John N. Loeb

The Influence of Hyperthyroidism and Hypothyroidism on the β-Adrenergic Responsiveness of the Turkey Erythrocyte

The mechanisms responsible for altered adrenergic tone in hyperthyroidism and hypothyroidism are not fully understood. To investigate these mechanisms, the beta-adrenergic receptor-cyclic AMP complex of the turkey erythrocyte was studied among groups of normal, hyperthyroid, and hypothyroid turkeys. In erythrocytes obtained from hypothyroid turkeys, there were fewer beta-adrenergic receptors than in normal cells as determined by the specific binding of [(125)I]iodohydroxybenzylpindolol, as well as associated decreases both in catecholamine-responsive adenylate cyclase activity and in cellular cyclic AMP content. In contrast, erythrocytes obtained from hyperthyroid turkeys contained the same number of beta-receptors and had the same catecholamine-responsive adenylate cyclase activity as cells from normal birds. Other characteristics of the beta-receptors in cells from hyperthyroid birds were indistinguishable from those present in normal erythrocytes. However, within the range of circulating catecholamine concentrations, 5-50 nM, the erythrocytes of the hyperthyroid turkeys generated substantially more cyclic AMP after exposure to isoproterenol than did normal cells. These results suggest that thyroid hormone affects beta-receptor-cyclic AMP interrelationships in the turkey erythrocyte by two distinct mechanisms: (a) In hypothyroidism, both beta-receptors and catecholamine-dependent cyclic AMP formation are coordinately decreased; (b) in hyperthyroidism, beta-receptors are unchanged but there is an amplification of the hormonal signal so that occupation of a given number of receptors at physiological concentrations of catecholamines leads to increased levels of cyclic AMP.

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte. Quantitative relation to ligand binding and cyclic AMP generation.

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K+ concentration on both [3H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between beta-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [3H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K+ concentrations, and at each K+ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K+ concentrations. In contrast to basal potassium influx, which is 50-70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K+, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to beta-adrenergic receptor occupany over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy beta-adrenergic receptor sites function independently of each other. Analysis of the relation of catecholamine-dependent potassium transport to the number of beta-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.

Differential sensitivity of nuclear and mitochondrial DNA synthesis to suppression by cortisone treatment

Abstract The administration of cortisone to young rats is known to result in a prompt suppression of liver DNA synthesis. The present study demonstrates that this suppression represents a selective inhibition of thymidine incorporation into nuclear DNA, and that after a single dose of cortisone to weanling animals the ratio of thymidine incorporated into nuclear DNA to that incorporated into mitochondrial DNA falls to 10–15 % of the ratio in control animals. The persistence of mitochondrial DNA synthesis in these animals despite nearly total suppression of nuclear DNA synthesis provides another example of mitochondrial autonomy. In older rats incorporation of thymidine into nuclear DNA is shown to be considerably less sensitive to inhibition than it is in more rapidly growing animals; even in the larger animals, however, it continues to be more easily suppressed than incorporation into mitochondrial DNA.

Stimulation of glucose transport in Clone 9 cells by insulin and thyroid hormone: role of GLUT-1 activation

Thyroid hormone (T3) and insulin are both shown to stimulate glucose transport in Clone 9 cells, a rat liver cell line in which the utilization of glucose is limited by transport rate and in which only the GLUT-1 transporter isoform is expressed. Pre-treatment of these cells with T3 moreover substantially enhances the stimulatory effect of insulin such that at maximally effective hormone concentrations the effects of T3 and insulin on glucose transport are more than additive and indeed nearly multiplicative, suggesting that the mechanisms mediating the enhancement of glucose transport differ between the two hormones. Cell surface biotinylation followed by Western-blot analysis of plasma membrane fractions showed that the stimulatory effects of T3 and insulin on glucose transport, whether acting singly or in combination, exceed the attendant increases in the abundance of GLUT-1 in the plasma membrane. It is suggested that activation of GLUT-1 molecules pre-existing in the plasma membrane plays a major role in mediating the stimulatory effects of T3 and insulin on glucose transport in this cell line.

Potassium fluxes in the rat reticulocyte. Ouabain sensitivity and changes during maturation.

K+ turnover is markedly enhanced in the rat reticulocyte, both influx and efflux rates being increased by factors of approximately 3 over the corresponding rates in adult cells. These accelerated fluxes are observed despite the absence of any appreciable change in intracellular K+ concentration during the course of maturation. Qualitative characteristics of the active transport process for K+ influx appear to be identical in reticulocytes and mature erythrocytes with regard both to K+ sensitivity, and to ouabain sensitivity as a function of external K+ concentration. The number of ouabain binding sites per unit volume of cells, however, is increased by a factor of approximately three in the reticulocyte and thus correlates well with the observed degree of enhancement of active K+ influx in these cells. Half-maximal rates of ouabain-sensitive K+ influx are observed at external K+ concentrations well below 1 mM for both reticulocytes and mature erythrocytes. It is concluded that the enhanced rate of K+ accumulation in the reticulocyte can be quantitatively attributed to an increased number of pump units which are qualitatively identical to those in the mature cell, and which function at a near-maximal rate at the ambient K+ concentration present in normal rat plasma.

Free and membrane-bound ribosomes in regenerating rat liver

Abstract There has heretofore been little agreement concerning the validity of earlier conjectured shifts in the distribution of free and membrane-bound ribosomes during the precess of hepatic regeneration. In order to provide a quantitative test of these conjectures a simple double-isotope technique is here described which permits a detailed comparison of free and membrane-bound ribosomes isolated from “co-homogenates” of livers from partially hepatectomized and sham-operated animals. It is shown that 48 h after partial hepatectomy, a time at which liver cell proliferation is proceeding at a maximal rate, there is indeed a striking shift in ribosome distribution resulting in about a 40% rise in the proportion of free ribosomes. Over the same interval the absolute concentration of hepatic ribosomes increases markedly with the result that the total number of free ribosomes per unit mass of regenerating tissue rises to nearly double that in normal liver. The observed shift to favor a substantial increase in the concentration of free ribosomes is consistent with an enhanced need in regenerating liver for the synthesis of proteins destined for intracellular use. It is suggested that the method described here, which permits sensitive and precise comparison of the distribution of free and membrane-bound ribosomes in paired tissue samples, may find useful application in other systems as well.

Beta-Adrenergic Receptors and Isoproterenol-stimulated Potassium Transport in Erythrocytes from Normal and Hypothyroid Turkeys: QUANTITATIVE RELATION BETWEEN RECEPTOR OCCUPANCY AND PHYSIOLOGIC RESPONSIVENESS

We have previously reported that in hypothyroid turkeys the number of beta-adrenergic receptors in intact erythrocytes is reduced by approximately 50% without any changes in the affinity of the receptor for the agonist, isoproterenol. In view of the physiological action of the catecholamines to stimulate bidirectional ion fluxes in these cells, we have now examined the possibility that the decrease in beta receptor number might be associated with concomitant changes in catecholamine-dependent potassium ion transport. Hypothyroid turkey erythrocytes display decreased sensitivity to isoproterenol-stimulated potassium influx. Half-maximal stimulation of potassium influx occurs at 9.2+/-1.7 nM in hypothyroid cells as opposed to only 3.8+/-0.4 nM in normal cells (P < 0.005). A maximal stimulatory concentration of isoproterenol (100 nM) leads to the same increment in ion flux in erythrocytes from hypothyroid and normal turkeys. Analysis of the quantitative relationship between isoproterenol concentration, receptor occupancy, and associated effects upon potassium influx shows that at low levels of isoproterenol, where occupancy is linear with agonist concentration, occupation of a given number of beta receptors leads to a stimulation of potassium transport that is identical in erythrocytes from normal and hypothyroid turkeys. Thus, decreased sensitivity to catecholamine-stimulated potassium transport in hypothyroidism can be attributed to the decrease in receptor number and the resulting two- to threefold higher isoproterenol concentration required for occupancy of the same number of beta receptors. Once a single receptor is occupied, however, the more distal components of the sequence of events mediating the physiological response to beta-adrenergic agonists in the hypothyroid cell function as they do under normal circumstances. It would appear, therefore, that the decrease in sensitivity to isoproterenol-dependent ion flux in the hypothyroid turkey erythrocyte can be accounted for solely by the decrease in receptor number. These changes are shown to occur in the absence of any modifications in the number of Na(+)-K(+) ATPase effector units per cell.

Effects of Cortisone on Thymidine Incorporation by Various Nonlymphoid Tissues of the Weanling Rat

Summary Acute effects of both high and low doses of cortisone upon the incorporation of radioactive thymidine have been examined in a variety of non-lymphoid tissues of the weanling rat. It is shown (a) that thymidine incorporation by different tissues varies over an enormous range in its sensitivity to suppression by cortisone administration, and (b) that at low doses of hormone all the tissues studied are clearly separable into two distinct classes: those in which cortisone produces a profound suppression of thymidine incorporation, and those which are completely unresponsive to the hormone. The pattern of response observed thus far suggests the generalization that thymidine incorporation is particularly susceptible to suppression in tissues which are composed of stable cell populations and in which DNA synthesis is primarily a concomitant of new cell accretion (“growth”) rather than one of cell renewal. The phenomenon of cortisone-induced suppression of thymidine incorporation in individual tissues sensitve to low doses of hormone may provde a convenient model for studies of biochemical mechanisms underlying the well-known suppression of somatic growth which occurs at comparable doses both in experimental animals and in children.

Leucine and phenylalanine incorporation by rat liver microsomes containing endogenous template RNA: A kinetic study of the effect of polyuridylic acid

Abstract Amino acid incorporation by rat liver microsomes in vitro is accompanied by a proportional loss of messenger RNA. The present study shows that in the presence of saturating levels of polyuridylic acid the rate of polyuridylic acid-directed phenylalanine incorporation at any given time is linearly related to the cumulative amino acid incorporation due to natural messenger RNA. A kinetic analysis of the data strongly supports the hypothesis that phenylalanine incorporation by microsomes becomes susceptible to stimulation by polyuridylic acid only after the loss of endogenous template RNA, and hence that, under carefully controlled conditions, polyuridylic acid susceptibility may be used as a valid measure of the number of messenger-free ribosomes present.

Kallikrein Inhibitory Capacity of α2-Macroglobulin Subunits

Summary Human α2-macroglobulin can be dissociated by dialysis against 3 M urea into two 11S subunits which remain stable under physiologic conditions. These subunits retain kallikrein inhibitory capacity albeit at a reduced level when compared with the native protein. They furthermore protect bound kallikrein from further inhibition by native α2-macroglobulin. Bound kallikrein can not be dissociated from either the subunits or the native protein by Sephadex G-200 gel filtration.