Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. I. Exchange and hydrolysis kinetics at millimolar nucleotide concentrations.

Parallel measurements in steady-state of ATP hydrolysis rate (vhydr) and the simultaneous reverse reaction, i.e., the ADP-ATP exchange rate (vexch), allowed the determination of a kinetic parameter, KE, containing only the four rate constants needed to characterize the enzyme intermediates involved in the sequence (Formula: see text). In order to compare the properties of these enzyme intermediates under different sets of conditions, KE was measured at varying K+ and Na+ concentrations in the presence of millimolar concentrations of ATP, ADP and MgATP, using an enzyme preparation that was partially purified from bovine brain. (1) In the presence of Na+ (150 mM), K+ (20-150 mM) was found to increase the exchange rate and decrease the ATP hydrolysis rate at steady-state. As a result, KE increased at increasing K+. However, the value of KE found by extrapolation to K+ = 0 was 7-times lower than the value actually measured in the absence of K+. This finding indicates that one of the intermediates, EATP or EP, or both, when formed in the presence of Na+ alone, are different from the corresponding intermediate(s) formed in the presence of Na+ + K+ (at millimolar substrate concentration). (2) In the presence of 150 mM K+, Na+ (5-30 mM) was found to increase the ADP/ATP exchange as well as the ATP hydrolysis rate at steady-state. The ratio of the two rates was constant. This finding, when interpreted in terms of KE, indicates that Na+ does not have to leave the enzyme for ATP release to be accelerated by K+ in the backward reaction. This also is in opposition to the usual versions of the Albers-Post model, which does not have simultaneous presence of Na+ and K+.

Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. II. Exchange and hydrolysis kinetics at micromolar nucleotide concentrations.

The ATP hydrolysis rate and the ADP-ATP exchange rate of (Na+ + K+)-ATPase from ox brain were measured at 10 microM Mg2+free and at micromolar concentrations of free ATP and ADP. (1) In the absence of K+, substrate inhibition of the hydrolysis rate was observed. It disappeared at low Na+ and diminished at increasing concentrations of ADP. This was interpreted in terms of free ATP binding to E1P. In support of this interpretation, free ATP was found to competitively inhibit ADP-ATP exchange. (2) In the presence of K+, substrate activation of the hydrolysis rate was observed. Increasing (microM) concentrations of ADP did not give rise to competitive inhibition in contrast to the situation in the absence of K+ (cf. 1, above). This was interpreted to show that at micromolar substrate, some low-affinity, high-turnover Na+ + K+ activity is possible, provided the Mg2+ concentration is low. (3) While small concentrations of K+ increased the hydrolysis rate (cf. 2) they decreased the rate of ADP-ATP exchange. To elucidate this phenomenon, parallel measurements of exchange and hydrolysis rates were performed over a wide range of ATP and ADP concentrations, with and without K+. If, in the presence and absence of K+, ADP (and ATP competing) are binding to the same phosphorylated intermediate for the backward reaction, it places quantitative restrictions on the ratio of rate constants with and without K+. The results did not conform to these restrictions, and the discrepancy is taken as evidence for the necessity for a bicyclic scheme for the action of the (Na+ + K+)-ATPase. (4) An earlier statement concerning the nature of the phosphoenzyme obtained in the presence of Na+ and K+ is amended.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity.

A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain.

The expressions for the kinetic constants corresponding to the steady state model for hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase proposed recently are analyzed with the object of determining the rate constants. The theoretical background for the necessary procedures is described. The results of this analysis are: (1) A small class (four) of rate constants are determined directly by the previously published values of the kinetic constants. (2) For a somewhat larger class of rate constants upper and lower bounds may be established. For several rate constants the upper and lower bounds differ by less than a factor 1.6 (for the "(Na+ + K+)-enzyme", i.e. the enzyme activity with K+ and millimolar substrate concentration) and 1.2 (for the "Na+-enzyme",i.e. the activity at micromolar substrate concentrations). (3) Experiments on inhibition by K+ of the Na+-enzyme at various Mg2+ concentrations are reported and analyzed. With the additional assumption that the rate constants governing the addition to ATP of Mg2+ is independent of whether or not ATP is bound to an enzyme molecule, a set of consistent values for all the 23 rate constants in the mechanism may be obtained. (4) The values of some rate constants lend further support to the contention discussed in a previous paper that the enzyme hydrolyzes ATP along two kinetically distinct pathways, depending on the presence of K+ and on the concentration of substrate, without the necessity of having more than one active substrate site per enzyme unit at any time. (5) The results show that while the two enzyme forms, the "Na+-enzyme" E1 and the "K+-enzyme" E2K, add substrate with (second order) rate constants of the same order of magnitude (differing only by a factor of four in favor of the former), the rate constants for the reverse processes differ by a factor of 100, being largest for the K+-enzyme. This is the main reason for the large difference in the Michaelis constants for the two forms reported previously. (6) Compatibility of the model with the well-known rapid dephosphorylation of the phosphorylated enzyme in the presence of K+ requires the presence, at non-zero steady state concentration, of an enzyme-potassium-phosphate intermediate, which is acid labile and is therefore not detected as a phosphorylated enzyme using conventional methods.

Kinetics of oligomycin inhibition and activation of Na+/K(+)-ATPase.

Oligomycin inhibition of the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase was studied at varying NaCl concentrations and it was found that for a given amount of live enzyme, the observed inhibition of a particular total oligomycin concentration decreased as the amount of added, (heat-) denatured enzyme increased. In the present article we derive a scale factor for the oligomycin concentration, i.e., the fraction of the total concentration of oligomycin which is free in solution, as a function of the enzyme concentration used. This fraction decreased linearly with the protein concentration and may attain quite small values. We also study the Na(+)-dependence of the hydrolysis rate at saturating substrate concentrations ([Mg2+] = [ATP] = 3 mM), in the presence as well as the absence of KCl, at various concentrations of oligomycin. These data may be explained if it is assumed that the sole effect of oligomycin is to confer upon the enzyme an increased affinity for Na+, i.e., oligomycin merely enhances the inhibitory effect of Na+ on the (maximal) activity seen at high Na(+)-concentrations. The increased Na(+)-affinity in the presence of oligomycin should result in activation of the hydrolysis rate measured under conditions where Na(+)-activation is predominant, i.e., at low Na(+)-concentration and sub-saturating substrate concentrations. This prediction is verified for both Na(+)-ATPase and for Na+/K(+)-ATPase. This proposed action of oligomycin seems to be corroborated also by other evidence discussed in the text.

Kinetics of Na+-ATPase: influence of Na+ and K+ on substrate binding and hydrolysis.

An analysis of the influence of Na+ and K+ on the kinetics of Na+-ATPase in broken membrane preparations from bovine brain is presented with particular emphasis on the effect of the cations on the binding and splitting of the substrate MgATP and on the derivation of a detailed kinetic model for that interaction. It was found that the enzyme in the absence of Na+ and K+, but in the presence of 7 mM free Mg2+, at pH 7.4 (37 degrees C) exhibits an ouabain-sensitive ATPase activity. The simplest model quantitatively compatible with all the data involves two different, interconvertible (conformational) forms of the enzyme, E1 and E'1, with the following properties: The E1 form does not bind K+ but has three independent and equivalent high-affinity sites (Kd = 5.6 mM) for Na+. It binds and hydrolyzes substrate only when two or three sodium ions are bound to it. The E'1 form binds and hydrolyzes the substrate only in the absence of monovalent cations. It is competitively inhibited by K+ (Kd = 0.23 mM), and this inhibition is further enhanced by binding of Na+ to the K+-bound form at two equivalent, independent sites (Kd = 12 mM). It is suggested that the E'1 form is the Mg2+-induced conformational state of the enzyme observed by others, which differs from the usually encountered E1 and E2 forms. The model allows the calculation of ATP-binding and ADP-releasing rate constants for the E1-form for later comparison with corresponding rate constants for the (na+ + K+)-ATPase (following paper).

Kinetics of (Na+ + K+)-ATPase: analysis of the influence of Na+ and K+ by steady-state kinetics.

The influence of Na+ and K+ on the steady-state kinetics at 37 degrees C of (Na+ + K+)-ATPase was investigated. From an analysis of the dependence of slopes and intercepts (from double-reciprocal plots or from Hanes plots) of the primary data on Na+ and K+ concentrations a detailed model for the interaction of the cations with the individual steps in the mechanism may be inferred and a set of intrinsic (i.e. cation independent) rate constants and cation dissociation constants are obtained. A comparison of the rate constants with those obtained from an analogous analysis of Na+-ATPase kinetics (preceding paper) provides evidence that the ATP hydrolysis proceeds through a series of intermediates, all of which are kinetically different from those responsible for the Na+-ATPase activity. The complete model for the enzyme thus involves two distinct, but doubly connected, hydrolysis cycles. The model derived for (Na+ + K+)-ATPase has the following properties: The empty, substrate free, enzyme form is the K+-bound form E2K. Na+ (Kd = 9 mM) and MgATP (Kd = 0.48 mM), in that order, must be bound to it in order to effect K+ release. Thus Na+ and K+ are simultaneously present on the enzyme in part of the reaction cycle. Each enzyme unit has three equivalent and independent Na+ sites. K+ binding to high-affinity sites (Kd = 1.4 mM) on the presumed phosphorylated intermediate is preceded by release of Na+ from low-affinity sites (Kd = 430 mM). The stoichiometry is variable, and may be Na:K:ATP = 3:2:1. To the extent that the transport properties of the enzyme are reflected in the kinetic ATPase model, these properties are in accord with one of the models shown by Sachs ((1980) J. Physiol. 302, 219-240) to give a quantitative fit of transport data for red blood cells.

Diethyl pyrocarbonate inactivates CD39/ecto-ATPDase by modifying His-59.

Diethyl pyrocarbonate (DEPC) in conditions that favour carbethoxylation of histidyl residues strongly inactivated E-type ATPase activity of a rat lung membrane preparation, as well as ecto-ATPase activity of rat vessels and human Epstein-Barr virus-transformed B lymphocytes. Inactivation of the enzyme (up to 70%) achieved at concentrations of DEPC below 0.5 mM could be fully reversed by 200 mM hydroxylamine at pH 7.5, thus confirming histidine-selective modification. UTP effectively protected the enzyme activity from DEPC inactivation. This was taken to indicate that the conformation adopted by the enzyme molecule upon substrate binding was not compatible with DEPC reaching and/or modifying the relevant histidyl residue. Substrate activation curves were interpreted to show the enzyme molecule to be inactive, at all substrate concentrations tested, when the target histidyl residue had been modified by DEPC. Comparison of known sequences of CD39-like ecto-ATP(D)ases with the results on inactivation by DEPC revealed His-59 and His-251 (according to the human CD39 sequence) as equally possible targets of the inactivating DEPC modification. Potato apyrase lacks a homologue for the former residue, while the latter is preserved in the enzyme sequence. Therefore, this enzyme was exposed to DEPC, and since hydrolysis of ATP and ADP by potato apyrase was insensitive to modification with DEPC, it was concluded that His-59 is the essential residue in CD39 that is affected by DEPC modification in a way that causes inactivation of the enzyme.

[32P]ATP synthesis in steady state from [32P]Pi and ADP by Na+/K(+)-ATPase from ox brain and pig kidney. Activation by K+.

The ouabain-sensitive synthesis of [32P]ATP from [32P]Pi and ADP (vsyn) was measured in parallel with the ouabain-sensitive hydrolysis of [32P]ATP (vhy) at steady state, at varying concentrations of sodium, potassium, magnesium, inorganic phosphate, ADP, ATP and oligomycin, and at varying pH. Na+ was necessary for ATP synthesis, but vsyn was decreased by high sodium concentrations. Oligomycin, depending on the Na+ concentration, either decreased or did not affect vsyn. Potassium, at low concentrations (1-5 mM) increased vsyn at all magnesium and sodium concentrations tested, lower potassium concentrations being needed to activate vsyn at lower sodium concentrations. vsyn was optimal below pH 6.7, decreasing abruptly at higher values of pH. At pH 6.7, vsyn was a hyperbolic function of the concentration of inorganic phosphate. In the presence of potassium, half-maximal rate was obtained at [Pi] congruent to 40 mM, whereas a higher concentration was needed to obtain half-maximal rate in the absence of K+. In contrast, increasing the concentration of ADP caused a nonhyperbolic activation of vsyn, the pattern obtained in the presence of potassium being different from that obtained in its absence. Increasing the ATP concentration above 0.5 mM decreased vsyn. The data are used to elucidate (1) which reaction steps are involved in the ATP-synthesis catalysed by the Na+/K(+)-ATPase at steady state in the absence of ionic gradients and (2) the mechanism by which K+ ions stimulate the reaction.

Characterisation of Ca2+ or Mg(2+)-dependent nucleoside triphosphatase from rat mesenteric small arteries.

When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent Km in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF4-, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N'-dicyclohexylcarbodiimide caused significant (greater than 30%) inhibition. In the presence of micromolecular concentrations of Ca2+ and Mg2+, small (less than 20 mM) concentrations of Na+, K+, Rb+, Cs+ and choline+, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. III. A minimal model.

A steady-state kinetic investigation of the effect of K+ on the Na+-enzyme activity of the (Na+ + K+)-ATPase in broken membrane preparations is reported. Analysis of the kinetic patterns obtained, together with the results reported in the first two articles of this series permit the following conclusions. 1. K+ inhibits the Na+-enzyme (the enzyme activity measured at micromolar substrate concentrations in the presence of Na+). The inhibition of non-competitive at low and competitive at higher K+ concentrations and is enhanced by free Mg2+. 2. The results indicate that the Na+-enzyme at steady-state tends to be accumulated in an enzyme-potassium complex when K+ is added. 3. The enzyme-potassium complex, in turn, binds Mg2+ in a dead-end fashion. The dissociation constant for the enzyme-K-Mg complex, estimated from the data, is 7.2 mM. The same value was obtained earlier for the Mg2+ inhibition constant of the substrate-free form of the (Na+ + K+)-enzyme (the enzyme activity measured with Na+ and K+ and at millimolar substrate concentrations) suggesting that the two constants describe the same equilibrium. 4. On the basis of the known (optimal) activity of the (Na+ + K+)-ATPase, relative to that of the Na+-ATPase, a rate constant condition is found which must be met if the Post-Albers kinetic scheme is to satisfy the data. Kinetic data for the phosphoenzyme indicate that this condition is not satisfied. 5. On the basis of the kinetic results a model for the hydrolytic action of (Na+ + K+)-ATPase is proposed. This model encompasses the Post-Albers scheme but contains two distinctive hydrolysis cycles (an 'Na+-enzyme cycle' and a '(Na+ + K+)-enzyme cycle') with widely different affinities for the substrates. Only one of the cycles (the Na+-enzyme cycle) involves acid-stable phosphorylated enzyme intermediates at discernible steady-state concentrations. Which of the two main cycles is predominant in any particular system is determined by the concentration of ligands and substrates. 6. According to this scheme, an enzyme preparation may exhibit both a high (Na+-enzyme) and a low ((Na+ + K+)-enzyme) substrate affinity, without the necessity of assigning more than one substrate site to a particular enzyme unit at any one time.

Nucleotide hydrolytic activity of isolated intact rat mesenteric small arteries.

Segments of isolated intact rat mesenteric small arteries were incubated in physiological bicarbonate buffer in the presence of nano- to millimolar concentrations of ATP. ATP was hydrolysed, and when the vessel was transferred from one incubation to another, the enzyme activity was transferred with the vessel, consistent with the presence of an ecto-ATPase. The substrate, ATP, was shown to induce a modification of the hydrolytic activity which occurred the more rapidly the higher the concentration of ATP. The modified system hydrolysed ATP with a decreased substrate affinity. As the substrate induced a modification of the hydrolytic activity, steady-state velocity measurements for determination of kinetic parameters could not be obtained. Nevertheless, it was possible to compare the modification caused by ATP and UTP, and to compare the hydrolysis rates measured with [32P]ATP, [32P]UTP and [32P]GTP. It was concluded that the hydrolytic activity of the vessels did not distinguish between the nucleoside triphosphates (NTPs). In a histidine buffer, the activity was shown to be activated by micromolar concentrations of either Ca2+ or Mg2+, and not to be influenced by inhibitors of P-type, F-type and V-type ATPases. Functional removal of the endothelium before assay did not reduce the measured NTP hydrolysis. At millimolar concentrations of trinucleotide the hydrolysis rate was 10-15 mumol per min per gram of tissue or 0.11-0.17 mumol per min per 10(6) vascular smooth muscle cells. This value is equivalent to the maximal velocity obtained for the Ca2+ or Mg(2+)-dependent NTPase released to the medium upon 2 s of sonication of the vessels (Plesner, L., Juul, B., Skriver, E. and Aalkjaer, C. (1991) Biochim. Biophys. Acta 1067, 191-200). Comparing the characteristics of the released NTPase to the characteristics of the activity of the intact vessel, they showed a strong resemblance, but the substrate-induced modification of the enzyme was seen only in the intact preparation.

Ecto-ATPases: identities and functions.

Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.

Rate and duration of net glycogen synthesis following glucose administration to fasted human leukocytes.

When glucose was added to fasted human leukocytes in a final concentration of 0.5-5 mM there was a phase of glycogen synthesis followed by a phase of glycogen breakdown. The duration of the phase of net glycogen synthesis increased with increasing concentrations of glucose applied, but the net rate of glycogen synthesis was inversely related to this figure and decreased from approx. 7 nmol/10(7) cells per min at 0.5 mM glucose to an average of 4 nmol/10(7) cells per min at 5 mM glucose.

Facilitated transport of 3-O-methyl-D-glucose in human polymorphonuclear leukocytes.

Transport of the nonmetabolizable hexose analogue 3-O-methyl-D-glucose (30MG) was measured in human polymorphonuclear leukocytes at 37 degrees C, pH 7.4. 3OMG at very low concentration (0.05 mM) equilibrated with the intracellular water with a rate constant of about 0.08 s-1. Transport of 3OMG in the presence of 20 microM cytochalasin B and transport of L-glucose were insignificant. Countertransport of 14C-labelled 3OMG was demonstrated. Exchange of 3OMG between the extracellular and intracellular water showed saturation with a Km of about 4 mM. Thus, the transport of 3OMG is mediated almost exclusively by facilitated diffusion.

The influence of membrane charge on the kinetic properties of Na,K-ATPase.

Lipophilic ions modify the affinity of the cation binding sites of the membrane-bound Na,K-ATPase. We studied the effect of the lipophilic ions tetraphenyl-phosphonium (TPP+) and tetraphenylboron (TPB-) on the binding of Na+ and K+ to the cation site(s) that are exhibited by the enzyme during the catalytic cycle: the high-affinity (inside) Na-binding site, site I, the low-affinity (outside) Na-leaving site, site II, and the high-affinity (outside) K-site, site III. Site I: In the presence of TPP+ (positive charge added to the lipid environment) a higher Na(+)-concentration was needed to obtain phosphorylation of the enzyme, whereas in the presence of TPB- (negative charge added to the lipid environment) phosphorylation was obtained at a lower Na(+)-concentration, but the change in apparent K0.5 for Na+ was small, (K0.5Na,TPP = 0.180 mM and K0.5Na,TPB = 0.07 mM), indicating only a minor influence of membrane charge on Na+ binding to site I. Site II: Compared to control conditions, more Na+ was required to inhibit ATP-hydrolysis and to increase the steady-state level of ADP-sensitive phosphoenzyme when TPP+ was present, and the opposite was observed with TPB-, indicating a strong influence of membrane charge on the Na+ occupancy of site II. Site III: TPP+ induced a significant decrease both in the rate of K-dependent dephosphorylation of preformed E32P and in the K+ affinity. The effect of TPP+ on the ATP hydrolysis rate strongly resembled the effect of decreasing [KCl]. The results indicated a pronounced effect of adding positive charge to the lipid environment of site III.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ATP and related nucleotides on the tone of isolated rat mesenteric resistance arteries.

The relationship between ecto-ATPase activity and the vasoactive effect of ATP is unclear. Previously we have characterized the ectonucleoside triphosphatase activity of isolated rat mesenteric small arteries and now characterize the effect of nucleotides on the tone of these arteries. In resting arteries, ATP caused concentration-dependent contractions that were transient and could not be reproduced within 2 h. Transient contractions in response to ATP also were elicited in arteries precontracted with norepinephrine, but the potency of ATP was increased and responses to repeated stimulations could be obtained. Contractions were followed by relaxation. The response to ATP was unaffected by 100 microM theophylline, 1 microM propranolol or removal of the endothelium. Transient contractions followed by relaxation were caused also by ADP, 2-methyl-thio-ATP (2meSATP) and alpha, beta-methylene-ATP (alpha, beta-meATP). UTP caused sustained contractions, whereas GTP and ITP had little effect. The rank order of potency (alpha, beta-,mATP > ATP > ADP) suggested that P2x purinoceptors were responsible for the contractions, whereas the rank order of potency for the relaxation (alpha, beta-meATP > or = ATP > 2meSATP) was not consistent with the relaxation being mediated by P2Y purinoceptors as defined originally. Desensitization of the contractile response to ATP by alpha, beta-meATP was variable. In contrast, inhibition of the response to ATP was obtained consistently and dose-dependently with GTP.

Effects of ATP and UTP on [Ca2+]i, membrane potential and force in isolated rat small arteries.

We have investigated excitation-contraction coupling mechanisms associated with the activation of purinoceptors and putative pyrimidinoceptors by assessing the effects of ATP and UTP on cytoplasmic Ca2+ activity ([Ca2+]i), membrane potential (Em) and force in rat mesenteric small arteries. UTP induced a sustained concentration-dependent contractions, closely associated with concentration-dependent increases in [Ca2+]i. Superfusion with 0.1 mM UTP caused a sustained depolarisation of 12 +/- 1 mV (SE, n = 8). In Ca(2+)-free medium, the increase in [Ca2+]i and the contraction obtained with UTP (1 mM) were both transient and were inhibited by prior exposure to noradrenaline (NA). In vessels depolarised with KCl, UTP caused no change in Em, but a sustained increase in force and a transient increase in [Ca2+]i were induced, leading to an increased force/[Ca2+]i ratio. Similar effects on [Ca2+]i, Em and force were observed with ATP; but the effect of ATP on force was transient, whereas the effect on [Ca2+]i and Em declined only slowly. There was no crosstachyphylaxis between the responses to ATP and UTP: in the presence of 1 mM of either, the other drug induced contractions in low concentrations, as if they acted through distinct receptors. The results suggest that both UTP and probably ATP release intracellular Ca2+, possibly from the stores emptied by NA. The sustained response to UTP appears to be due to an influx of extracellular Ca2+. UTP but not ATP was found to enhance the force-generating effect of [Ca2+]i.

Influence of intramembrane electric charge on Na,K-ATPase.

Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-), on interactions of Na+ and K+ with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland. Na+ and K+ interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions. Phosphoenzyme interactions with extracellular cations were evaluated through K(+)-, ADP-, and Na(+)-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP-insensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na+ affinity and the rate coefficient for Na(+)-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP+. TPB- had opposite effects. Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na+ was replaced by N-methyl-D-glucamine or Tris+, but were unaffected by the replacement of Cl- by other anions. Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions. Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field.