Joseph Segal

Specific Binding Sites for Triiodothyronine in the Plasma Membrane of Rat Thymocytes CORRELATION WITH BIOCHEMICAL RESPONSES

As a prerequisite to studies of whether the plasma membrane of the rat thymocyte contains specific, saturable binding sites for the thyroid hormone 3,5,3-triiodothyronine (T(3)), a method was developed for the isolation of a plasma membrane fraction from these cells. As judged from both electron microscopic and marker enzyme studies, the fraction was composed principally of plasma membrane vesicles, was free of nuclear contaminants, and was only slightly contaminated with other subcellular components. At 37 degrees C and pH 7.4, binding of [(125)I]T(3) by the fresh membrane preparation was rapid, reaching a maximum at 5 min and then declining with time, so that by 60 min binding was virtually nil. Decreased binding with time was due to a loss of functional binding sites, but did not reflect desensitization, since the decrease in binding activity with time was independent of the presence or absence of T(3). Scatchard analysis of saturation studies revealed the presence of two binding sites, one with an apparent dissociation constant (K(d)) of 0.95 nM and a maximum capacity of 5.3 x 10(10) sites/100 mug protein, and the other with an apparent K(d) of 25 nM and a binding capacity of 1.4 x 10(12) sites/100 mug protein. Measurement of the ability of several thyronine analogues to inhibit the binding of [(125)I]T(3) revealed the following rank order of potency: l-T(3) > l-T(4) > d-T(3) = d-T(4) > l-3,5-T(2) > rT(3) > d,l-thyronine. Binding of T(3) was inhibited by the omission of calcium from the medium or by the addition of the beta adrenergic antagonist alprenolol. As judged from studies of the lower affinity binding site, these manipulations decreased the affinity, but not the number, of binding sites for T(3). The relative potencies of thyronine analogues to inhibit the binding of [(125)I]T(3) were generally parallel to their previously reported potencies in stimulating the uptake of the sugar analogue 2-deoxy-glucose (2-DG) in intact rat thymocytes in vitro. Further, the inhibition of T(3)-binding produced by l-alprenolol or by excluding calcium from the medium resembled the previously reported inhibition that these manipulations produce with respect to T(3)-induced enhancement of 2-DG uptake. These findings suggest that the binding sites for T(3) present in the plasma membrane of rat thymocytes act as functional receptors linked to the stimulation of 2-DG uptake that T(3) induces in these cells.

Stimulation by Triiodothyronine of the In Vitro Uptake of Sugars by Rat Thymocytes

Studies were conducted to ascertain the in vitro effect of 3,5,3-triiodothyronine (T(3)) on the accumulation of the glucose analogue, 2-deoxyglucose (2-DG), by thymocytes freshly isolated from weanling rats. At a concentration of 1 muM, T(3) stimulated the 15-min uptake of (3)H-2-DG after cells had been exposed to T(3) for only 30 min. Significant stimulation of 2-DG accumulation was produced by 1 nM T(3), with increasing stimulation at doses ranging up to 10 muM. T(3) did not alter the fraction of accumulated 2-DG that was phosphorylated, and kinetic studies indicated that its effect was associated with a significant increase in the apparent V(max) of 2-DG accumulation, but not the apparent K(m). T(3) also enhanced the accumulation by thymocytes of the nonmetabolized glucose analogue, 3-O-methylglucose (3-O-MG), an effect that was evidently the result of an increase in 3-O-MG transport into the cell, because it was seen in cells incubated with (3)H-3-O-MG for only 30 s. The proportionate increase in 2-DG accumulation produced by T(3) was not altered by preincubating cells with concentrations of puromycin or cycloheximide sufficient to reduce [(3)H]-leucine incorporation by 95%, and T(3) over a period of >2 h had no effect on [(3)H]leucine incorporation itself. These results indicate that T(3) stimulates the uptake of sugars in rat thymocytes in vitro by an effect on their inward transport. The promptness of the effect and its failure to be inhibited during profound inhibition of protein synthesis further indicate that this effect of T(3) is not mediated through a nuclear-dependent mechanism. Rather, the properties of this response, and of the increases in amino acid and 2-DG accumulation produced by T(3) in other tissue preparations, strongly suggest that these effects of T(3) are mediated at the level of cell membrane.

Action of the Thyroid Hormone at the Level of the Plasma Membrane

In this presentation, I present evidence indicating a direct action of thyroid hormone at the level of the plasma membrane. Characteristically, the plasma membrane-mediated effects of thyroid hormones are prompt in onset, independent of new protein synthesis, and are associated with changes in the transmembrane transport of ions and substrates. The presence of specific binding sites for thyroid hormone in plasma membrane of various tissues and species, although inconclusive in itself, provides additional support for the direct action of thyroid hormone on the plasma membrane. A model for the mechanism of action of thyroid hormone at the plasma membrane level to increase sugar uptake by rat thymocytes is delineated, and the physiological role of the plasma membrane-mediated action of thyroid hormone is discussed.

Lanthanum increases the rat thymocyte cytoplasmic free calcium concentration by enhancing calcium influx

In the present study, I have examined the effect of lanthanum (La3+) on cytoplasmic free calcium concentration in isolated rat thymocytes employing the quin2 technique. As with its effect on 15Ca accumulation in rat thymocytes (Segal, J. and Ingbar, S.H. (1984) Endocrinology, 115, 160-166), La3+ produced a concentration-related increase in thymocyte cytoplasmic free calcium concentration. This effect of La3+ was very prompt in onset, evident within about 30 s from the time of addition of La3+. The lowest effective concentration of La3+ was 6 microM (+22.7% above control), and it increased progressively to reach maximal values at 25 microM (+100% above control). La3+ added to quin2-loaded thymocytes suspended in a calcium-free medium was without effect. In addition, La3+ had no significant effect on 45Ca efflux, and La3+ did not inhibit calcium-ATPase activity in the rat thymocytes. These results demonstrate that in rat thymocytes La3+ increases cytoplasmic free calcium concentration by increasing the extracellular calcium influx into the cell rather than the release of calcium from an intracellular pool.

Direct and Synergistic Interactions of 3,5,3′-Triiodothyronine and the Adrenergic System in Stimulating Sugar Transport by Rat Thymocytes

Isolated rat thymocytes were preincubated with various catecholamines, alone and together with 3,5,3-triiodothyronine (T(3)), and the accumulation of the glucose analogues, 2-deoxy-d-glucose (2-DG) and 3-O-methylglucose (3-O-MG), was then measured. Epinephrine induced a time- and dose-dependent increase in the 15-min accumulation of 2-DG; at a concentration of 100 muM epinephrine, the effect was evident after a preincubation period of only 5 min. The lowest concentration of epinephrine at which a significant effect was evident was 1 muM. Epinephrine also produced a dose-dependent increase in the accumulation of 3-O-MG, and the lowest concentration at which a significant effect was evident was again 1 muM. Isoproterenol, a beta-adrenergic agonist, like epinephrine, increased the accumulation of 2-DG, whereas the alpha-agonist, phenylephrine, had no effect. The response to epinephrine was inhibited by the beta-antagonist, alprenolol, but the alpha-antagonist, phentolamine, had no effect. As previously demonstrated, T(3) increased 2-DG accumulation, and like epinephrine, its effect was blocked by alprenolol. Neither T(3) (0.1 nM) nor epinephrine (0.1 muM) had any effect when acting alone, but when added together at these concentrations, they significantly increased the accumulation of both 2-DG and 3-O-MG. Neither T(3) with isoproterenol nor T(3) with phenylephrine produced a comparable synergistic effect. But T(3) (0.1 nM) acting with isoproterenol (0.1 muM) and phenylephrine (0.1 muM) together, synergistically increased 2-DG accumulation. In addition, the alpha-antagonist, phentolamine, which alone had no effect, inhibited the synergistic effect induced by T(3) and epinephrine. The effects of epinephrine and T(3) alone, as well as their combined synergistic effect on 2-DG accumulation, were not blocked by the inhibitor of protein synthesis, puromycin. FROM THESE RESULTS WE CONCLUDE THE FOLLOWING: (a) the stimulatory effect of the catecholamines on the accumulation of 2-DG and 3-O-MG reflects an action at the beta-receptor; (b) the synergistic interaction between T(3) and epinephrine requires the participation of both beta- and alpha-adrenergic components; (c) T(3) and epinephrine act on 2-DG and 3-O-MG accumulation through a common mechanism or inter-related mechanisms, probably mediated at the beta-adrenergic site; and (d) these effects of T(3) and epinephrine, alone and together, are independent of new protein synthesis. These results suggest that, with respect to the response we are describing, T(3) and epinephrine do not act on nuclear mechanisms, but may act instead at the level of the plasma membrane.

A postulated mechanism for the coordinate effects of ionophore A23187 on calcium uptake and cell viability in rat thymocytes

At the physiological concentration of Ca2+, the presumed calcium ionophore A23187 produced dose-related increases in 45Ca uptake by rat thymocytes and decreases in cell viability, effects that displayed a strong linear correlation. In media containing a very low concentration of Ca2+ (7.10(-6) M), in contrast, ionophore A23187 had no specific effect on either 45Ca uptake or cell viability. The calcium-dependent cytotoxicity of ionophore A23187 resembles that of other agents that are not ionophores, but that are known to perturb the plasma membrane. Consequently, we suggest that, in the rat thymocyte, ionophore, ionophore A23187 may not act as a true ionophore, but may perturb the cell membrane, allowing Ca2+ to pass freely through the membrane along its electrochemical gradient.

Studies on the age-related decline in the response of lymphoid cells to mitogens: Measurements of Concanavalin A binding and stimulation of calcium and sugar uptake in thymocytes from rats of varying ages

We have previously demonstrated that mitogenic response of rat thymocytes to concanavalin A (Con A) declines with age (Segal, Troen and Ingbar, Thymus, in press). To elucidate the mechanism underlying this process, we have examined the effect of age on Con A binding and stimulation of calcium and sugar uptake in thymocytes from rats varying in age from 10 to 360 days. Binding of Con A by thymocytes remained unchanged with advancing age. Basal uptake of the glucose analogue 2-[3H]deoxyglucose (2-DG) by rat thymocytes declined with age, becoming significantly lower than maximal values (26 days) at 4 months of age. While the proportionate increase in thymocyte 2-DG uptake produced by Con A remained essentially unchanged. However, because of the decline in basal 2-DG uptake, total uptake of 2-DG in the presence of Con A decreased with age becoming significantly lower than maximal values at 4 months. Basal calcium-45 uptake by thymocytes was practically the same in all the age-groups studied, except at 21 days, where, as with basal 2-DG uptake, it was markedly smaller, But the stimulatory effect of Con A on 45Ca uptake declined progressively with age and was nil at 360 days. From these observations I suggest that the age-related decline in the responsiveness of rat thymocytes to Con A does not result from a change in the binding of Con A by the lymphoid cell, but from, at least in part, a decrease in its cellular stimulation of calcium and sugar uptake.

Beta-Adrenergic Potentiation of the Increased In Vitro Accumulation of Cycloleucine by Rat Thymocytes Induced by Triiodothyronine

We have previously demonstrated that 3,5,3-triiodothyronine (T(3)), whether administered in vivo or added to suspending media in vitro, promptly stimulates the in vitro accumulation of the nonmetabolized amino acids, alpha-aminoisobutyric acid, and cycloleucine (CLE) by thymocytes isolated from weanling rats. In these studies, we have examined the in vitro interaction between catecholamines and T(3) with respect to this effect. The previously reported enhancement of CLE accumulation in thymocytes by T(3) in vitro (1 muM) was confirmed. When added alone in concentrations ranging between 10 nM and 0.1 mM, the adrenergic agonists, epinephrine and norepinephrine, had no effect on CLE accumulation. At a concentration of 1 muM, isoproterenol, terbutaline, and phenylephrine were also without effect. However, the effect of T(3) was clearly potentiated by the concomitant addition of epinephrine, norepinephrine, and possibly isoproterenol, whereas terbutaline and phenylephrine were without effect. Neither basal nor T(3)-enhanced CLE accumulation was affected by the addition alone of the adrenergic blocking agents, propranolol (0.1 mM), phentolamine (10 muM), or practolol (0.1 mM). Nevertheless, the beta(1)- and beta(2)-antagonist, propranol, and the beta(1)-antagonist, practolol, blocked the increment in CLE accumulation produced by epinephrine; the alpha-antagonist, phentolamine, was without effect. The enhancement of CLE accumulation that occurred in the presence of T(3), with or without epinephrine, was seen to be a result of an inhibition of CLE efflux, because T(3) alone inhibited CLE efflux, and this effect was increased when epinephrine was also present. On the other hand, neither T(3) alone nor T(3) plus epinephrine appreciably altered the rate of inward transport of CLE. As judged from studies of the ability of thymocytes to exclude trypan blue, neither T(3) alone nor T(3) plus epinephrine either enhanced or impaired viability of cells during 3-h periods of incubation. Cell water content, measured with [(3)H]urea, was unaffected by T(3), either alone or in the presence of epinephrine. In confirmation of previous results, the stimulatory effect of T(3) on CLE accumulation was unaffected by concentrations of puromycin sufficient to inhibit protein synthesis by at least 95%, and the potentiating action of epinephrine on the response to T(3) was similarly unaffected. From these findings, it is concluded that the effect of T(3) to increase CLE accumulation by thymocytes in vitro, though itself independent of adrenergic mediation, is potentiated by beta(1)-adrenergic stimulation. This interaction appears distinctly different from other thyroid hormone-catecholamine interactions, in which thyroid hormones enhance physiological responses to catecholamines. Its mechanism remains unclear, but the properties of the T(3) effect, and possibly the interaction itself, suggest that T(3) enhances CLE accumulation by an action at the level of the cell membrane.

Calmodulin modulates thymocyte adenylate cyclase activity through the guanine nucleotide regulatory unit

We have previously demonstrated in rat thymocyte plasma membranes that adenylate cyclase activity and its stimulation by 3,5,3-triiodothyronine (T3) are influenced by calmodulin, and that these effects of calmodulin require calcium. In the present study, the mechanism by which calmodulin exerts its action was examined, in situ, in fresh plasma membranes isolated from rat thymocytes. Adenylate cyclase activity was potentiated by guanyl nucleotides, NaF and forskolin. Calmodulin did not affect basal adenylate cyclase activity. However, calmodulin influenced the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity, but had no effect on the fluoride-stimulated enzyme activity. This was evident from experiments with inhibitors of calmodulin: trifluoperazine, calmidazolium, and antibodies against calmodulin. The three inhibitors did not change basal adenylate cyclase activity, but all produced a marked decrease in the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity. The inhibitory effect of all three agents was reversed completely by the addition of calmodulin. The three inhibitors, however, failed to affect the fluoride-stimulated adenylate cyclase activity. In addition, T3, like the calmodulin inhibitors, did not change basal adenylate cyclase activity, increased the guanyl nucleotide- and forskolin-stimulated enzyme activity, but had no effect on the fluoride-stimulated enzyme activity. From these results I suggest that in the rat thymocyte calmodulin activation, and thereby T3 stimulation of the calcium-sensitive adenylate cyclase system is mediated through the guanine nucleotide regulatory unit.