Loss of glucose transport in developing avian red cells.

Although red cells are generally associated with significant glucose transport and dependence on glycolysis, the mature red cells of some species (e.g. pig) show very low glucose transport. The generally low level of glucose transport in mature mammalian red cells is the result of maturational development, since it has been shown that even in red cells which have negligible glucose transport (e.g. pig red cells) the corresponding reticulocytes have significant glucose transport activity. The reticulocytes of the chicken, however, show minimal glucose transport activity. But this also is the result of maturational development, since chicken bone marrow red cells do transport glucose which diminishes upon cell maturation in vitro. The erythroblast chicken cell line, HD3, has high glucose transport activity which is lost upon induction to the red cell phenotype. Growing HD3 cells have much higher levels of transport than native chicken bone marrow cells and this is associated in part with elevation of glucose transporter (GLUT) mRNAs as a consequence of the expression of the v-erbA and v-erbB oncogenes. Both native bone marrow red cells and HD3 cells, when incubated in vitro under conditions where maturation occurs, show substantial losses of GLUT mRNA and GLUT proteins. To assess whether the inducers of maturation (hemin and butyrate) affect only the normally expressed GLUTs, chicken GLUT3 expressed from a different promoter was introduced into the HD3 cell by retroviral infection. Both the endogenous and exogenous transporters were lost upon cell differentiation and maturation, leaving a cell with low glucose transport activity. Conversely, in growing cells, butyrate had a pronounced effect on the elevation of the GLUT3 mRNA, especially on the exogenous GLUT3 mRNA, and elevated glucose transport prior to differentiation. These results are consistent with the conclusion that chicken red cell development involves a requirement to reduce glucose transport activity. The near absence of glucose transport in the embryonic chicken red cell is thus due to a loss of this transporter during early development which occurs at an earlier developmental stage in the chicken red cell than in the mammalian red cell.

Alteration in liver plasma membrane phospholipids and protein kinase activities during the development of chick embryo.

The changes in phospholipid compositions, membrane fluidity and protein kinase A, protein kinase C, tyrosine and casein kinase activities in chick embryo liver plasma membranes during development have been investigated. The percentage participation of sphingomyelin increased while that of phosphatidylserine decreased during chick embryo development. The alterations in membrane sphingomyelin accompanied an increase of steady-state fluorescence anisotropy (rs) of membrane bilayer. Regression analysis indicated positive linear correlations between the percentage participation of sphingomyelin in total membrane phospholipids and (i) protein kinase C (r = 0.903); (ii) casein kinase (r = 0.936); (iii) protein kinase A (r = 0.850); (iv) tyrosine kinase (r = 0.960) activities. We suggest that sphingomyelin might be an specific activator for all types of protein kinase activities investigation.

Casein and tyrosine kinase activities of developing chick embryo liver. Effect of triiodothyronine.

Cytosol cAMP-independent quercetin-inhibited protein kinase of developing chick embryo liver was measured at three embryonic ages (days 12, 14 and 18) in the presence of casein and poly (Glu-Na, Tyr) 4:1 as substrates. In the early embryonic stages the tyrosine kinase was almost as active as casein kinase, but on day 18 the tyrosine phosphorylation was only 25% of the casein phosphorylation. Both kinase activities strongly increased by the end of embryonic development: 7-fold with casein and 2.6-fold with poly (Glu-Na, Tyr) 4:1. Triiodothyronine caused twice the stimulation of casein and tyrosine phosphorylation on day 12, but had no effect on day 18.

Effect of membrane phospholipid composition and fluidity on rat liver plasma membrane tyrosine kinase activity.

1. The effect of membrane phospholipid composition and fluidity on tyrosine kinase activity was investigated in rat liver plasma membranes. 2. The phospholipid composition has been modified by in vitro enrichment of plasma membranes with different phospholipids in the presence of lipid transfer proteins and by partial delipidation with exogenous phospholipases A2, C and D and subsequent enrichment with phosphatidylglycerol. 3. Phosphatidylglycerol and dioleoylglycerophosphocholine caused dramatic elevation of this activity, while phosphatidylserine and phosphatidylethanolamine were less effective. Enrichment with dipalmitoylglycerophosphocholine and sphingomyeline reduced tyrosine kinase activity.

Effect of a sunflower oil-supplemented diet on protein kinase activities of rat liver plasma membranes.

1. The effect of a sunflower oil-enriched diet on plasma membrane-bound protein kinase C, protein kinase A, casein and tyrosine kinase activities was studied. 2. The diet induced an increase in the content of linoleic acid and a decrease in the content of palmitic acid. The anisotropy parameter (rs) of the fluorescence probe DPH and SDPH decreased strongly in the experimental group. 3. Protein kinase C was stimulated more than two times. Tyrosine kinase, protein kinase A and casein kinase activities were increased by 65, 57 and 40%, respectively. 4. We suggest that a more fluid lipid environment favours higher plasma membrane-bound protein kinase activities.

Sphingomyelin-metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterol-supplemented diet.

The effect of cholesterol-supplemented diet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcholine:ceramide-phosphocholine transferase, and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10, 20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcholine:ceramide-phosphocholine transferase, and r = 0.998 for protein kinase C and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide.

Effect of triiodothyronine on cAMP-dependent and cAMP-independent protein kinase activities in developing chick embryo liver.

1. Changes in liver cytosol cAMP-dependent kinase and cAMP-independent growth-related quercetin-inhibited casein kinase activities during chick embryo development were studied. 2. Both kinase activities were found to increase continuously during the experimental period. 3. Upon treatment of embryos with triiodothyronine, an activation of cAMP-dependent kinase A and cAMP-independent casein kinase was observed which was most pronounced on days 12 and 14.

Phospholipid-dependence of rat liver plasma membrane protein kinase activities--a new approach.

The influence of the phospholipid composition and fluidity on protein kinase A and protein kinase C activities in rat liver plasma membranes was studied. We observed that enrichment of membranes with phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and dioleoylphosphatidylcholine caused activation of both protein kinases. Phosphatidylglycerol was found to be most effective activator. The enrichment of plasma membranes with dipalmitoylphosphatidylcholine and sphingomyelin led to decrease in protein kinase A and C activities. The stimulatory effect of phosphatidylglycerol was confirmed in plasma membranes pretreated with exogenous phospholipases A2, C and D, and subsequently enriched with phosphatidylglycerol. We suggest that besides the specific presence of definite phospholipids protein kinases A and C require a more fluid membrane lipid bilayer to display an optimal activity.

Protein phosphorylation in erythroid cell development.

Changes in the phosphorylation of proteins during erythroid cell development have been investigated by assaying the activity of three protein kinases in circulating reticulocytes, and dividing and non-dividing erythroblasts obtained from the bone marrow of anaemic rabbits. Kinase activities decreased during erythroid cell development, but protein phosphorylation was generally limited by substrate availability rather than enzyme activity. Using permeabilized cells some changes in the patterns of proteins phosphorylated by [gamma-32P]ATP were observed during erythroid cell development.

Forskolin as an activator of adenylate cyclase complex of differentiating erythroid bone-marrow cells.

The study concerns the manner in which forskolin activates the adenylate cyclase system of differentiating rabbit bone-marrow erythroid cells. The results presented show that forskolin can stimulate the basal activity of adenylate cyclase in the absence of guanine nucleotides in an in vitro assay containing plasma membranes derived from both dividing and non-dividing cells. In the presence of guanine nucleotide the activation of adenylate cyclase by forskolin is increased, but the effect is not additive and is abolished by the beta-thio analogue of GDP. Addition of forskolin to cell cultures causes a transient increase in the activity of adenylate cyclase, which is maximal by 30 minutes and disappears within 24 hours. The conclusion is made that the effect of forskolin on adenylate cyclase complex of differentiating rabbit bone-marrow erythroblasts is similar to the effect of erythropoietin (Bonanou-Tzedaki et al., 1986) and is transdusing via stimulatory guanine nucleotide-regulatory protein.

Adenylate cyclase system of differentiating erythroid cells.

The review provides a survey of current knowledge about the changes in hormone-sensitive adenylate cyclase complex of erythroid cells. The basal enzyme activity decreases continuously during differentiation and maturation. Guanine nucleotides (GTP and GMP-P (NH)P) increase the adenylate cyclase activity of both early and late rabbit bone marrow erythroblasts. The stimulating effect of the beta 2-adrenergic drugs such as L-isoprenaline is limited to the immature cells. L-noradrenaline, a beta 1-agonist is inactive. The lack of response of non-dividing rabbit erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors during differentiation, but to a decrease in the effectiveness of the coupling between the components of the system: receptor-guanine nucleotide regulatory protein-catalytic subunit. Prostaglandins E1 and E2 consistently enhance adenylate cyclase activity of erythroblasts on different stages of development. Erythropoietin (0.2 U/ml) causes a transient increase in the activity of adenylate cyclase, which is maximal by 20 min incubation of the cells in the presence of the hormone and disappears within 4 hours. The magnitude of the response to erythropoietin depends on the stage of erythroid cell development and is inverse related to the extent of previous hormonal stimulation of the cell.

Independent activation of adenylate cyclase by erythropoietin and isoprenaline.

The possibility that catecholamines modulate the erythropoietin-induced increase in production of cyclic AMP was investigated by examining the effect of erythropoietin and/or L-isoprenaline on the activity of the plasma membrane adenylate cyclase of anaemic rabbit bone marrow erythroblasts. Membranes isolated from cells cultured in the presence of both hormones exhibited both the transient stimulation of basal activity characteristic of erythropoietin action and the loss of the in vitro response to L-isoprenaline, concomitant with the loss of beta-adrenergic receptors, characteristic of L-isoprenaline stimulation. The presence of erythropoietin during cell culture with L-isoprenaline had no effect on the desensitization or number of beta-adrenergic receptors. The stimulation of adenylate cyclase by erythropoietin was observed also in the presence of the beta-antagonist propranolol, when both were added either to whole cells or to isolated membranes. We conclude that these two hormones activate adenylate cyclase independently of each other, via different receptors, with little evidence of cross-modulation.

The effect of erythropoietin on the adenylate cyclase activity of rabbit bone marrow erythroblasts.

The involvement of adenylate cyclase in the response elicited by erythropoietin was investigated in fractionated erythroblasts obtained from anaemic rabbit bone marrow. Addition of 0.2 U/ml erythropoietin to cell cultures caused a transient increase in the activity of plasma membrane adenylate cyclase, which was observed within 5 minutes, was maximal by 20 minutes and disappeared within 4 hours. The magnitude of the response to hormonal stimulation depended on the stage of erythroid cell development and was greater in the more immature cells. Erythropoietin could also stimulate the basal activity of adenylate cyclase in an in vitro assay containing plasma membranes of immature, but not mature, erythroid cells. The degree of activation was hormone-concentration dependent, was maximal at 0.2-0.5 U/ml erythropoietin (5-12 nM) and was observed in the absence of exogenous guanine nucleotides. The in vitro effect of erythropoietin, however, was abolished by GDP (S) and extensive washing of the membranes made hormone action GTP-dependent. The ability of the hormone to stimulate adenylate cyclase activity in vitro was inversely related to the extent of hormonal stimulation in vivo. This desensitization was observed within 20 minutes and persisted for many hours. It is suggested that erythropoietin activates the adenylate cyclase of immature erythroblasts via a receptor and a guanine nucleotide-binding protein with high affinity for GTP.

Classification of beta-adrenergic subtypes in immature rabbit bone marrow erythroblasts.

The beta-adrenergic receptors of immature rabbit bone marrow erythroid cells (proerythroblasts and basophilic erythroblasts) were identified. [125I]iodocyanopindolol bound to membrane preparations derived from these erythroblasts in a rapid, reversible and saturable manner. Scatchard analysis of binding data revealed a single class of binding sites (Hill coefficient of 0.954) with an apparent equilibrium dissociation constant (Kd) of 8 pM, and a density of binding sites (Bmax) of 1.53 pM/10(6) cells, corresponding to 920 receptors per cell. The binding of [125I]iodocyanopindolol was inhibited stereospecifically by concentrations of (-)-propranolol 2 orders of magnitude lower than by the (+)-isomer. Only L-isoprenaline and L-adrenaline activated the adenylate cyclase of immature rabbit erythroblasts, while L-noradrenaline, a beta 1-adrenergic agonist, was inactive. The order of potency of different agonists for displacement of bound [125I]iodocyanopindolol was: isoprenaline greater than adrenaline greater than noradrenaline with respective EC50 (concentration required for half maximal inhibition of binding) of 7.9 X 10(-7) M, 1.5 X 10(-5) M and 7.9 X 10(-5) M. This agonist potency series did not change with differentiation of rabbit bone marrow erythroblasts. The inhibition of specific [125I]iodocyanopindolol binding to immature cells by beta 1- and beta 2-selective drugs (noradrenaline, practolol, procaterol and butoxamine) resulted in linear Hofstee plots. The inhibition curves obtained with procaterol and butoxamine, with apparent Kd values of 3.1 X 10(-9) M and 4.9 X 10(-9) M, further evidence that the high-affinity binding sites correspond to a homogeneous beta 2-receptor subtype.

Stimulation of the adenylate cyclase activity of rabbit bone marrow immature erythroblasts by erythropoietin and haemin.

The effect of two agents of erythroid cell differentiation on the adenylate cyclase activity of fractionated rabbit bone marrow erythroblasts has been investigated. Addition of 0.2U/ml erythropoietin to cell cultures causes a transient increase in the activity of plasma membrane adenylate cyclase, which is maximal by 20 min and disappears within 4 h. The magnitude of the response to hormonal stimulation depends on the stage of erythroid cell development and is greater in the more immature cells. Addition of 50 microM haemin to cultures of erythroblasts also causes an increase in the activity of adenylate cyclase, which differs from the effect of erythropoietin in kinetics and specificity of target cells. With immature cells the haemin-induced stimulation starts after the first hour and continues to increase up to 20 h of culture. Erythropoietin but not haemin can stimulate the basal activity of adenylate cyclase in an in vitro assay containing plasma membranes of immature erythroid cells. The degree of activation depends on the concentration of erythropoietin and is maximal with 0.2-0.5 U/ml hormone (5-12 nM). In the presence of guanine nucleotides the activation of adenylate cyclase by erythropoietin is increased further but the effect is not additive. With respect to the basal and the guanine-nucleotide-stimulated activities of adenylate cyclase erythropoietin acts differently from the beta-agonist l-isoprenaline. The in vitro effect of erythropoietin is abolished by the beta-thio analogue of GDP, GDP[beta S], and extensive washing of membranes makes hormone action GTP-dependent. The stimulation of adenylate cyclase by the addition of erythropoietin to the reaction mixture is inversely related to the extent of previous hormonal stimulation of the cells from which the membranes were prepared. This loss of hormonal responsiveness is due to desensitization or receptor down-regulation and persists for up to 20 h. We conclude that in immature erythroblasts erythropoietin acts via a receptor and a guanine nucleotide-binding protein with high affinity for GTP (EC50 less than 10 nM), whereas haemin appears to activate adenylate cyclase indirectly, as a consequence of progressive perturbations of the plasma membrane.

Characteristics of the beta-adrenergic adenylate cyclase system of developing rabbit bone-marrow erythroblasts.

After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells.

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having beta 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of beta-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.

Characteristics of the adenylyl cyclase system of differentiating rabbit bone marrow erythroblasts.

Changes in the cellular content of cyclic AMP and in the activities of adenylyl cyclase, cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinases during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic erythroblasts, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Adenylyl cyclase activity was found to decrease continuously as the cells developed, from approx. 180 pmoles cyclic AMP formed/mg of protein/20 min in proerythroblasts to 10 pmoles in circulating reticulocytes. The proerythroblasts were the richest cells in cyclic AMP which is present at a cellular concentration of approx. 1.4 microM. In basophilic cells the cyclic AMP content was about 80% lower than in proerythroblasts. No further changes in cyclic AMP levels were observed after the final cell division. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity occurred. In polychromatic cells there was a further drop in phosphodiesterase activity and after the last cell division the enzyme activity was constant and very low. Both cytosolic cyclic AMP-binding capacity and cytosolic cyclic AMP-dependent protein kinase activity decreased in dividing rabbit bone marrow erythroblasts when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative but only quantitative changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent Kd for the interaction of binding proteins with cyclic AMP was 4 . 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and their affinity for cyclic AMP. The phosphorylation of rabbit erythroblast plasma membrane proteins by membrane-associated protein kinase(s) was found to be cyclic AMP-dependent in dividing cells during the early stages of differentiation. When the erythroid cells reach the non-dividing stage in their development, autophosphorylation of membrane ghosts was no longer stimulated by cyclic AMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Characteristics of the adenylate cyclase system of differentiating rabbit bone marrow erythroblasts.

Adenylate cyclase activity of rabbit bone marrow erythroblasts decreased continuously as the cells developed. The proerythroblasts were the richest cells in cAMP. No changes in cAMP levels were observed after the final cell division. cAMP-phosphodiesterase activity declined rapidly during the early period of erythroid cell development and remained constant but extremely low after condensation of the nucleus. Both cytosolic cAMP-binding capacity and cAMP-dependent protein kinase activity decreased in dividing erythroblasts when calculated in terms of cell number but remained constant per cell volume. Membrane-associated protein kinase was found to be cAMP-dependent only in the dividing cells. The adenylate cyclase activity of both early and late erythroblasts was stimulated by GTP and p (NH)ppG, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing cells. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind 125I-iodohydroxybenzylpindolol with equal affinities. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p (NH) ppG during erythroblast development was observed.