Inositol phosphates modulate human red blood cell Ca(2+)-adenosine triphosphatase activity in vitro by a guanine nucleotide regulatory protein.

D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] inhibits human red blood cell (RBC) Ca(2+)-stimulable, Mg(2+)-dependent adenosine triphosphatase (Ca(2+)-ATPase) activity in vitro. Because we have previously shown that adrenergic receptors exist on the human mature RBC membrane and can modulate Ca(2+)-ATPase activity, we examined the possibility that a guanine nucleotide regulatory protein (G protein) mediated the Ins(1,4,5)P3 effect. Guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) 10(-4) mol/L also inhibited RBC Ca(2+)-ATPase activity. Pertussis toxin 200 ng/mL blocked the effects of both Ins(1,4,5)P3 and GTP gamma S on Ca(2+)-ATPase activity. In separate studies, pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation was shown to occur in RBC membranes under conditions in which measurements of Ca(2+)-ATPase activity were performed. When Ins(1,4,5)P3 10(-7) mol/L and GTP gamma S 10(-6) mol/L were added to membranes concurrently, their inhibitory actions on the enzyme were additive. At greater concentrations of Ins(1,4,5)P3 (10(-6) to 10(-5) mol/L) and GTP gamma S (10(-4) mol/L), the inositol phosphate reversed the inhibitory effect of GTP gamma S. These observations indicate that the novel effect of Ins(1,4,5)P3 on the activity of a plasma membrane Ca(2+)-ATPase depends at least in part on the action of a pertussis toxin-susceptible G protein.

Structure-activity relationships of milrinone analogues determined in vitro in a rabbit heart membrane Ca(2+)-ATPase model.

The cardiac activity of a series of analogues of the positive inotropic bipyridines amrinone (5-amino-[3,4'-bipyridin]-6(1H)-one) and milrinone (2-methyl-5-cyano-[3,4'-bipyridin]-6(1H)-one) was evaluated in vitro in a rabbit myocardial membrane Mg(2+)-dependent, Ca(2+)-stimulable adenosine triphosphatase (Ca(2+)-ATPase) model and structure-activity relationships were compared for nine closely related derivatives. In the present studies, a 5-bromo analogue of milrinone stimulated myocardial membrane Ca(2+)-ATPase significantly (10(-7) M; P < 0.001 vs control, with 67% of the activity of milrinone), whereas a 2'-methyl-2H-milrinone derivative was inactive. Although amrinone was inactive in this assay, its 2-methyl analogue was stimulatory. However, analogues lacking a 2-substituent (with or without a 5-cyano group) or with the 3-N position blocked by a methyl group did not stimulate myocardial membrane Ca(2+)-ATPase activity. Structural data for these bipyridines show that those with either a 2- or 2'-methyl substituent have a twist conformation, whereas those without are nearly planar. Activity data reveal that those bipyridines with a nonplanar conformation are more active in the Ca(2+)-ATPase assay. Further study of milrinone analogues with a 2'-methyl substituent shows that even though the effect on the twist angle is equivalent to that of 2-methyl substitution, these analogues are less potent. Data for this series reveal that the prerequisites for Ca(2+)-ATPase stimulation include not only a 2-methyl to maintain a twist conformation but also a free 3-N position and a 5-substituent. This model for optimal activity in the myocardial membrane Ca(2+)-ATPase system differs from those proposed for phosphodiesterase enzyme receptor recognition only in the requirement for a nonplanar molecule. We have previously shown that milrinone, but not amrinone, shares structural homology with thyroxine and was able to stimulate myocardial membrane Ca(2+)-ATPase activity in a manner similar to the thyroid hormone. Additionally, milrinone, but not amrinone, was an effective competitor for thyroxine binding to the serum transport protein transthyretin. Analysis of the milrinone-transthyretin crystal complex confirms the structural homology between milrinone and thyroid hormone which is not shared by amrinone. Modeling studies of the binding interactions of milrinone analogues indicate that the 2-desmethylmilrinone analogue, the most inhibitory analogue, lacks the hydrophobic contacts present in milrinone in its transthyretin-bound complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Rabbit skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase activity: stimulation in vitro by thyroid hormone analogues and bipyridines.

Sarcoplasmic reticulum-enriched membranes from rabbit skeletal muscle contained Ca(2+)-ATPase activity which was significantly enhanced (26% increase, P < 0.001) in vitro by physiological concentrations (10(-10) M) of L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3). In contrast, the biologically inactive iodothyronine analogues D-T4 and 3,3',5,5'-tetraiodothyroacetic acid (Tetrac) (10(-10) M) were without effect on enzyme activity. 3,5-Dimethyl-3'-isopropyl-L-thyronine (Dimit), a bioactive analogue, was highly effective as a Ca(2+)-ATPase stimulator, increasing enzyme activity by 43% (P < 0.02 vs. T4 effect). A bipyridine cardiac inotropic agent, milrinone, has been reported to be thyromimetic in a myocardial membrane Ca(2+)-ATPase system, and in concentrations from 10(-10) to 10(-5) M enhanced skeletal muscle SR membrane Ca(2+)-ATPase activity in vitro (P < 0.001). Milrinone analogues which have been previously shown to enhance rabbit myocardial membrane Ca(2+)-ATPase activity, and which have a twist relationship of the pyridine rings, were also striated muscle Ca(2+)-ATPase stimulators. We conclude that (1) striated muscle is a mammalian tissue in which physiological levels of biologically relevant thyroid hormone analogues, particularly Dimit, stimulate Ca(2+)-ATPase activity in vitro by a non-genomic mechanism; (2) cardiac bipyridine analogues which are thyromimetic in vitro in rabbit heart, and which have structural homologies with thyroid hormone, are stimulators of rabbit striated muscle sarcoplasmic reticulum Ca(2+)-ATPase activity.

Inositol phosphates modulate binding of thyroid hormone to human red cell membranes in vitro.

D-Myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and D-myo-inositol 4,5-bisphosphate (10(-6) mol/L) displaced specifically bound L-[125I] T4 from human erythrocyte membranes in vitro by up to 80%. D-Myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1-monophosphate, and D-myo-inositol 1,4-bisphosphate were ineffective in decreasing thyroid hormone binding to membranes. The effect of Ins(1,4,5)P3 on high affinity binding reflected a change in Kd (5.8 x 10(-11) vs. 1.5 x 10(-11) mol/L) and binding capacity (15 vs. 2 fmol/mg membrane protein) in the absence and presence of Ins(1,4,5)P3, respectively. Ins(1,4,5)P3 also displaced T3 from red cell membranes. Thus, selected inositol phosphates regulate the abundance of sites available for binding of thyroid hormone by human red cell membranes. This stereospecific action of inositol phosphates is among several plasma membrane effects recently described for these members of the signal-transducing phosphoinositide pathway.

Thyroid hormone stimulates release of calmodulin-enhancing activity from human erythrocyte membranes in vitro.

1. Thyroid hormone (L-thyroxine, 10(-10) mol/l) incubated in vitro with human erythrocyte membranes induced the release of a soluble calmodulin-like material, the 3':5'-cyclic nucleotide phosphodiesterase-stimulating activity of which was at least six-fold greater than its concentration measured by a specific calmodulin radioimmunoassay. 2. The material had the characteristics of calmodulin in that it stimulated both phosphodiesterase and erythrocyte Ca(2+)-ATPase activities, cross-reacted with and was neutralized by anti-calmodulin antibody, was adsorbed by phenothiazine-Sepharose and was heat-stable. Control supernatant from the incubation of membranes in the absence of thyroxine contained calmodulin, the bioactivity of which was not enhanced beyond that predicted from radioimmunoassay. Subsequent addition of thyroxine did not increase calmodulin bioactivity. Calmodulin-agarose removed calmodulin-enhancing activity from the supernatant. 3. Thus, the enhancing factor(s) appears to interact directly with calmodulin. These observations indicate that thyroid hormone promotes the release from human erythrocyte membranes of a soluble factor (or factors) which binds to calmodulin and significantly increases its bioactivity. This enhancing activity is similar to that of a calmodulin activator described in a rat model of hypertension (S.-L. Huang et al., J Clin Invest 1988; 82: 276-81).

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.

Hexose-specific inhibition in vitro of human red cell Ca(2+)-ATPase activity.

In a concentration-dependent manner (5.5-27.5 mmol/l), D-glucose incubated in vitro with human erythrocyte membranes at 37 degrees C for 1 h inhibited membrane Ca(2+)-ATPase activity by up to 75%. The IC50 was 11 mmol/l. L-Glucose was ineffective, as were 3-O-methylglucose, 2-deoxyglucose, sorbitol and myo-inositol. In contrast, D-fructose decreased Ca(2+)-ATPase activity nearly as effectively as D-glucose and mannose and galactose at 11 mmol/l were less than 50% as effective as D-glucose. Tunicamycin (12 pmol/l), but not 10 mmol/l aminoguanidine, progressively antagonized in vitro the D-glucose effect on the enzyme. Erythrocyte membrane Ca(2+)-ATPase activity may be regulated by glycosylation, rather than nonenzymatic glycation.

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.

Structure-activity relationships of retinoids as inhibitors of calmodulin-dependent human erythrocyte Ca(2+)-ATPase activity and calmodulin binding to membranes.

All-trans retinoic acid displaces the binding of radiolabelled calmodulin to human erythrocyte membranes, and inhibits the activity of plasma membrane Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase; EC 3.6.1.3). This enzyme is dependent upon the action of calmodulin. In this study we explored the structural attributes of the retinoids which confer this ability to inhibit enzyme activity and calmodulin binding. With respect to the fatty acid side-chain, a clear requirement for inhibition is a trans-configuration of the polar end-group. The importance of the ring structure is indicated by the ineffectiveness of polyprenoic acid and a benzene ring retinoid analogue as inhibitors of enzyme activity and calmodulin binding. There was good correlation between the relative potencies of the analogues as enzyme inhibitors and as inhibitors of calmodulin binding. The ability of selected retinoid analogues, at physiological concentrations with respect to all-trans retinoic acid, to inhibit erythrocyte Ca(2+)-ATPase activity and membrane binding of calmodulin underscores the structurally specific effects of these compounds on the interaction of calmodulin with the membrane-bound enzyme.

Effects of caloric restriction and aging on erythrocyte membrane Ca(2+)-ATPase activity in specific pathogen-free Fischer 344 rats.

Dietary caloric restriction extends life span in the Fischer 344 rat. The interaction of aging and caloric restriction was examined at the level of the plasma membrane transport-associated enzymes, Ca(2+)-adenosine triphosphatase (ATPase) and Na,K-ATPase, in the Fischer rat. Animals were in four age groups, ranging from 6.1 to 25.0 months, and were specific pathogen-free (SPF, barrier-raised). Results from male and female animals raised on an ad libitum diet were compared with those from rats that received 60% of the age-specific caloric intake of their ad lib littermates. The responses of erythrocyte membrane Ca(2+)-ATPase activity in vitro to thyroid hormone (L-thyroxine [T4]; 3,5,3'-triiodothyronine [T3]) and to purified calmodulin, a Ca(2+)-binding protein activator of Ca(2+)-ATPase, were measured. Erythrocyte membrane Na,K-ATPase was also compared in the two diet groups, as was plasma glucose. Plasma membrane Ca(2+)-ATPase activity in the absence of added thyroid hormone and calmodulin was significantly reduced in calorically restricted rats (-39%, P less than .001), compared with ad lib-fed animals, and the response was similar in the four age groups aged 6.1, 12.7, 17.0, and 25.0 months. In contrast, pooled (all ages) Ca(2+)-ATPase response in vitro to T4 and to T3 in calorically restricted animals was enhanced compared with the ad lib group (+62% and +58%, P less than .001, respectively). Calmodulin responsiveness of the enzyme was increased by 45% (P less than .001) in calorie-deprived animals, similar to the change in T4 and T3 responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex-dependent inhibition by retinoic acid of thyroid-hormone action on rabbit reticulocyte Ca2(+)-ATPase activity.

The interaction was examined in vitro of retinoic acid and thyroid hormone with rabbit reticulocyte Ca2(+)-ATPase. L-Thyroxine (T4) (0.1 nM) stimulated female-source Ca2(+)-ATPase activity (+21%; P less than 0.03) and inhibited male-source enzyme (-20%; P less than 0.05). Addition of retinoic acid (10 nM-1 microM) did not influence T4-inhibitable male-source Ca2(+)-ATPase, but caused a 52% loss of T4 effect on the female-source enzyme. Incubation of female-source membranes with testosterone caused the enzyme response to T4 and retinoic acid to become that of male-source membranes, and the male-source enzyme response was converted into the 'female' pattern by exposure to 17 beta-oestradiol. We postulate that a membrane-associated sex-steroid-dependent factor imparts a gender-specific interaction of thyroid hormone and retinoic acid on Ca2(+)-ATPase, and that ultimately the factor is shed during erythrocyte maturation.

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.

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.

Analogue-specific action in vitro of atrial natriuretic factor on human red blood cell Ca2+-ATPase activity.

Specific atrial natriuretic factor (ANF) analogues have been found to have inhibitory activity in vitro in a calmodulin-dependent, human red blood cell membrane Ca2+-adenosine triphosphatase (ATPase) model. Studied at 10(-8) to 10(-6) M concentrations, atriopeptin I (residues 127-147 of rat prepro-ANF sequence) and atriopeptin III (residues 127-150) progressively inhibited Ca2+-ATPase activity by up to 20% (p less than 0.001). This degree of inhibition was consistent with activities of other (calmodulin-independent) enzyme inhibitors in this model. Therefore, the C-terminal Phe-Arg-Tyr sequence (residues 148-150) is unnecessary for atriopeptin action on Ca2+-ATPase. Human and rat atrial peptides with amino acids 123-150 were inactive, indicating that the 123-126 sequence (Ser-Leu-Arg-Arg) must be cleaved to activate atriopeptins in this system. Human ANF fragment 129-150 also had no effect on Ca2+-ATPase, defining the importance of residues 127-128 (Ser-Ser) proximal to the disulfide bridge (joining 129 to 145). The addition of purified calmodulin to red blood cell membranes in the presence of inhibitory ANF did not restore Ca2+-ATPase activity to normal levels, indicating that the ANF effect on this enzyme is calmodulin-independent. Atriopeptin I and atriopeptin III had no effect on red blood cell Na+, K+-ATPase activity in vitro. Thus, the structure-activity relationships of ANF analogues in this novel human cell membrane model are highly specific. Although the inhibitory action of ANF analogues on Ca2+-ATPase, a calcium pump-associated enzyme, may be unique to the red blood cell, the calcium dependence of the gluconeogenic effects of ANF in the kidney would be supported by inhibition of this ATPase.

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity.

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.

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.

Early abnormal development of calmodulin gene expression and calmodulin-resistant Ca2+-ATPase activity in avian dystrophic muscle.

We have reported previously that the pectoralis muscle from three month-old dystrophic chickens with signs of myopathy exhibits increased calmodulin content, elevated calmodulin-specific mRNA (Biochem. Biophys. Res. Commun. 137:507-512, 1986), and reduced sarcoplasmic reticulum (SR) Ca2+-ATPase activity in response to calmodulin exposure in vitro (Clin. Res. 34: 725A, 1986). To determine the early time sequence for development of these abnormalities, we have studied muscle from embryos and post-hatched chickens at various ages. Quantitated by dot blot analysis, there was an approximate two-fold increase in calmodulin-specific mRNA in dystrophic muscle as early as 13 days ex ovo which was maintained throughout development up to three months ex ovo. Similarly, Ca2+-ATPase activity measured in SR membranes from chickens as early as 13 days post-hatch was also found to be resistant to stimulation in vitro by exogenous calmodulin, whereas the enzyme from normal muscle was calmodulin-stimulable. These findings suggest that the genetic lesion expressed in the avian dystrophic animal model involves the loss of normal control of intracellular calcium metabolism early in the maturation of the affected musculature and prior to appearance of disease signs.

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.

Action of erythropoietin in vitro on rabbit reticulocyte membrane Ca2+-ATPase activity.

The mechanism of action of erythropoietin is thought to require specific interaction with the target cell surface and involve alteration of cellular calcium metabolism. Using the rabbit reticulocyte membrane as a model of the immature red cell membrane, we investigated the effects of human recombinant erythropoietin on membrane Ca2+-ATPase (calcium pump) activity in vitro. Erythropoietin in a concentration range of 0.025 to 3.0 U/ml progressively decreased membrane Ca2+-ATPase activity by up to 64% (P less than 0.01). These concentrations have been shown by others to stimulate in vitro erythroid growth. The action of erythropoietin on reticulocyte Ca2+-ATPase required an incubation time of 1 h before enzyme assay for maximum effect and was neutralized by antierythropoietin antiserum. Other nonhemopoietic growth factors (epidermal growth factor, insulin) had no effect in this assay. Ca2+-ATPase activity of membranes prepared from rabbit mature red blood cells was not inhibited by erythropoietin. The novel effect of erythropoietin on reticulocyte membrane Ca2+-ATPase activity is a mechanism by which erythropoietin can influence cellular Ca2+ metabolism.

Donor age-dependent decline in response of human red cell Ca2+-ATPase activity to thyroid hormone in vitro.

The effect of increasing donor age on the susceptibility of human red blood cell Ca2+-ATPase activity to stimulation in vitro by thyroid hormone was studied in 26 normal subjects, aged 15-81 yr. Basal enzyme activity (no added thyroid hormone) was unaffected by donor age. Group analysis, young (less than or equal to 50 yr) vs. elderly (greater than 60 yr old), revealed a 23% decrease in responsiveness of the enzyme to L-T4 (P less than 0.001). Regression analysis confirmed an age-dependent decline in thyroid hormone stimulability of Ca2+-ATPase [r = -0.42 (T4 effect) and -0.38 (T3 effect); P less than 0.01]. Red cell membrane Na,K-ATPase activity was not affected by donor age. Plasma T4 and T3 concentrations in these normal subjects also did not change with age. Possible contributions of the following mechanisms to this age-correlated change in enzyme activity were examined: altered responsiveness to calmodulin of membrane Ca2+-ATPase; membrane content of endogenous calmodulin, endogenous plasma T4 and T3 concentrations, and plasma glucose concentrations. Calmodulin responsiveness is required for iodothyronine action on the enzyme, but the calmodulin responsiveness of cells from elderly donors was not significantly different from that of cells from younger donors (P greater than 0.10). There was no relationship between membrane immunoassayable calmodulin and donor age or membrane calmodulin and Ca2+-ATPase activity. There were positive correlations between donor plasma T4 level and basal enzyme activity (P less than 0.05) and between donor plasma T3 concentration and hormone-responsive Ca2+-ATPase (P less than 0.01), but these did not contribute to the age effect. Plasma glucose previously was found to modulate red cell Ca2+-ATPase activity, but did not correlate with decreased hormone responsiveness of the enzyme in elderly donors. In conclusion, we found that the susceptibility of human red cell Ca2+-ATPase to in vitro thyroid hormone stimulation declined significantly with advancing donor age. Several possible calmodulin-dependent mechanisms for this age-dependent change were excluded, and thus, we postulate that the altered hormone sensitivity of the enzyme is membrane phospholipid mediated.