Selective and reversible inhibition of heart sarcolemmal (Na+ + K+)-ATPase by p-bromophenyl isothiocyanate. Evidence for a sulfhydryl group in the ATP-binding site of the enzyme

Isothiocyanates are potent modifiers of thiol groups, and they have been successfully applied in studying the active site structure of renal (Na+ + K+)-ATPase. However, very little has been known on interactions of isothiocyanates with myocardial sarcolemmal ATPases. In the present study the mode of interaction and inhibitory effect of p-bromophenyl isothiocyanate (BPITC) on isolated rat heart sarcolemmal preparation ATPase activities not exhibiting (Mg-Ca)-ATPase activity was investigated. BPITC in concentrations of 10(-7)-10(-4) mol . l-1 inhibited selectively and non-competitively the (Na+ + K+)-ATPase activity in the sarcolemma with an ID50 around 2.10(-7) mol . l-1. The non-specific interaction of BPITC with bivalent cations, namely with Mg2+ and Ca2+, in the reaction system was eliminated by preincubation of membranes with BPITC keeping the ratio of inhibitor to membrane protein concentration constant. Under these conditions no considerable inhibitory effects were observed on Mg2+-ATPase or the low-affinity Ca2+-ATPase of sarcolemma. Preincubation of membranes with 2 mmol . l-1 ATP protected (Na+ + K+)-ATPase activity against inhibition by BPITC. The interaction of BIPTC with the sarcolemma proved to be reversible in the presence of beta-mercaptoethanol or dithiothreitol.     

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue

1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.     

Hydrolytic properties of the (Na+ + K+)-ATPase isozymes for beta-(2-furyl)acryloyl phosphate, a pseudosubstrate for the sodium pump

The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), a synthetic substrate for the (Na+ + K+)-ATPase by the partially purified enzyme from rat brain and rat kidney, has been assessed. Using previously determined FAPase reaction conditions, it was discovered that the KI for ouabain of the alpha 2/3 isozyme of the (Na+ + K+)-ATPase was approximately 10(-5) M, while for the alpha 1 isozyme the KI was approximately 10(-3) M. These values were an order of magnitude higher (lower affinity) than the KI's for ouabain as determined when using ATP in a coupled assay for (Na+ + K+)-ATPase activity: approximately 10(-6) M and approximately 10(-4) M for the alpha 2/3 and alpha 1 isozymes, respectively. This discrepancy was alleviated by altering established reaction conditions. Previously published FAPase studies have overlooked this fact, since either the properties of the isozymes of the (Na+ + K+)-ATPase were unknown at that time, or ouabain titration profiles were never performed.     

The changes in conformation of (Na+ K+)-ATPase from rat brain membranes are accompanied by changes of protein segment movements in the nanosecond range

Differential polarized phase fluorometry of fluorescein-5-isothiocyanate (FITC) showed that the activation of (Na,K)-ATPase in crude plasma membranes from rat brain by 10 mmol.l-1 K+ and 100 mmol.l-1 Na+ significantly increased the rotational relaxational rate (R) of enzyme-bound FITC. This increase was blocked by both ouabain (0.1 mmol.l-1) and vanadate (0.1 mmol.l-1). In the absence of ATP, R was increased less after adding of 10 mmol.l-1 K+ to the membranes. The shifts in the nanosecond movements of the protein segments measured as R during the activation of (Na,K)-ATPase suggest that this type of movement might be of some functional importance.     

Heart sarcolemmal (Na+ + K+)-ATPase has an essential amino group in the potassium binding site on the enzyme molecule

Selective modification of primary amino groups of (Na+ + K+)-ATPase by trinitrobenzene sulfonic acid (TNBS) resulted in a considerable inhibition of the specific activity of the enzyme. Investigation by means of enzyme and sorption kinetics of activation of heart sarcolemmal (Na+ + K+)-ATPase by its monovalent cationic ligands added simultaneously with TNBS revealed: a considerable competition between K+-ions and TNBS for the potassium binding site on the enzyme molecule; a non-competitive type of inhibition of Na+-induced activation of the enzyme. Both, potassium and sodium ions depressed, and magnesium ions enhanced the initial rate of TNBS-sorption; however, none of the above cations influenced the equilibrium value of TNBS sorption onto isolated sarcolemmal membranes. Ouabain, on the other hand, did not inhibit the initial rate and decreased the equilibrium value of TNBS sorption onto the membranes. The results obtained enabled the identification of an essential amino group in the potassium binding site of the (Na+ + K+)-ATPase molecule.     

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.     

Gill (Na+,K+)-ATPase in diadromous, freshwater palaemonid shrimps: species-specific kinetic characteristics and alpha-subunit expression

To better comprehend physiological adaptation to dilute media and the molecular mechanisms underlying ammonia excretion in palaemonid shrimps, we characterized the (Na+,K+)-ATPase from Macrobrachium amazonicum gills, disclosing high- (K(0.5) = 4.2+/-0.2 micromol L(-1); V = 33.9+/-1.9 U mg(-1)) and low-affinity (K(0.5) = 0.144+/-0.010 mmol L(-1); V = 232.9+/-15.3 U mg(-1)) ATP hydrolyzing sites. Stimulation by Na+ (K(0.5) = 5.5+/-0.3 mmol L(-1); V = 275.1+/-15.1 U mg(-1)), Mg2+ (K(0.5) = 0.79+/-0.06 mmol L(-1); V = 261.9+/-18.3 U mg(-1)), K+ (K(M) = 0.88+/-0.04 mmol L(-1); V = 271.8+/-10.9 U mg(-1)) and NH4(+) (K(M) = 5.0+/-0.2 mmol L(-1); V = 385.9+/-15.8 U mg(-1)) obeys single saturation curves, activity being stimulated synergistically by NH4(+) and K+. There is a single K+ binding site, NH4(+) binding to a second, exclusive site, stimulating activity by 33%, modulating K+ affinity. (Na+,K+)-ATPase activity constitutes approximately 80% of total ATPase activity (K(Iouabain) = 147.5+/-8.9 micromol L(-1)); Na+-, K+-, Ca2+-, V- and F(o)F(1)-ATPases are also present. M. amazonicum microsomal fractions possess approximately 2-fold less (Na+,K+)-ATPase alpha-subunit than M. olfersi, consistent with a 2.6-fold lower specific activity. These differences in (Na+, K+)-ATPase stimulation by ATP and ions, and specific activities of other ATPases, suggest the presence of distinct biochemical adaptations to life in fresh water in these related species.     

FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli

The KdpFABC complex of Escherichia coli, which belongs to the P-type ATPase family, has a unique structure, since catalytic activity (KdpB) and the capacity to transport potassium ions (KdpA) are located on different subunits. We found that fluorescein 5-isothiocyanate (FITC) inhibits ATPase activity, probably by covalently modifying lysine 395 in KdpB. In addition, we observed that the KdpFABC complex is able to hydrolyze p-nitrophenyl phosphate (pNPP) in a Mg(2+)-dependent reaction. The pNPPase activity is inhibited by FITC and o-vanadate. Low concentrations of ATP (1-30 microM) stimulate the pNPPase activity, while concentrations of >500 microM are inhibitory. This behavior can be explained either by a regulatory ATP binding site, where ATP hydrolysis is required, or by proposing an interactive dimer. The notion that FITC inhibits pNPPase and ATPase activity supports the idea that the catalytic domain of KdpB is much more compact than other P-type ATPases, like Na(+),K(+)-ATPase, H(+),K(+)-ATPase, and Ca(2+)-ATPase.     

Diffusion-limited kinetics of immobilized myosin ATPase

To test the possibility that ATP diffusion limits the kinetics of myosin ATPase (EC. 3.6.1.3) in situ, myosin was covalently bound to the surface of 2 kinds of films: collagen and Immunodyne. On collagen films, it was bound either with 1-ethyl-3 (3 dimethyl-aminopropyl)carbodiimide (EDC) or with dimethyl-3,3'-dithiobis(propionimidate) (DTP). The apparent Km for K+-ATP rose from 0.26 mM for free myosin in solution to 2-5 mM for covalently bound myosin, and maximum K+-ATPase activity was very low. With the other film, Immunodyne from Pall, the maximum activity of bound myosin was 170 nmol per min per 1.5 cm2 film. The apparent Km for K+-ATP was 2.1 mM when the incubation mixture was vigorously stirred, and the effect of stirring indicated that the kinetics of K+-ATP hydrolysis are limited by external diffusion. The large amount of myosin bound per unit of Immunodyne film surface permitted the study of Mg2+-ATPase activity, although it was 400-500 times less than the K+-ATPase activity. The apparently non-Michaelian kinetics of Mg2+-ATP hydrolysis are attributable to the external diffusion. The apparent Michaelis constant observed at low Mg2+-ATP concentrations rose from 0.27 microM for myosin in solution to 5 microM for myosin bound to Immunodyne film.     

The fluorescein isothiocyanate-binding site of the plasma-membrane H+-ATPase of Neurospora crassa

The mammalian (Na+,K+), Ca2+-, and (H+,K+)-ATPases contain a well-characterized lysine residue that reacts with fluorescein 5'-isothiocyanate (FITC); enzymatic activity is protected by ATP, suggesting that the residue is located in or near the nucleotide-binding domain. In this study, the plasma-membrane H+-ATPase of Neurospora crassa is also shown to be sensitive to FITC. The reaction occurs with pseudo first-order kinetics, has a pKa of 8.0, and is stimulated by Mg2+. Enzymatic activity is protected by MgADP with a Kd of 0.2-0.3 mM, close to the Ki with which MgADP serves as a competitive inhibitor of ATP hydrolysis. A tryptic peptide labeled with FITC in the absence, but not in the presence, of MgADP has been isolated and sequenced. The FITC-sensitive residue is Lys474, located in a region that exhibits significant homology with the mammalian cation-transporting ATPases.     

Further biochemical characterization of an Na+ pump inhibitor purified from human urine

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.     

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.     

Localization of a human Na+,K+-ATPase alpha subunit gene to chromosome 19q12----q13.2 and linkage to the myotonic dystrophy locus

The gene coding for a Na+,K+-ATPase alpha subunit (ATP1A3) has been localized to the q12----q13.2 region of human chromosome 19, potentially close to the myotonic dystrophy (DM) gene. In view of previous studies implicating a Na+,K+-ATPase in the pathology of DM, we have examined the possibility that ATP1A3 is a candidate for the DM locus. Although linked, several clear instances of recombination between ATP1A3 and DM rule out the possibility that mutations in ATP1A3 cause the disease. Examination of multiply informative pedigrees indicates the gene order DM-APOC2-ATP1A3.     

K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.     

TXB2: cardiostimulant effect that involves beta-adrenoceptor and Na+ + K+-ATPase activity

The biological properties of Thromboxane B2 (TXB2) on isolated rat heart were studied. Its actions were compared with U-46619 a Thromboxane A2 mimetic compound and with isoproterenol. TXB2 induced a concentration-dependent increase in contractility, that was non-competitively antagonized by propranolol. In addition TXB2 inhibited Na+ + K+-ATPase activity at the same concentrations that influenced the mechanical activity. Inhibition of beta-adrenoceptors efficiently blocked the inhibitory action of TXB2 upon Na+ + K+-ATPase-activity. Isoproterenol simulated the positive inotropic effect and the inhibitory action of TXB2 on Na+ + K+-ATPase-activity. In contrast, U-46619 did not alter the basal dF/dt, neither the enzyme activity. The foregoing results suggest that TXB2 resembles the biological effect of catecholamines-inducing stimulation of myocardial contractility and inhibition of Na+ + K+-ATPase activity.     

The effect of ouabain on sensory neuron neurite growth in organotypic culture

Ouabain, a known inhibitor of Na+, K(+)-ATP, taken in a wide range of concentrations, was investigated in organotypic tissue culture of dorsal root ganglia cells of 10-11 day old chick embryos. Ouabain inhibited neurite growth in a dose-dependent manner. The Hill coefficient was defined as 1, and the Kp value was estimated as 1 x 10(-10) M. At inhibitor concentrations exceeding 1 x 10(-9) M, the growth of neurites was totally inhibited. It is assumed that Na+, K(+)-ATPase may play an important role in regulation of the process of neurite growth in sensory neurones.     

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.     

5-HT uptake blockade prevents the increasing effect of K(ATP) channel blockers on electrically evoked [3H]-5-HT release in rat and mouse neocortical slices

To explore if prolonged--as opposed to acute--5-HT uptake blockade can lead to changes in the function of ATP-dependent potassium (K(ATP)) channels, we investigated in rat and mouse neocortical slices the effects of K(ATP) channel blockers on electrically evoked [3H]-serotonin ([3H]-5-HT) release after short- and long-term exposure to 5-HT uptake blockers. Glibenclamide (1 microM), a K(ATP) channel blocker, enhanced the electrically evoked [3H]-5-HT release by 66 and by 77%, respectively, in rat and in mouse neocortex slices. This effect was confirmed in the rat by tolbutamide (1 microM), another K(ATP) channel antagonist. After short-term blockade (45 min) of 5-HT uptake, glibenclamide still increased the release of [3H]-5-HT in the rat. Glibenclamide, however, failed to enhance [3H]-5-HT release after long-term uptake blockade (210 min). In the mouse, however, both short- and long-term inhibition of 5-HT reuptake by citalopram (1 microM) prevented the facilitatory effect of glibenclamide. The Na(+)/K(+)-ATPase inhibitor ouabain (3.2 microM) abolished the glibenclamide-induced increase in [3H]-5-HT release in both rat and mouse, suggesting that an operative Na(+)/K(+)-ATPase is a prerequisite for activation of K(ATP) channels. The terminal 5-HT(1B) autoreceptor-mediated feedback control was involved in the glibenclamide-induced increase in [(3)H]-5-HT release only in mouse neocortical tissue, as evident from the use of the 5-HT(1B) autoreceptor ligands metitepin (1 microM) and cyanopindolol (1 microM). These results suggest that in the rat long-term uptake blockade leads to an impaired activity of the Na(+)/K(+)-ATPase, which increases intracellular ATP and consequently closes K(ATP) channels. In the mouse, however, short-term uptake blockade seems to already reduce the activity of the Na(+)/K(+)-ATPase and thereby the consumption of ATP. Blockade of 5-HT transporters thus may close K(ATP) channels through increased intracellular ATP. The following slight depolarisation seems to facilitate 5-HT release. These results may contribute to a better understanding of the mechanisms involved in the clinical time latency of antidepressant efficacy of monoamine uptake blockers.