Inhibition by quercetin of thyroid hormone stimulation in vitro of human red blood cell Ca2+-ATPase activity

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations of thyroid hormone. Quercetin, a flavonoid that inhibits several membrane-linked ATPases, suppressed thyroid hormone action on red cell Ca2+-ATPase activity and also interfered with binding of the hormone by red cell membranes. These effects of quercetin were dose-dependent over a range of concentrations (1-50 microM). In contrast, in the absence of thyroid hormone, quercetin at low concentrations stimulated Ca2+-ATPase activity and at 50 microM inhibited the enzyme. The effects of quercetin at low concentrations (1-10 microM), namely, stimulation of Ca2+-ATPase and inhibition of membrane-binding of thyroid hormone, mimic those of thyroid hormone and are consistent with the thyronine-like structure of quercetin. At high concentrations, quercetin is generally inhibitory of Ca2+-ATPase activity. Chalcone, fisetin, hesperetin and tangeretin are other flavonoids shown to reduce susceptibility of membrane Ca2+-ATPase to hormonal stimulation.     

Bepridil and cetiedil reversibly inhibit thyroid hormone stimulation in vitro of human red cell Ca2+-ATPase activity

Thyroid hormone (10(-11) to 10(-10) M) stimulates plasma membrane Ca2+-ATPase activity in vitro in various tissues, including the human red cell (RBC), by a calmodulin-requiring mechanism. Bepridil and cetiedil are Ca2+ antagonists with an intracellular (calmodulin-antagonist) site of action, as well as an effect on the calcium channel in excitable tissues. We have studied the actions of bepridil and cetiedil on Ca2+-ATPase in a channel-free membrane (RBC) to determine effectiveness of these agents as inhibitors of thyroid hormone action on the enzyme. Dose-response studies showed that thyroid hormone stimulation of Ca2+-ATPase activity in vitro was significantly inhibited by as little as 2 x 10(-5) M bepridil and cetiedil. IC50 values of bepridil and cetiedil for thyroid hormone response of the enzyme were 5 x 10(-5) and 2 x 10(-5) M, respectively, whereas IC50s of these agents for enzyme activity in the absence of thyroid hormone were both 10(-4) M. Progressive addition of purified rat testis calmodulin in vitro (10-150 ng calmodulin/mg membrane protein) restored hormone responsiveness in the presence of bepridil and cetiedil. Binding of labeled thyroid hormone by RBC membranes was unaffected by bepridil and cetiedil (up to 2 x 10(-4) M). Thus, bepridil and cetiedil are Ca2+ antagonists that reversibly inhibit thyroid hormone action on human RBC Ca2+-ATPase by a calmodulin-dependent mechanism. Thyroid hormone effect on Ca2+-ATPase is more susceptible to bepridil and cetiedil inhibition than is basal enzyme activity.     

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17beta-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca2+-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10-11-10-6 M, showed an inverted U-shape potency in the regulation of Ca2+-ATPase activity. At physiologically relevant concentrations (10-8-10-9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10-11-10-6 M) and 17beta-estradiol (10-12-10-9 M) caused a dose-dependent increase in the hydrolytic ability of Ca2+-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.     

Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone

The in vitro stimulation of human and rabbit erythrocyte membrane Ca2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5'-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 +/- 3.3 mumol Pi mg membrane protein-1 90 min-1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to L-thyroxine (T4) (10(-10)M) increased Ca2+-ATPase activity to 29.2 +/- 3.8 mumol Pi (P less than 0.001). Dose-response studies conducted with T4 showed that maximal stimulatory response was obtained at 10(-10) M). Hormonal stimulation was comparable for L-T4 and triiodo-L-thyronine (T3) (10(-10) M). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and D-T4, each at 10(-10) M, significantly decreased enzyme activity compared to control (basal) levels. The action of L-T4 on myocardial membrane Ca2+-ATPase activity was inhibited by trifluoperazine (100 microM) and the naphthalenesulfonamide W-7 (50-100 microM), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 micrograms/mg membrane protein-1) in the myocardial membrane fraction and 0.35 micrograms/mg-1 in cytosol. Myocardial Ca2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.     

The effect of thyroid hormone on the high affinity Ca2+-ATPase in rat liver plasma membrane

The effect of thyroid hormone on the high affinity Ca2+-ATPase activity in rat liver plasma membrane was studied. The high affinity Ca2+-ATPase activity in plasma membrane was activated by 10(-7)-10(-5) M of Ca2+ and was inhibited by 70 microM trifluoperazine. Thyroidectomy of rats was associated with an increase in the activity of high affinity Ca2+-ATPase. The increased enzyme activity was normalized by T4 administration to the animals. On the other hand, Na+-K+-ATPase activity in the membrane was decreased by thyroidectomy and the decreased enzyme activity was normalized by T4 administration. The results suggest that thyroid hormone inhibits the Ca2+ extrusion system by inhibiting calmodulin-independent high affinity Ca2+-ATPase in liver plasma membrane.     

Studies on the mechanism of thyroid hormone stimulation in vitro of human red cell Ca2+-ATPase activity

The stimulation in vitro of human red blood cell Ca2+-ATPase activity by thyroxine (T4) and triiodothyronine (T3) in physiological concentrations is shown to depend upon binding of iodothyronines to red cell membranes. Calmodulin enhances the activity of thyroid hormone in this model system but there is no direct interaction of calmodulin and hormone.     

Retinoic acid is a modulator of thyroid hormone activation of Ca2+-ATPase in the human erythrocyte membrane

The thyroid hormones thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) stimulate plasma membrane Ca2+-ATPase (EC 3.6.1.3) activity in human erythrocytes by a mechanism independent of the cell nucleus. The current studies were conducted to determine the effect of retinoic acid on the extranuclear activation by T4 and T3 of Ca2+-ATPase in the human red cell. The retinoid inhibited basal and T4-stimulatable activity of that enzyme in a dose-dependent manner. At the highest tested concentration (10(-6) M), retinoic acid inhibited basal enzyme activity by 25% and T4-stimulated activity by 72%. A concentration as low as 5 x 10(-10) M retinoic acid shifted the dose-response curve of both T4 and T3 so that the concentration of each associated with maximal enzyme stimulation was 10(-9) M instead of 10(-10) M. Retinoic acid displaced [125I]T4 binding to red cell membranes as effectively as unlabeled T4. Retinol failed to influence either basal or T4-stimulated enzyme activity or to displace T4 binding. These results indicate that retinoic acid can partially block the T4 and T3 stimulation of Ca2+-ATPase in human red cell membranes and suggest a physiologic role for the retinoid as a modulator of this peripheral action of thyroid hormone. They suggest that the red cell membrane is an important site of action for this active retinoid.     

Stimulation by thyroid hormone analogues of red blood cell Ca2+-ATPase activity in vitro. Correlations between hormone structure and biological activity in a human cell system

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations (10(-10) M) of L-thyroxine (L-T4) and 3,5,3'-triiodo-L-thyronine (L-T3). This human cell system has been utilized to examine a series of iodothyronine and iodotyrosine analogues for structure-activity relationships. Analogue purity was verified by high pressure liquid chromatography. Analogues were studied at a concentration of 10(-10) M and the stimulatory effect of each analogue was compared with that of L-T4 in this system. Essential to Ca2+-ATPase stimulation were occupation of the 3 and 5 phenyl positions by iodide, bromide, or methyl groups, the L-configuration of the alanine side chain, side chain length equal to that of alanine, and a perpendicular (skewed) conformation of the two rings. The 4'-hydroxyl group is not essential to Ca2+-ATPase stimulation in this model system. T3 was 76% as active as T4 in stimulating Ca2+-ATPase activity. The stimulatory effect of 3,5-dimethyl-3'-isopropyl-L-thyronine and 3,5,3',5'-tetrabromo-L-thyronine approximated that of L-T4. Selected tyrosine analogues also stimulated the enzyme. The bioactivities of hormone analogues in this human model of extra-nuclear thyroid hormone action differ in several ways from results obtained previously in other animal model systems in vitro and in vivo.     

Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms

Steroid hormones are lipophilic suggesting they intercalate into the bilayer of target cell plasma membranes, potentially altering the fluidity and function of the membrane. The present study measured the effects of steroidal exposure on both phospholipid fluidity and integral protein mobility. Studies were performed on the effects of a variety of steroids on phosphatidylcholine liposomes, synaptosomal plasma membranes and sarcoplasmic reticulum membranes. Progesterone decreased the lipid fluidity, whereas testosterone had no effect on lipid movement. The estrogen, 17 beta-estradiol, an aromatised metabolite of testosterone, increased lipid mobility. In each case, the steroid action was concentration-dependent. The steroids all increased the activity of the Ca2+ ATPase of SR membrane, in keeping with their effects on this enzyme's aggregation state. The results suggest that, although lipid fluidity is a factor influencing protein activity, their mobility within the bilayer is the primary determinant of enzyme activity in the membrane for most proteins.     

The alpha 1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca(2+)-ATPase.

Membrane Ca(2+)-ATPase activity was stimulated in vitro separately by T4 (10(-10) M) and by epinephrine (10(-6) M). In the presence of a fixed concentration of T4, additions of 10(-8) and 10(-6) M epinephrine reduced the T4 effect on the enzyme. beta-Adrenergic blockade with propranolol (10(-6) M) prevented stimulation by epinephrine of Ca(2+)-ATPase activity, but did not prevent the suppressive action of epinephrine on T4-stimulable Ca(2+)-ATPase. In contrast, alpha 1-adrenergic blockade with unlabelled prazosin restored the effect of T4 on Ca(2+)-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulation by T4 of red cell Ca(2+)-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (Kd, 1.2 x 10(-8) M; Bmax, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains alpha 1-adrenergic receptor sites that are capable of regulating Ca(2+)-ATPase activity.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Action of long-chain fatty acids in vitro on Ca2+-stimulatable, Mg2+-dependent ATPase activity in human red cell membranes.

Human red cell membrane Ca2+-stimulatable, Mg2+-dependent adenosine triphosphatase (Ca2+-ATPase) activity and its response to thyroid hormone have been studied following exposure of membranes in vitro to specific long-chain fatty acids. Basal enzyme activity (no added thyroid hormone) was significantly decreased by additions of 10(-9)-10(-4) M-stearic (18:0) and oleic (18:1 cis-9) acids. Methyl oleate and elaidic (18:1 trans-9), palmitic (16:0) and lauric (12:0) acids at 10(-6) and 10(-4) M were not inhibitory, nor were arachidonic (20:4) and linolenic (18:3) acids. Myristic acid (14:0) was inhibitory only at 10(-4) M. Thus, chain length of 18 carbon atoms and anionic charge were the principal determinants of inhibitory activity. Introduction of a cis-9 double bond (oleic acid) did not alter the inhibitory activity of the 18-carbon moiety (stearic acid), but the trans-9 elaidic acid did not cause enzyme inhibition. While the predominant effect of fatty acids on erythrocyte Ca2+-ATPase in situ is inhibition of basal activity, elaidic, linoleic (18:2) and palmitoleic (16:1) acids at 10(-6) and 10(-4) M stimulated the enzyme. Methyl elaidate was not stimulatory. These structure-activity relationships differ from those described for fatty acids and purified red cell Ca2+-ATPase reconstituted in liposomes. Thyroid hormone stimulation of Ca2+-ATPase was significantly decreased by stearic and oleic acids (10(-9)-10(-4) M), but also by elaidic, linoleic, palmitoleic and myristic acids. Arachidonic, palmitic and lauric acids were ineffective, as were the methyl esters of oleic and elaidic acids. Thus, inhibition of the iodothyronine effect on Ca2+-ATPase by fatty acids has similar, but not identical, structure-activity relationships to those for basal enzyme activity. To examine mechanisms for these fatty acid effects, we studied the action of oleic and stearic acids on responsiveness of the enzyme to purified calmodulin, the Ca2+-binding activator protein for Ca2+-ATPase. Oleic and stearic acids (10(-9)-10(-4) M) progressively inhibited, but did not abolish, enzyme stimulation by calmodulin (10(-9) M). Double-reciprocal analysis of the effect of oleic acid on calmodulin stimulation indicated noncompetitive inhibition. Addition of calmodulin to membranes in the presence of equimolar oleic acid restored basal enzyme activity. Oleic acid also reduced 125I-calmodulin binding to membranes, but had no effect on the binding of [125I]T4 by ghosts. The mechanism of the decrease by long chain fatty acids of Ca2+-ATPase activity in situ in human red cell ghosts thus is calmodulin-dependent and involves reduction in membrane binding of calmodulin.     

Observations on the (Ca2+ plus Mg2+)-ATPase activator found in various mammalian erythrocytes.

1. A soluble activator of membrane (Ca2+ plus Mg2)-ATPase is present in hemolysates of the newborn calf and cow, the new born and adult pig as well as human erythrocytes. 2. The activator is also found in reticulocytes of the adult pig. 3. The activator obtained from any of the above species is capable of stimulating the membrane (Ca2+ plus Mg2+)-ATPases of the other species, regardless of the age of the animals. 4. The results obtained from density fractionation of human erythrocytes revealed that the soluble factor has little simulatory effect on membranes of young erythrocytes from which it is derived but caused a marked stimulation on (Ca2+ plus Mg2+)-ATPase activity of the intermediate aged and old erythrocyte membranes. 5. The above observations support the following conclusions: (a) the extremely low levels of (Ca2+ plus Mg2+)-ATPase in cow erythrocytes is not due to the lack of a (Ca2+ plus Mg2+)-ATPase activator; (b) the distribution of (Ca2+ plus Mg2+)-atpase activator is not species specific and the differences in the level of membrane (Ca2+ plus Mg2+)-ATPase activity in various species of cells is an inherent property of that particular membrane (c) the (Ca2+ plus Mg2+)-ATPase activator is present at least from the time of reticulocyte formation and remain during tthe life span of the erythrocyte.     

The (Ca + Mg)-ATPase of reticulocytes

Rabbits were made reticulocytotic by repeated bleeding or by injection of phenylhydrazine. Up to reticulocyte levels of 70%, the baseline activity of magnesium-dependent calcium-stimulated adenosine triphosphatase ( ( Ca + Mg)-ATPase, EC 3.6.1.3.) of the red cell plasma membrane was not significantly changed. Maximal activity in the presence of calmodulin was significantly reduced, the stimulation by the activator falling from about 350% to about 50%.     

Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes)

Vesicles are released during the in vitro culture of sheep reticulocytes which can be harvested by centrifugation at 100,000 X g for 90 min. These vesicles contain a number of activities, characteristic of the reticulocyte plasma membrane, which are known to diminish or disappear upon reticulocyte maturation. The activities include acetylcholinesterase, cytochalasin B binding (glucose transporter) nucleoside binding (i.e. nucleoside transporter), Na+-independent amino acid transport, and the transferrin receptor. Enzymes of cytosolic origin are not detectable or are present at low activity in the vesicles. Cultures of whole blood, mature red cells, or white cells do not yield comparable levels of these activities, supporting the conclusion that the activities arise from the reticulocytes. In addition, the lipid composition of the vesicles shows the high sphingomyelin content characteristic of sheep red cell plasma membranes, but not white cell or platelet membranes, also consistent with the conclusion that the vesicles are of reticulocyte origin. It is suggested that vesicle externalization may be a mechanism for shedding of specific membrane functions which are known to diminish during maturation of reticulocytes to erythrocytes.     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from ratcerebellum by calmodulin affinity chromatography. The enzyme was identifiedas plasma membrane Ca2+-ATPase by its interaction with calmodulin andmonoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, andby thapsigargin insensitivity. The purpose of the study was to establishwhether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinaseC (PKC), affect the Ca2+-ATPase isolated from excitable cells and whethertheir effects are comparable to those on the RBC Ca2+-ATPase. We found thatcalmodulin and PKC activated both enzymes. There were significantquantitative differences in the phosphorylation and activation of the SMversus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SMCa2+-ATPase was approximately 3 fold lower and significantly less stimulatedby calmodulin. The initial rate of PKC catalyzed phosphorylation (in thepresence of 12-myristate 13-acetate phorbol) was approximately two timesslower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approachedmaximum level at around 5 min, comparable level of phosphorylation of SMCa2+-ATPase was observed only after 30 min. The PKC-catalyzedphosphorylation resulted in a statistically significant increase inCa2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase.The differences may be associated with diversities in Ca2+-ATPase functionin erythrocytes and neuronal cells and different isoforms composition.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Specific inositol phosphates inhibit basal and calmodulin-stimulated Ca(2+)-ATPase activity in human erythrocyte membranes in vitro and inhibit binding of calmodulin to membranes.

D-Myo-inositol 1,4,5-trisphosphate (Ins[1,4-,5]P3) inhibits rat heart sarcolemmal Ca(2+)-ATPase activity (T. H. Kuo, Biochem. Biophys. Res. Commun. 152: 1111, 1988). We have studied the effect and mechanism of action of Ins(1,4,5)P3 and related inositol phosphates on human red cell membrane Ca(2+)-ATPase (EC 3.6.1.3) activity in vitro. At 10(-6) M, Ins(1,4,5)P3 and D-myo-inositol 4,5-bisphosphate (Ins[4,5]P2) inhibited human erythrocyte membrane Ca(2+)-ATPase activity in vitro by 42 and 31%, respectively. D-Myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 1-phosphate were not inhibitory. Enzyme inhibition by Ins(1,4,5)P3 was blocked by heparin. Exogenous purified calmodulin also stimulated red cell membrane Ca(2+)-ATPase activity; this stimulation was inhibited by Ins(1,4,5)P3. Ins(4,5)P2 and Ins(1,4,5)P3, but not Ins(1,4)P2, inhibited the binding of [125I]calmodulin to red cell membranes. Thus, specific inositol phosphates reduce plasma membrane Ca(2+)-ATPase activity and enhancement of the latter in vitro by purified calmodulin. The mechanism of these effects may in part relate to inhibition by inositol phosphates of binding of calmodulin to erythrocyte membranes.