Susan D. Blas

In vitro stimulation of human red blood cell Ca2+-ATPase by thyroid hormone☆

Abstract Ca2+-ATPase activity in human erythrocyte ghosts previously washed to remove endogenous thyroid hormone is stimulated in vitro by physiologic concentrations of thyroxine (T4) and triiodothyronine (T3). Two- to three-fold increases (P

The Effect of In Vivo Glucose Administration on Human Erythrocyte Ca2+-ATPase Activity and on Enzyme Responsiveness In Vitro to Thyroid Hormone and Calmodulin

To characterize endogenous control mechanisms for human erythrocyte membrane Ca2+-ATPase (“calcium pump”) activity, we studied the effect of changes in blood glucose concentration in vivo within the physiologic range on Ca2+-ATPase activity in red cells. Red cells obtained in the course of induced hyperglycemia were also studied to determine susceptibility of membrane Ca2+-ATPase to stimulation in vitro by thyroid hormone and calmodulin, both of which have been shown previously to enhance Ca2+-ATPase activity. Oral glucose administration (75 g) to eight healthy, adult subjects induced predictable increases in concentrations of blood glucose and immunoreactive insulin. Basal levels of activity of Ca2+-ATPase in red cells obtained after glucose ingestion fell 55% (P < 0.025) by 30 min after glucose with recovery of enzyme activity to levels not significantly different from basal by 60 min. Activity of red cell Ca2+-ATPase at time zero was significantly stimulated in vitro by thyroxine (T4, 10−10 M), triiodo-L-thyronine (T3, 10−10 M), and calmodulin (100 ng/mg membrane protein). In vivo glucose administration led to depression of red cell enzyme responsiveness in vitro to T4 and T3; recovery from this effect did not occur by 120 min after oral administration of glucose. Calmodulin responsiveness of the enzyme in vitro was less significantly reduced in red cells obtained after glucose ingestion. Intravenous (i.v.) glucose administration (20 g) to five subjects also led to decreased basal enzyme activity (61% of fasting level at 20 min). A significant decrease in response of enzyme to T4 was achieved by 8 min after glucose administration (P < 0.02), with recovery by 60 min. Calmodulin responsiveness showed an insignificant downward trend. Exposure of red cell membranes to glucose (200 mg/ dl) or insulin (100 nil/ml) in vitro resulted in 26% and 17% reductions, respectively, in basal Ca2+-ATPase activity (P < 0.001), together with a reduction in thyroid hormone stimulation of the enzyme. Tunicamycin, an inhibitor of enzymatic protein glycosylatioh, reversed these in vitro effects of glucose, but not of insulin, on erythrocyte Ca2+-ATPase activity. These observations are relevant to the interpretation of clinical studies of Ca2+-ATPase activity and to the physiology of the enzyme. Glucose administration predictably decreases Ca2+-ATPase in the human red cell, an alteration in calcium pump activity that would allow pulsing Of Ca2+ into the cell.

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.

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca2+-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.

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.

Interaction of thyroid hormone and sex steroids at the rabbit reticulocyte membrane in vitro: Control by 17β-estradiol and testosterone of thyroid hormone-responsive Ca2+-ATPase activity

Physiological concentrations (10(-10) M) of L-thyroxine and triiodo-L-thyronine were found in vitro to enhance Ca2+-ATPase activity in reticulocyte-enriched red cell membranes from female rabbits and to inhibit this enzyme in the male reticulocyte. Cross-incubation experiments with reticulocyte-enriched red cells and plasma from the opposite sex demonstrated that this sex-specific membrane response to thyroid hormone was transferable by plasma. Similar experiments with intact reticulocytes exposed to physiological concentrations (10(-11) M) of testosterone and 17 beta-estradiol indicated that the plasma factors were the sex steroids. That is, incubation in vitro with testosterone converted female-source reticulocytes to male-type responsiveness to thyroid hormone (inhibition of Ca2+-ATPase activity); incubation with estradiol converted male-source reticulocyte-enriched red cells to female-type responsiveness (stimulation by iodothyronines of membrane Ca2+-ATPase activity). Similar results were obtained when reticulocyte ghosts were incubated with testosterone and 17 beta-estradiol prior to determination of membrane enzyme activity. Etiocholanolone (5 beta-androstan-3 alpha-ol-17-one) and testosterone were equipotent, but 5 alpha-dihydrotestosterone had little activity in this system. Estrone and estradiol were equipotent, but estriol had no permissive effect on the stimulation by iodothyronine of reticulocyte membrane Ca2+-ATPase activity. Expression of thyroid hormone action in vitro on Ca2+-ATPase activity in the rabbit reticulocyte is determined at the membrane level by testosterone and estrogen. The structure-activity relationships of the sex steroids for this membrane action are different than those reported for nuclear actions of the steroids.

Calcium channel blocker inhibition of the calmodulin-dependent effects of thyroid hormone and milrinone on rabbit myocardial membrane Ca2+-ATPase activity

The Ca2+-ATPase activity of rabbit myocardial membranes is stimulated in vitro by L-thyroxine and by milrinone, a bipyridine. These effects are concentration dependent and calmodulin requiring. The calcium channel blockers nifedipine and verapamil have been reported to have anti-calmodulin effects in other assay systems. In this study we have examined the effects of nifedipine and verapamil on rabbit myocardial membrane Ca2+-ATPase activity, in the absence (basal activity) and presence of exogenous L-thyroxine (T4), 10(-10) M, and milrinone, 10(-7) M. Basal enzyme activity was inhibited by a minimum of 10(-6) M nifedipine (IC50 of 3.4 X 10(-5) M) and 10(-5) M verapamil (IC50 of 1.5 X 10(-4) M). Both calcium antagonists inhibited enzyme stimulation by T4 and milrinone, with half-maximal inhibition of T4 and milrinone effects, respectively, at 2.9 X 10(-5) M and 9.0 X 10(-6) M nifedipine and 3.0 X 10(-5) M and 5.2 X 10(-5) M verapamil. The addition of exogenous purified calmodulin, 40 ng/micrograms membrane protein, in the presence of 10(-5) M nifedipine or verapamil restored T4-stimulated enzyme activity. Nifedipine and verapamil, each at a concentration of 10(-6) M, significantly inhibited binding of radioiodinated calmodulin to rabbit heart membranes in vitro. These studies provide evidence that nifedipine and verapamil have an anti-calmodulin effect in this myocardial enzyme system. Through interaction with calmodulin, the channel blockers inhibit thyroid hormone and milrinone stimulation of myocardial membrane Ca2+-ATPase.

Differential Activities of Tolbutamide, Tolazamide, and Glyburide In Vitro on Rabbit Myocardial Membrane Ca2+-Transporting ATPase Activity

At clinically achievable concentrations (10−9 to 5 × 10−6 M), tolbutamide and tolazamide are in vitro inhibitors of Ca2+-transporting ATPase activity in sarcolemma-enriched rabbit myocardial membranes (sulfonylurea ICM, 10−7 M). Thyroid hormone stimulation of this calcium pump-associated enzyme in vitro has been previously reported;5 in our study, this hormonal action was shown to be inhibited by tolbutamide and tolazamide. In contrast to these two sulfonylureas, glyburide (up to 5 × 10−6 M) had no effect on basal or thyroid hormone-stimulable Ca2+-ATPase activity in vitro. Studies of binding of radiolabeled purified calmodulin to heart membranes showed that tolbutamide and tolazamide inhibited this interaction, whereas glyburide had no effect on calmodulin binding. Addition of purified calmodulin (5–40 ng/μg membrane protein) to myocardial membranes incubated with 10−7 M tolbutamide or tolazamide restored Ca2+-ATPase activity and thyroid hormone responsiveness of the enzyme. Inhibition by tolbutamide and tolazamide of myocardial sarcolemmal Ca2+-ATPase is a mechanism by which these two sulfonylureas may at least transiently raise resting sarcoplasmic Ca2+ concentration. This effect of sulfonylureas on Ca2+-ATPase is not expressed in the presence of the benzamide side chain of glyburide. The inhibitory action of certain sulfonylureas on Ca2+-ATPase is mediated by interference of the agents with the binding of calmodulin to cardiac membranes.

Interaction of amiodarone and its analogs with calmodulin

Benzofurans have important actions on the electrical properties of myocardium; the biochemical basis of those actions is not known. Crystallographic examination of these compounds has revealed that benzofurans share structural homologies with the traditional calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene and trifluoperazine. In the present study, the ability of amiodarone, desethylamiodarone, and benziodarone to displace the fluorescent ligand 8-anilino-1-naphthalene sulfonic acid (ANS) from calmodulin, to modulate the fluorescence emission of dansylcalmodulin, and to inhibit the activation by calmodulin of bovine brain cyclic nucleotide phosphodiesterase and human erythrocyte membrane Ca2+-ATPase were investigated at concentrations ranging from 10(-8) to 10(-6) M. These benzofurans displaced ANS from calmodulin with nearly equal efficiency upon forming a 1:1 complex with that protein. Each of these compounds also produced a decreased fluorescence emission of dansylcalmodulin, but with relative efficiencies being desethylamiodarone greater than amiodarone greater than benziodarone. Amiodarone and desethylamiodarone inhibited calmodulin-stimulable phosphodiesterase activity with similar potencies. Amiodarone and benziodarone inhibited calmodulin-stimulable Ca2+-ATPase activity equally, but desethylamiodarone had no effect. The observed differential effects of the amiodarone analogs suggest that calmodulin may possess multiple benzofuran-binding sites that are recognized by specific targets and ligands of this Ca2+-binding protein and that the cellular action of amiodarone and its analogs may reflect calmodulin antagonism.

Retinoic acid inhibits calmodulin binding to human erythrocyte membranes and reduces membrane Ca2(+)-adenosine triphosphatase activity.

Ca2(+)-ATPase activity in human red cell membranes is dependent on the presence of calmodulin. All trans-retinoic acid inhibited human red cell membrane Ca2(+)-ATPase activity in vitro in a concentration-dependent manner (10(-8) to 10(-4) M). In contrast, retinol, retinal, 13-cis-retinoic acid and the benzene ring analogue of retinoic acid did not alter enzyme activity. Purified calmodulin (up to 500 ng/ml, 3 X 10(-8) M) added to red cell membranes, in the presence of inhibitory concentrations of retinoic acid, only partially restored Ca2(+)-ATPase activity. 125I-Calmodulin bound to red cell membranes was displaced by unlabeled retinoic acid (50% reduction at 10(-8) M retinoic acid), as effectively as by unlabeled calmodulin. Another calmodulin-stimulable enzyme, bovine brain cyclic nucleotide phosphodiesterase, was unaffected by retinoic acid. 8-Anilino-1-naphthalene sulfonic acid bound to calmodulin, studied spectrofluorometrically, was not displaced by retinoic acid. Thus, retinoic acid inhibits calmodulin binding to red cell membranes, reducing calmodulin-stimulable Ca2(+)-ATPase activity. Retinoic acid does not directly interact with calmodulin, but rather exerts its effect by interfering with calmodulin access to the membrane enzyme. These effects occur at physiological concentrations of the retinoid.