Hepatic and renal phosphomonoesterases and adenosine triphosphatases in chronic hyperprolactinaemic rats.

Influence of hyperprolactinaemia, induced endogenously by anterior pituitary transplantation on rat hepatic and renal cortical and medullary phosphomonoesterases and adenosine triphosphatases (ATPases) has been investigated. Although prolactin has a stimulatory effect on phosphomonoesterases and ATPases, it exhibits a specific and temporal influence on each subtype of hepatic and renal ATPases and phosphomonoesterases at different durations of pituitary transplantation. The specific activities of alkaline phosphatase and Na(+)-K+ dependent ATPases are activated in all the regions of different durations of experimentation. However, acid phosphatases, Ca2+ and Mg2+ dependent ATPases exhibit a differential response to prolactin in renal cortex, medulla and liver. Direct influence of prolactin on hepatic and renal phosphomonoesterases and ATPases is thus suggested.     

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.     

Stimulation of Na+,K+-ATPase activity in certain membranes of the rat central nervous system (CNS) by acute administration of desipramine (DMI)

1. The activities of ATPase in rat CNS were studied 3 hr after administration of the noradrenaline uptake inhibitor, desipramine (DMI: 10 mg.kg-1, i.p.). Na+K+-ATPase activity significantly increased after DMI in the whole particulate from hypothalamus and mesencephalus but no changes in frontal cortex or in pons-medulla oblongata areas were found. This increase was prevented when the animals were pretreated with the noradrenergic neurotoxic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). 2. Purified membrane fractions from hypothalamus were obtained by differential and sucrose gradient centrifugation (0.8-1.2 M sucrose). It was observed that after DMI, Na+,K+-ATPase activity increased only in the membranous fraction lying at 0.9 M sucrose. 3. Mg2+- or Ca2+-ATPase activities were not modified by DMI treatment. 4. Citalopram, a specific serotonergic uptake inhibitor, did not affect ATPase activities. 5. The results obtained could indicate that DMI acute administration selectively stimulates Na+,K+-ATPase activity of certain membranes of the CNS after an increase in the concentration of the noradrenergic neurotransmitter in the synaptic gap.     

Effects of aspirin and paracetamol on ATPases of human fetal brain: an in vitro study

In vitro effects of aspirin and paracetamol at the doses 200, 400, 600, 800 nmole/mg protein on ATPases activity were studied in the cerebrum and cerebellum of human fetus covering the age range from 10 weeks to 32 weeks of gestation. Both aspirin and paracetamol inhibit Na+K+ ATPase and Mg2+ ATPase in a dose dependent manner. The inhibition of Na+K+ ATPase and Mg2+ ATPase activity which may affect the release and uptake of biogenic amines in CNS, hinders the maturation of human fetal brain.     

Protective Role of Cynodon dactylon in Ameliorating the Aluminium-Induced Neurotoxicity in Rat Brain Regions

Cynodon dactylon (Poaceae) is a creeping grass used as a traditional ayurvedic medicine in India. Aluminium-induced neurotoxicity is well known and different salts of aluminium have been reported to accelerate damage to biomolecules like lipids, proteins and nucleic acids. The objective of the present study was to investigate whether the aqueous extract of C. dactylon (AECD) could potentially prevent aluminium-induced neurotoxicity in the cerebral cortex, hippocampus and cerebellum of the rat brain. Male albino rats were administered with AlCl(3) at a dose of 4.2?mg/kg/day i.p. for 4?weeks. Experimental rats were given C. dactylon extract in two different doses of 300?mg and 750?mg/keg/day orally 1?h prior to the AlCl(3) administration for 4?weeks. At the end of the experiments, antioxidant status and activities of ATPases in cerebral cortex, hippocampus and cerebellum of rat brain were measured. Aluminium administration significantly decreased the level of GSH and the activities of SOD, GPx, GST, Na(+)/K(+) ATPase, and Mg(2+) ATPase and increased the level of lipid peroxidation (LPO) in all the brain regions when compared with control rats. Pre-treatment with AECD at a dose of 750?mg/kg b.w increased the antioxidant status and activities of membrane-bound enzymes (Na(+)/K(+) ATPase and Mg(2+) ATPase) and also decreased the level of LPO significantly, when compared with aluminium-induced rats. The results of this study indicated that AECD has potential to protect the various brain regions from aluminium-induced neurotoxicity.     

Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

Influence of gramicidin S on cardiac membrane Ca2+/Mg2+ ATPase activities and contractile force development

The effects of gramicidin S (GS), an antibiotic, on the rat heart membrane ATPases and contractile activity of the right ventricle strips were investigated. GS inhibited sarcolemmal Ca2+-stimulated ATPase (IC50 = 3 microM), Ca2+/Mg2+ ATPase which is activated by millimolar Ca2+ or Mg2+ (IC50 = 3.4 microM), and sarcoplasmic reticulum Ca2+-stimulated ATPase (IC50 = 6 microM). The type of inhibition for the sarcolemmal Ca2+/Mg2+ ATPase by GS was apparently uncompetitive, while that for Ca2+-stimulated ATPases in sarcolemma or sarcoplasmic reticulum was of mixed type. Other ATPases, including mitochondrial ATPase, sarcolemmal Na+-K+ ATPase, and myofibrillar ATPase, were not inhibited by this agent. GS also decreased the rat right ventricle maximum force development (half-maximal inhibitory concentration was 2-4 microM), maximum velocity of contraction, and maximum velocity of relaxation. The resting tension was increased by GS to over 200%. The contractile actions of GS were mostly irreversible upon washing the muscle 3 times over a 10-min period. Decreased Ca2+, Mg2+, Na+, K+ concentrations in the perfusate increased the effects of GS. These findings showed that GS was a potent inhibitor of divalent cation ATPases of heart sarcolemma and sarcoplasmic reticulum and it is suggested that these membrane effects may explain the cardiodepressant action of this agent.     

Genetic Study of Cationic ATPase Activities and Audiogenic Seizure Susceptibility in Recombinant Inbred and Congenic Strains of Mice

Total, Mg2+, and Na+, K+ ATPase activities were studied in fresh brain homogenates of the audiogenic seizure (AGS)-resistant C57BL/6J (B6) and AGS-susceptible DBA/2J (D2) inbred strains and in 13 B6 X D2 (BXD) recombinant inbred (RI) strains. These activities were also studied in the D2.B6-Iasb congenic mice, that are similar genetically to D2 mice, except for the Iasb gene which inhibits the spread of AGS activity. The total and Mg2+ ATPase activities of the brainstem were significantly lower in the D2 than in the B6 mice at 21 days of age. No differences were found between these strains for Na+,K+ ATPase activity. The total, Mg2+, and Na+,K+ ATPase activities in the B6 brainstem did not change noticeably from 21 to 80 days of age. In the D2 brainstem, however, the Mg2+ activity increased with age, and the Na+,K+ ATPase activity decreased from 30 to 80 days of age. No genetic associations could be found between AGS susceptibility and total or Mg2+ ATPase activities in the D2.B6-Iasb mice or among the 13 BXD RI strains. Hence, differences in genetic background, rather than differences in AGS susceptibility, can account for the lower ATPase activities in 21-day-old D2 mice. Further, the Mg2+ and Na+,K+ ATPase activities appear to be regulated by more than one gene. This study emphasizes the utility of RI and congenic strains for testing the biochemical basis of AGS susceptibility in mice.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

Ca2+-Mg2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca2+ +Mg2+ dependent ATPase activities (about 9 mumol/h per mg protein). The Na+ +K+ +Mg2+ dependent ATPase activity was 3.2 mumol/h per mg (+/- 1.0, S.D., n = 3) when microvesicles were prepared according to (1) and 1.5 mumol/h per mg (+/- 1.0, S.D., n = 3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca2+ transport in mitochondria and erythrocyte membranes had no effect on Ca2+ +Mg2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca2+ +Mg2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na+ +K+ +Mg2+ ATPase was decreased. Na+ +K+ +Mg2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na+ +K+ +Mg2+ dependent ATPase activity in the eluted coated microvesicle fraction. It was concluded that Ca2+ +Mg2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.

The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence. No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl. Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM). By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched. The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+. The ATPase activity of M. gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5). Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5. In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein. In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol. The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity. Further evidence that two distinct ATPases exist was obtained by showing that M. gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies. It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.     

Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+-ATPase activity

The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+,K+-ATPase assay (Beltowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+-stimulated and Na+-independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain-sensitive H+,K+-ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H+,K+-ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+,K+-ATPase was stimulated by K+ with Km of 0.26 +/- 0.04 mM and 0.69 +/- 0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 +/- 0.3 microM in the renal cortex and 1.9 +/- 0.4 microM in the renal medulla) and by Sch 28080 (Ki of 1.8 +/- 0.5 microM and 2.5 +/- 0.9 microM in cortex and medulla, respectively). We found that ouabain-sensitive H+,K+-ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+,K+-ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+,K+-ATPase activity by 32.1% at 1 h after injection but had no effect on H+,K+-ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.     

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.     

Half of the (Na+ + K+)-transporting-ATPase-associated K+-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior.

Ouabain inhibited 86RbCl uptake by 80% in rabbit gastric superficial epithelial cells (SEC), revealing the presence of a functional Na+,K+-ATPase [(Na+ + K+)-transporting ATPase] pump. Intact SEC were used to study the ouabain-sensitive Na+,K+-ATPase and K+-pNPPase (K+-stimulated p-nitrophenyl phosphatase) activities before and after lysis. Intact SEC showed no Na+,K+-ATPase and insignificant Mg2+-ATPase activity. However, appreciable K+-pNPPase activity sensitive to ouabain inhibition was demonstrated by localizing its activity to the cell-surface exterior. The lysed SEC, on the other hand, demonstrated both ouabain-sensitive Na+,K+-ATPase and K+-pNPPase activities. Thus the ATP-hydrolytic site of Na+,K+-ATPase faces exclusively the cytosol, whereas the associated K+-pNPPase is distributed equally across the plasma membrane. The study suggests that the cell-exterior-located K+-pNPPase can be used as a convenient and reliable 'in situ' marker for the functional Na+,K+-ATPase system of various isolated cells under noninvasive conditions.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

Calcium-Activated ATPases in Presynaptic Nerve Endings

We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.     

Effects of L-phenylalanine on acetylcholinesterase, (Na+,K+)-ATPase and Mg2+-ATPase activities in adult rat whole brain and frontal cortex

The effect of different L-phenylalanine (Phe) concentrations (0.12-12.1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase activities was investigated in homogenates of adult rat whole brain and frontal cortex at 37 degrees C. AChE, (Na+,K+)-ATPase and Mg2+-ATPase activities were determined after preincubation with Phe. AChE activity in both tissues showed a decrease up to 18% (p<0.01) with Phe. Whole brain Na+,K+-ATPase was stimulated by 30-35% (p<0.01) with high Phe concentrations, while frontal cortex Na+,K+-ATPase was stimulated by 50-55% (p<0.001). Mg2+-ATPase activity was increased only in frontal cortex with high Phe concentrations. It is suggested that: a) The inhibitory effect of Phe on brain AChE is not influenced by developmental factors, while the stimulation of Phe on brain Na+,K+-ATPase is indeed affected; b) The stimulatory effect of Phe on rat whole brain Na+,K+-ATPase is decreased with age; c) Na+,K+-ATPase is selectively more stimulated by high Phe concentrations in frontal cortex than in whole brain homogenate; d) High (toxic) Phe concentrations can affect Mg2+-ATPase activity in frontal cortex, but not in whole brain, thus modulating the amount of intracellular Mg2+.     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+ -ATPase and calmodulin insensitive (Na+ +K+)- and Mg2+ -ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca2+ -ATPase and calmodulin-insensitive (Na+ +K+)- and Mg2+ -ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca2+ -ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca2+ -ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na+ +K+)-ATPase and Mg2+ -ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Mechanism of regulation of phosphorylation-dephosphorylation of Ca2+, Mg2+ and Ca2+ -Atpases by modulator proteins isolated from rat brain cytosol

Two proteins of molecular mass 13 kDa, a specific inhibitor of Na+, K+ -ATPase and another of 12 kDa, which can distinguish between Ca2, Mg2+ and Ca2+ -ATPase activities have been obtained from the pooled fractions isolated from rat brain, using Sephadex G-100 chromatography. In order to determine the key step(s), which is affected by the modulators, we have designed an in vitro experiment of phosphorylation and dephosphorylation of these ATPases in the absence and presence of the modulators. The results suggest that the phosphorylation step of Mg2+ -independent Ca2+ -ATPase is inhibited, while in Mg2+ -dependent Ca2 -ATPase, the dephosphorylation step is stimulated by the modulators. The findings support our earlier observation that the modulators are able to distinguish between Mg2+ -independent and dependent Ca2+ -ATPases activities.