.5 microg/g body weight of glucocorticoids did not evoke any response on the activity of the enzyme. Progesterone (0.125 and 0.25 microg/g body weight) administration significantly decreased the enzyme activity by 21-36% and high dose (0.5 microg/g body weight) was ineffective. Testosterone exhibited a biphasic effect on Na(+)-K+ ATPase activity--a low dose stimulated by 14% while middle and high doses inhibited it by 19-24%. The results seem to be the first report on the effect of steroids on brain ATPase activity in a teleost. When 0.25microg/g body weight of actinomycin D or puromycin was administered prior to the treatment of similar doses of hormones, the inhibitors significantly inhibited the effect of the hormones by 24-52%. This clearly shows that the effect of the hormones was sensitive to the action of inhibitors suggesting a possible genomic mode of action under long-term treatment. The results suggest that cortisol, corticosterone and DES may possibly stimulate the co-transport of glucose and excitation of membrane potential while progesterone and testosterone inhibit them in the brain of O. mossambicus by regulating the activity of Na(+)-K+ ATPase.     

Na+,K+-ATPase and Mg2+-ATPase Activities in Different Regions of Rat Brain During Alloxan Diabetes

The effect of alloxan diabetes on the activities of Na+,K+-ATPase and Mg2+-ATPase was studied in three regions of rat brain at various time intervals after the onset of diabetes. It was observed that Na+,K+-ATPase activity increased at early time intervals after diabetes, followed by a recovery to near control levels in all three regions of the brain. There was an overall increase in Mg2+-ATPase activity in all the regions. A reversal of the effect was observed with insulin administration to the diabetic rats.     

Rat myometrial Na/K ATPase is increased by serum but not by isoproterenol and relaxin

1. Na/K ATPase activity in rat myometrial cells in culture exhibited a Kapp of 0.93 mM for Rb+ and a Ki of 31 microM for ouabain with respect to Rb+. 2. 86Rb+ uptake was stimulated by serum and monensin but was not affected by the uterine relaxants isoproterenol and relaxin in 0.5-7.5 mM Rb+. Nonetheless, these relaxants elicited significant increases in 45Ca2+ efflux under similar conditions. 3. These data suggest that increased Na/Ca exchange resulting from a stimulation of Na/K ATPase is not involved in the mechanism of action of relaxin and isoproterenol in the uterus.     

Insulin stimulates renal glomerular sodium-potassium adenosine triphosphatase activity

The effect of insulin on total and ouabain-inhibited membrane-bound adenosine triphosphatase (ATPase) activity in renal glomeruli isolated from adult white rats was examined. In concentrations of 1-10 micrograms/ml, insulin significantly stimulated the ouabain-inhibited (Na+ + K+)-ATPase activity, without affecting total (composite) ATPase activity. These results, coupled with previous findings demonstrating that glomerular (Na+ + K+)-ATPase activity is reduced in acute streptozotocin diabetes, suggest that the renal glomerulus is a target tissue with respect to this biologic effect of insulin.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

Bleomycin stimulates both membrane (Na+-K+) ATPase and electrogenic (Na+-K+) pump and partially removes the inhibition by vanadium ions

Bleomycin 2 X 10(-6) and 6 X 10(-6) mol.1(-1) increased the activity of specific (Na+-K+) ATPase of the rat brain microsomes. It also stimulated the electrogenic (Na+-K+) pump in intact skeletal muscle cells. The blocking effect of vanadyl (+4V) on membrane (Na+-K+) ATPase was eliminated completely by the drug, but the action of vanadate (+5V) was counteracted only partially. Electron paramagnetic resonance spectra revealed the formation of a +4V - bleomycin complex which is still able to activate the (Na+-K+) ATPase.     

In vivo electrical stimulation alters sensitivity of the brain (Na+ + K+)ATPase toward inhibition by vanadate

It has recently been shown that electrical stimulation of the brain cortex in vivo blocks invasion of cortical spreading depression (SD) into the stimulated area. The effect has been interpreted as a result of activating a K+ pumping mechanism that prevents the accumulation of this ion in the extracellular space to the high levels required for SD propagation. In the present experiments (Na+ + K+)ATPase activity was determined in the electrically stimulated region of the rat brain cortex. When ATP preparations containing vanadate were used as substrate, elevation of K concentration in the assay medium from 2 to 20 mM inhibited enzyme activity in homogenates from the normal cortex but not that from homogenates of the electrically stimulated cortical region. With vanadate-free ATP (Boehringer) as a substrate, slight stimulation by 20 mM K+ has been observed in both cases. Vanadate (0.25 microM) added to the assay medium containing Boehringer ATP and 20 mM K+ inhibited ATPase activity from the normal cortex but not that from the stimulated cortical area. Electrical stimulation may activate (Na+ + K+)ATPase at least partly by diminution of its susceptibility toward the inhibitory action of vanadate.     

瓦氏雅罗鱼生殖洄游过程中离子调节相关生理变化研究

为了解达里湖瓦氏雅罗鱼(Leuciscus waleckii)生殖洄游过程中血清离子调节相关生理变化, 对比了达里湖和贡格尔河瓦氏雅罗鱼血清离子(Na+、K+、Cl?、Ca2+和Mg2+)水平, 鳃、肠和肾组织Na+/K+-ATPase和鳃Ca2+/Mg2+-ATPase活性、血清催乳素(PRL)、生长激素(GH)和类胰岛素生长因子-1(IGF-1)水平及鳃组织结构差异; 并利用实验生态学方法, 研究达里湖中瓦氏雅罗鱼转入贡格尔河水24h后上述生理参数的响应。研究结果显示, 与达里湖未洄游的瓦氏雅罗鱼相比, 洄游到贡格尔河后其血清Na+含量显著降低(P<0.05), Cl?含量显著升高(P<0.05), 肾脏和肠组织中Na+/K+-ATPase活性显著升高(P<0.05), 而鳃组织中Na+/K+-ATPase活性无显著变化; 血清K+、Ca2+、Mg2+水平和GH、IGF-1、PRL含量无显著变化。将达里湖瓦氏雅罗鱼转入河水中24h后, 其血清Cl?含量显著升高(P<0.05)、K+含量显著降低(P<0.05), 且在鳃、肠和肾组织中Na+/K+-ATPase及鳃Ca2+/Mg2+-ATPase活性均显著升高(P<0.05), 血清PRL和IGF-1水平显著升高(P<0.05); 比较湖中和河中瓦氏雅罗鱼鳃组织形态结构, 显示湖中瓦氏雅罗鱼鳃基底膜分布着大量黏液细胞, 洄游到河水中后黏液细胞数量明显减少, 鳃基底膜上氯细胞体积增大而数量未见明显变化。本研究结果表明: 瓦氏雅罗鱼从达里湖洄游到贡格尔河后通过提高血清PRL和IGF-1水平, 进而介导鳃、肠和肾组织中Na+/K+-ATPase活性增加, 从而维持鱼体较高或稳定的血清离子水平。     

Alloxan inhibition of a Ca2+- and calmodulin-dependent protein kinase activity in pancreatic islets

Alloxan was found to inhibit a Ca2+- and calmodulin-dependent protein kinase recently identified in pancreatic islets. This effect of alloxan may be specifically related to the inhibitory action of alloxan on insulin secretion from islets since: 1) in islet-cell subcellular fractions, alloxan at micromolar concentrations irreversibly inhibits the Ca2+- and calmodulin-dependent protein kinase activity; 2) pretreatment of intact islets with alloxan at concentrations that inhibit insulin secretion similarly inhibits the protein kinase activity; and 3) alloxan inhibition of both insulin secretion and protein kinase activity in intact islets can be prevented by D-glucose. This inhibition by alloxan appears to be a direct effect on the enzyme since alloxan treatment of either the islet homogenate or the microsomal fraction enriched in protein kinase activity inhibited the kinase activity with similar concentration dependence. These results suggest that alloxan-induced inhibition of a Ca2+- and calmodulin-dependent protein kinase may represent a critical inhibitory site which mediates alloxan-induced inhibition of insulin secretion.     

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

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

Effect of starvation, alloxan diabetes and adrenalectomy on Na+ K+-ATPase of the mucosa of the small intestine of rat.

The dietary stress conditions such as starvation influenced Na+K+-ATPase activity which increased steadily above normal fed levels between the starvation periods of 24--48 hr. Also, an increased enzyme level was observed in alloxan diabetic rats and administration of insulin to diabetic rats led to a tendency towards a lowering of Na+K+-ATPase. Adrenalectomy brought about a lowering of Na+K+-ATPase activity from those of normals while the administration of hydrocortisone induced an enhancement. The results indicate that both starvation and diabetic conditions might cause a stress-like activation of adrenal cortex resulting in increased levels of glucocorticoids which in turn activate the intestinal Na+K+-ATPase activity.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide.

In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na+ + K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or by p-nitrophenylphosphate. (ii) Protection by ATP of (Na+ + K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 micron Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (IV) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activities stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity.     

The effect of ionophores and related agents on the induction of doming in a rat mammary epithelial cell line

Addition of dimethyl sulfoxide (DMSO) and the mammotropic hormones prolactin, hydrocortisone, insulin, and estradiol to confluent cultures of the epithelial cell line Rat Mammary (Rama) 25 increases dramatically the formation of domes in the cell monolayer after 48-72 hr. Associated with the increase in doming is an increase of 24% in the activity of the Na+/K+ ATPase. Both Ca2+ (A23187) and Na+ (monensin, gramicidin J, melittin) ionophores can replace DMSO in inducing domes, whilst the K+ ionophore valinomycin inhibits doming. However, there are no synergistic nor additive effects, respectively, with suboptimal or optimal concentrations of A23187 and melittin together. Ouabain, at concentrations which inhibit the Na/K ATPase in vitro, and amiloride, at concentrations reported to inhibit the passive transport of Na+, both inhibit completely the formation of domes induced by DMSO, A23187, and melittin. EGTA, however, inhibits only the induction of doming by DMSO and A23187; it is without effect with melittin. A23187 and melittin induce the major polypeptide changes that occur in doming cultures with DMSO, and most of these changes are also inhibited with ouabain. It is suggested that one possible interpretation of the findings is that the induction of doming by DMSO in Rama 25 cells occurs by means of sequential increases in Ca2+ and Na+ influxes into the cell, and that the increased intracellular concentration of Na+ so produced stimulates the Na+/K+ ATPase, with a net effect of pumping liquid beneath the cellular monolayer.     

Properties and drug sensitivity of adenosine triphosphatases from Schistosoma mansoni

The hydrolysis of ATP was measured in the presence of schistosome homogenates and various cations. The enzyme was stimulated strongly by either Ca2+ or Mg2+. Na+ added to the activation by Ca2+. A minor (17%) component was Na+ + K+ + Mg2+-dependent and ouabain-sensitive. Praziquantel, niridazole, oxamniquine, and hycanthone had no direct effect on the ATPase activity of schistosome homogenates. When schistosomes were pretreated with these drugs in vitro, washed thoroughly, and then homogenized, hycanthone, praziquantel, and oxamniquine caused a reduction in ATPase content of the worms. Niridazole did not share this effect. These results suggest that antischistosomal drugs did not directly inhibit ATPase, but did reduce ATPase in whole worms, possibly by removing or damaging the tegument, which is thought to contain most of the ATPase activity. In vitro ATPase measurements may be a useful indicator of pharmacologic activity of some types of drugs.     

Effect of anthelmintics and phenothiazines on adenosine 5'-triphosphatases of filarial parasite Setaria cervi

S. cervi showed particulate bound Ca2+ ATPase and Na+,K(+)-ATPase activities while Mg2+ ATPase was detected in traces. ATPase of S. cervi was also differentiated from the nonspecific p-nitrophenyl phosphatase activity. Female parasite and microfilariae exhibited higher Ca2+ ATPase and Na+,K(+)-ATPase activities than the male adults and the enzyme Na+,K(+)-ATPase was mainly concentrated in the gastrointestinal tract of the filarial parasite. Na+,K(+)-ATPase of the filariid was ouabain-sensitive while Ca2(+)-ATPase activity was regulated by concentration of Ca2+ ions and inhibited by EGTA. Phenothiazines, viz. trifluoperazine, promethazine and chlorpromazine caused significant inhibition of Ca2+ ATPase and Na+,K(+)-ATPase. Diethylcarbamazine was a potent inhibitor of these ATPases. Mebendazole, levamisole and centperazine also caused significant inhibition of the ATPases indicating this enzyme system as a common target for the action of anthelmintic drugs.     

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.