Sarcolemmal glucose transport in Ca2+-tolerant myocytes from adult rat heart. Calcium dependence of insulin action

Cardiac myocytes were isolated from adult rat ventricles by a method which preserves their functional integrity, including long survival in physiological concentrations of Ca²⁺. Sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog3-O-methyl-d-glucose. Transport was saturable and showed competition byd-glucose and other features of chemical and stereo-selectivity. Transport was stimulated by insulin in a dose-dependent manner, resulting in an almost 5-fold increase inV_max, with little change inK_m. Stimulation of 3-methylglucose transport by insulin was largely Ca²⁺ -dependent. Omission of Ca²⁺ from the incubation medium caused a minor rise in basal 3-methylglucose uptake but the insulin-stimulated rise inV_max was only 30%. The Ca²⁺ antagonist D600 also antagonized stimulation of hexose transport by insulin. In all the above respects, 3-methylglucose transport in myocytes is identical to that in intact heart muscle. In addition, the decrease in insulin response by Ca²⁺ emission was partially reversed by subsequent return to a Ca²⁺ -containing medium. ATP levels remained stable in the absence of Ca²⁺, showing that the Ca²⁺ dependence did not reflect nonspecific cell damage.     

Insulin action on cardiac glucose transport Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na⁺/K⁺ pump. ⁸⁶Rb⁺-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na⁺/K⁺ pump. Ouabain (10^?3 mol/I) inhibited the steady-state uptake of ⁸⁶Rb⁺ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive ⁸⁶Rb⁺-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. ⁸⁶Rb⁺-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na⁺/K⁺ pump activity by ouabain and a concomitant shift in the intracellular Na⁺:K⁺ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na⁺/K⁺ pump and the glucose transport system.     

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.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca²⁺-dependent K⁺ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca²⁺, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K⁺ (balanced by the uptake of Na⁺) and a stimulation of both the influx and the efflux of ⁸⁶Rb. These effects were antagonized by quinine, a known inhibitor of the Ca²⁺-dependent K⁺ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca²⁺-dependent K⁺ channel whose characteristics are similar to those described in other cell systems.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (²H₂O) and dimethyl sulfoxide (Me₂SO) on [³H]ouabain binding to (Na⁺,K⁺)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg²⁺+ATP+Na⁺). In contrast, both solvents stimulated type II (i.e., Mg²⁺+P_i-, or Mn²⁺-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg²⁺+Na⁺+ATP, 75% in the Mg²⁺+P_i system, and in the presence of Mn²⁺ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, ²H₂O inhibition of type I ouabain binding was dependent on Na⁺ concentration in the reaction and was reduced as Na⁺ was elevated. Contact of the enzyme with Me₂SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na⁺+ATP first and then to Me₂SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me₂SO and ²H₂O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H₂O, and E₁ enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H₂O at the active enzyme center and E₂ enzyme conformation. This postulation of a role of H₂O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

The ability of the mitochondrial Ca2+-binding glycoprotein to restore Ca2+ transport in glycoprotein-depleted rat liver mitochondria

Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca²⁺-binding glycoprotein and its Ca²⁺-transporting activity may be as low as 10–20% of the starting value. Both the rate of Ca²⁺ uptake and the capacity to maintain a high Ca²⁺ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca²⁺-transporting ability. The active component in the supernatant is the Ca²⁺-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 μM Mg²⁺, the glycoprotein alone may restore fully the Ca²⁺-transporting ability of the particles. The maximal velocity is already reached at 0.1 μg glycoprotein/mg mitochondrial protein.     

Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance ofα1-adrenergic stimulation

We have previously demonstrated mobilization of Ca²⁺ in the efflux of Rb⁺ (K⁺) from isolated hamster brown adipocytes as a consequence of norepinephrine stimulation. We have now investigated the adrenoceptor subtype specificity of these responses and found them both to be of theα₁-subtype. Futher, we have found that the Rb⁺ (K⁺) effux was dependent upon a primary Ca²⁺mobilization event in response to the α₁-adrenergic stimulation, since the Rb⁺ efflux could also be demonstrated by the addition ionophore A23187 to the cells. The norepinephrine- and A23187-stimulated Rb⁺ effluxes were both inhibited by the Ca²⁺-dependent K⁺ -channel blocker apamin. Apamin also significantly attenuated Ca²⁺ mobilization in cells in response to a submaximal concentration of norepinephrine. We conclude that α₁-adrenergic stimulation of brown fat cells leads to a mobilization of intracellular Ca²⁺ which, in itself or via other mechanisms, leads to an increase in cytosolic Ca²⁺ concentration which, in turn, activates a Ca²⁺ -dependent K⁺ channel leading to a K⁺ release from these cells. A possible role for this channel to sustain and augment the response toα₁-adrenergic stimulation is discussed.     

Ca2+-dependent phosphorylation of rat ovary proteins

A 105,000 × g supernatant fraction from prepubertal rat ovaries was incubated in the presence of [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Inclusion of Ca²⁺ in the phosphorylation reaction promoted a selective ³²p incorporation into two proteins of M_r = 95,000 and 50,000. Inclusion of chlorpromazine with Ca²⁺ blocked the Ca²⁺-stimulated increase of ³²p incorporation. Our results demonstrate the presence of Ca²⁺-stimulated protein phosphorylation system capable of recognizing endogenous substrate proteins in the prepubertal rat ovary.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Mechanisms of activation of renal (Na+ + K+)-ATPase in the rat Effects of acute and chronic administration of dexamethasone

The mechanisms of activation of renal (Na⁺ + K⁺)-ATPase by administration of the synthetic glucocorticoid hormone, dexamethasone, have been investigated in adrenalectomized rats. Chronic treatment with dexamethasone (1–5 mg/100 g body wt. daily for 5 days) stimulated (Na⁺ + K⁺)-ATPase specific activity in crude homogenated and microsomal fractions of renal cortex (by approx. 100–150%) and renal medulla (by approx. 100%). Acute treatment with dexamethasone (0.5–10 mg/100 g body wt.) also stimulated enzyme activity in crude homogenates and microsomal fractions of renal cortex and medulla (by approx. 40–50%). Stimulation was dose dependent and occurred within 2h after hormone treatment. In vitro addition of dexamethasone (10^?4–10^?8 M) to microsomal fractions did not modify the specific activity of (Na⁺ + K⁺)-ATPase. Stimulation of (Na⁺ + K⁺)-ATPase activity by acute and chronic administration of the hormone was demonstrated whether specific activities were expressed as a function of cellular protein or cellular DNA. Dexamethasone treatment increased the ratios protein:DNA and, to a lesser extent, the ratios RNA:DNA. However, these effects were mainly due to a reduction in the renal contents of DNA, which suggests that the observed enzyme activation is not due to an action of the hormone on renal hypertrophy. Dexamethasone also reduced cellular DNA contents in the liver. The characteristics of the activation process were essentially similar after treatment with single or multiple doses of the hormone. There were increases in the value for Na⁺ (approx. 100%), K⁺ (approx. 40%) and ATP (approx. 160%). The K_m values for Na⁺ (approx. 17 mM) and K⁺ (approx. 1.8 mM) were unchanged and there was a small increase in the K_m value for ATP (0.7 mM as against 1.7 mM). There was no difference in the Hill coefficients for the three substrates. The levels of the high-energy P_i intermediate of the (Na⁺ + K⁺)-ATPase reaction were augmented by dexamethasone treatment and the increased levels were quantitatively correlated with the observed stimulation of (Na⁺ + K⁺)-ATPase specific activity. The apparent turnover numbers of the reaction remained unchanged. The specific activity of the ouabain-sensitive p-nitrophenylphosphatase increased proportionally to the increase in (Na⁺ + K⁺)-ATPase specific activity. Enzyme activation by acute dexamethasone treatment occurred in the absence of changes in glomerular filtration rate and tubular Na⁺ excretion.These results indicate that (Na⁺ + K⁺)-ATPase activation by acute and chronic dexamethasone treatment represents an increase in the number of enzyme units with little or no change in the kinetic properties (affinity, cooperativity) of the enzyme. In addition, the information presented suggests a direct regulatory effect of glucocorticoid hormones on the activity of renal (Na⁺ + K⁺)-ATPase and is inconsistent with the concept that changes in Na⁺ loads mediate the effects of these hormones on enzyme activity. Instead, the results suggests a primary role for glucocorticoid hormones in the renal regulation of Na⁺ homeostasis.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles I. Ca2+ outflow in the non-energized state of the calcium pump

Passive Ca²⁺ permeability of sarcoplasmic reticulum vesicles has been studied after maximal loading with Ca²⁺ (150–200 nmol/mg protein) in the presence of Ca²⁺, MgATP and an ATP generating system of limited capacity. Outflow of accumulated Ca²⁺ in the non-energized state of the system was studied by depletion of the medium of one of the substrates, either MgATP (by complete consumption) or Ca²⁺ (by complexation with EGTA). It was found that Ca²⁺ outflow under these conditions is relatively slow and independent of the medium concentration of Ca²⁺ (5·10^?9–5·10^?5 M) or MgATP (0.7–730 μM). Outflow curves were steep at the beginning of the outflow phase (30–60 nmol/min per mg protein), and outflow proceeded at a much lower rate below 100 nmol Ca²⁺/mg protein. Outflow could be completely inhibited by La³⁺. The Ca²⁺ release curves are not compatible with simple diffusion, and cannot be accounted for by Ca²⁺ binding inside the vesicles. Neither are our observations consistent with permeation mediated via the Ca²⁺ translocation sites involved in active transport. We suggest that non-energized Ca²⁺ outflow may proceed by a process of ion-exchange through negatively charged, water-filled channels in the membrane, the properties of which are altered by a high intravesicular concentration of Ca²⁺.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine

The presence of an Na⁺/Ca²⁺ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na⁺ gradient-dependent Ca²⁺ uptake and the inhibition of ATP-dependent Ca²⁺ accumulation by Na⁺. The presence of 75 mM Na⁺ in the uptake solution reduces ATP-dependent Ca²⁺ transport by 45%, despite the fact that Na⁺ does not affect Ca²⁺-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na⁺ inhibition of ATP-dependent Ca²⁺ uptake to 20%, apparently by preventing accumulation of Na⁺ in the vesicles realized by the Na⁺-pump. It was concluded that high intravesicular Na⁺ competes with Ca²⁺ for intravesicular Ca²⁺ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca²⁺ transport shows a sigmoidal dependence on the Na⁺ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na⁺ is between 15 and 20 mM. Uptake of Ca²⁺ in the absence of ATP can be enhanced by an Na⁺ gradient (Na⁺ inside > Na⁺ outside). This Na⁺ gradient-dependent Ca²⁺ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca²⁺ of this system is below 1 μM. In contrast to the ATP-dependent Ca²⁺ transport, there is no significant difference in Na⁺ gradient-dependent Ca²⁺ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca²⁺ uptake is 5-times greater than the Na⁺/Ca²⁺ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na⁺/Ca²⁺ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na⁺/Ca²⁺ exchange system while it doubles the ATP-driven Ca²⁺ pump activity.     

The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Concanavalin a binding and Ca2+ fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca²⁺ content of the cells. ⁴⁵Ca²⁺ fluxes were measured under conditions where artifacts due to Ca²⁺ binding to concanavalin A could be excluded. Both ⁴⁵Ca²⁺ influx into and efflux from these cells are significantly activated by the lectin. If ⁴⁵Ca²⁺ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in ⁴⁵Ca²⁺ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 μg/ml) no significant increase in ⁴⁵Ca²⁺ influx occurs but an increase in ⁴⁵Ca²⁺ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca²⁺ influx into the cells and that activation of Ca²⁺ efflux occurs as a response to an increase in the cytosolic Ca²⁺ activity. Thus, Ca²⁺ may well play a role in triggering lymphocyte activation.     

Mechanism of the involvement of monoamine oxidase in the regulation of (Na+ + K+)-ATPase in rat brain

Increasing concentrations of dopamine fail to give a biphasic response to (Na⁺ + K⁺)-ATPase activity in various subcellular fractions of rat brain preincubated with monoamine oxidase inhibitors, viz. 1·10^?4 M clorgyline and 1·10^?4 M deprenyl. The product of the monoamine-oxidase-catalysed reaction with dopamine as substrate is 3-methoxy-4-hydroxyphenylacetaldehyde. An analogue of this product is 3-methoxy-4-hydroxybenzaldehyde. This analogue, when incubated with the subcellular fractions which had been preincubated with monoamine oxidase inhibitors and dopamine, gave a more pronounced biphasic response to (Na⁺ + K⁺)-ATPase activity than that observed in the fractions incubated with dopamine alone.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.