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Several observations provide some clues as to the possible mode of the regulatory action of thyroid hormone (TH) in the heart, indicating delayed action at the level of the nucleus and acute effects on the plasma membrane. Here we present evidence for a direct and rapid stimulatory effect of TH in the intact normal heart. In the isolated perfused rat heart, 3,5,3'-tri-iodothyronine (T3) produced a positive inotropic effect increasing both the left ventricular peak systolic pressure (P) and +dP/dt values, but had no significant effect on heart rate. This effect of T3 was: (1) very rapid in onset (starting after 15 s) and transient, increasing gradually to reach a maximum (80% above control) at about 20 min, and declining progressively 20 to 30 min later; (2) dose-related and biphasic, occurring at physiological concentrations as low as 1 pM (+dP/dt) and 10 pM (P), reaching a maximum at 1 nM, and decreasing at higher concentrations; and (3) thyroid hormone specific, as shown by the effects of several TH analogs (L-T3 > L-thyroxine (T4) = D-T3 > D-T4; 3,3',5'-tri-iodothyronine (rT3), 3,5,-L-di-iodothyronine and DL-thyronine had no effect). The calcium blockers nifedipine and verapamil, at concentrations of 10(-8) - 10(-5) M given before or after the addition of T3 (10(-9) - 10(-6) M), inhibited the T3-induced increase in cardiac inotropic activity in a time- and dose-related fashion. We suggest that the acute effect of TH in the heart is plasma membrane-mediated and calcium-dependent.

Background: Triiodothyronine (T3) administration can improve postischemic myocardial recovery. Heparin can interfere with cellular binding of T3. Introduction of heparin into an isolated heart model may interfere with this effect., Methods: Four groups of 8 rat hearts were placed on a modified Langendorff apparatus. All groups underwent 15 minutes of perfusion with modified Krebs-Henseleit solution (KH), followed by 20 minutes of normothermic global ischemia and 30 minutes of reperfusion. Group I underwent reperfusion with KH. Group II underwent reperfusion with KH and 1 x 10(-6) mol/L of T3. In group III, hearts underwent preischemic perfusion with heparinized KH (1,000 U/L) and reperfusion with KH containing 1 x 10(-6) mol/L of T3 and 1,000 U/L of heparin. In group IV, rats were given heparin at 2,000 IU/kg 30 minutes before sacrifice, and isolated hearts were reperfused with KH and 1 x 10(-6) mol/L of T3. A latex balloon in the left ventricle monitored hemodynamic variables., Results: Left ventricular developed pressure throughout postischemic reperfusion was greater in all the groups receiving T3 when compared with group I. Group II showed significantly greater recovery than either group III (p < 0.05) or group IV (p < 0.05)., Conclusions: Addition of T3 to the reperfusate enhances postischemic myocardial recovery in the isolated heart model, whereas addition of heparin reduces this effect.

Calcium has been shown in vitro to serve as the first messenger for the rapid effect of thyroid hormone at the level of the plasma membrane. In the present study the physiological relevance of this mechanism is examined in the whole animal. To this end, the effect of T3 on 45calcium uptake and sugar [2-deoxyglucose (2-DG]) uptake, an effect that requires extracellular calcium, and the influence of calcium blockers thereon were measured in ventricles, atria, diaphragm, fat, and liver in the rat. In the first three tissues, T3 produced comparable changes in 45Ca uptake and 2-DG uptake (T3 increased 2-DG uptake in fat, where 45Ca uptake was undetected, and had no effect in liver); this activity was blocked by the calcium channel blocker cadmium. The effect of T3 on 45Ca uptake, like its effect on the in vivo uptake of 2-DG described previously, was biphasic and time related; at physiological doses of 0.01 and 0.1 micrograms/100 g BW, T3 increased 45Ca uptake, whereas at greater (pharmacological) doses of 1 and 100 micrograms/100 g BW, T3 was without effect or inhibited 45Ca uptake. In ventricles and atria, the stimulatory effect of T3 on 45Ca uptake was very rapid [within 2 min, at which time it was at or near maximum (50-90% above control] and then declined gradually and was not seen after 10-20 min. Of the several calcium blockers employed, verapamil (organic) and cadmium (inorganic) were found to be the most effective. Verapamil and cadmium produced a rapid, transient, and dose-related inhibition of 45Ca uptake in the tissues examined (except fat tissue where, under the experimental conditions employed, 45Ca uptake was undetected). Verapamil, given iv (200 micrograms/100 g BW) or ip (1 mg/100 g BW), reduced tissue 45Ca uptake by 50-90% within 2 or 10 min, respectively, and then its inhibitory effect diminished rapidly and was not seen after 20-30 min. Cadmium, given iv (was ineffective when given ip) produced a rapid and dose-related decrease in 45Ca uptake, being most effective (40-90% inhibition) 1-10 min after its administration. Cadmium (1 mg/100 g BW) injected into the tail vein 2 min before T3 produced a comparable inhibition of the stimulatory effect of T3 on 45Ca uptake and 2-DG uptake. Verapamil did not change the T3 effect.(ABSTRACT TRUNCATED AT 400 WORDS)

The thyroid hormone T3 produced a very rapid and transient increase in 45calcium uptake by freshly isolated rat heart slices, which was seen already 15 sec after the addition of the hormone, reached a maximum at 30 sec, and then progressively declined and returned to control values after 10 min. This effect of T3 was independent of extracellular calcium, concentration related (evident at a physiological concentration of 10 pM, reached maximum of about 75% above control at 1 nM, and was smaller at greater concentrations), and thyroid hormone specific, as judged from the order of potency of several thyroid hormone analogs: L-T3 greater than L-T4 greater than or equal to D-T3 greater than 3'-isopropyl-3,5-L-diiodothyronine greater than D-T4 greater than 3,5-L-diiodothyronine greater than r-L-T3 greater than D,L-thyronine. The inorganic calcium channel blockers La3+, Cd2+, and Mn2+ inhibited, in a concentration-related fashion, basal and T3-induced increases in 45Ca uptake in the cardiac slices. The organic calcium channel blockers verapamil, nifedipine, and diltiazem were without effect, indicating that in the quiescent cardiac slice the effect of T3 on 45Ca uptake is independent of sarcolemmal depolarization. Additional studies demonstrated that the stimulatory effect of T3 on 2-deoxyglucose uptake by the cardiac slices required extracellular calcium and was inhibited by the calcium channel blockers La3+, Cd2+, and Mn2+. The present study provides conclusive evidence for two central issues: that calcium is the first messenger for the prompt, plasma membrane-mediated action of thyroid hormone to increase cellular sugar uptake, and that thyroid hormone produces an acute increase in calcium uptake by the heart, an effect that is demonstrable at physiological concentrations and is thyroid hormone specific and, therefore, points to a physiological relevance for this action.

We have shown that 3,5,3'-tri-iodothyronine (T3) produces a prompt increase in sugar transport in rat thymocytes by increasing the maximal velocity without changing the Michaelis-Menten constant of the plasma membrane sugar transport system. To elucidate further the mechanism of this effect, we have now assessed the influence of T3 on the number and affinity of sugar transporters in thymocytes, measured as the sugar (2-deoxyglucose; dGlc)-displaceable binding of cytochalasin B. Cytochalasin B inhibited in a dose-related manner the uptake of dGlc by rat thymocytes with inhibition constant values of 0.19 and 0.22 mumol/l in the presence and absence of T3 respectively. Binding of cytochalasin B by the sugar-displaceable sites was rapid and saturable, demonstrating a single class of sites having an apparent dissociation constant of 0.33 +/- 0.02 (S.D.) mumol/l and maximal binding capacity of 3.73 +/- 0.48 pmol/20 x 10(6) cells (11.2 +/- 1.4 x 10(4) sites/thymocyte). In the rat thymocyte, sugar transporters were found to be located in two major subcellular pools, the plasma membrane and microsomes, the latter being about twice the size of the former. In these subcellular compartments, as well as in the intact cell, binding of [3H]cytochalasin B by the sugar-displaceable sites constituted about 40% of total cytochalasin B binding. 3,5,3'-Tri-iodothyronine in concentrations that stimulated uptake of dGlc by thymocytes had no effect on [3H]cytochalasin B binding (total and sugar-displaceable) in the intact cell and in the plasma membrane and microsomal compartments, nor did it influence the affinity and number of sugar transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

In view of our previous demonstration that T3 promptly increases the cAMP concentration in freshly isolated rat thymocytes in vitro, we studied the effects of T3 on adenylate cyclase activity in a crude thymocyte plasma membrane preparation. In common with adenylate cyclase in other tissues, the enzyme in rat thymocytes was activated by NaF, GTP, 5'-guanylylimidodiphosphate, and beta-adrenergic agonists and was inhibited by high concentrations of calcium. In the presence of 1 microM Ca+2, T3 induced a time-dependent increase in adenylate cyclase activity that was statistically significant between 1 and 2 min and maximum between 2 and 5 min after hormone addition. As judged from observations made at 5 min, the effect of T3 was dose dependent over the range 1 nM to 1 microM. The stimulatory effect of T3 was calcium dependent, since it was abolished by EGTA at a concentration (0.5 mM) that did not alter basal enzyme activity, and the effect of T3 in the presence of EGTA was restored by the addition of either 0.1 or 1 mM Ca+2. As judged from the lack of hydrolysis of added cAMP, phosphodiesterase activity in the assay mixture was nil in both the presence and absence of T3. Both epinephrine and the specific beta-adrenergic agonist isoproterenol, but not the alpha-agonist phenylephrine, increased adenylate cyclase activity, and their effects appeared to be additive to that of T3. The beta-adrenergic antagonist L-alprenolol, in doses that did not influence basal adenylate cyclase activity, produced a dose-related inhibition of the stimulatory effect of T3 and of the effects of epinephrine and isoproterenol as well. Neither D-alprenolol nor the alpha-antagonist phentolamine had any effect. Various thyronine analogs displayed a rank order of potency in stimulating adenylate cyclase activity very similar to their relative potencies in increasing cAMP concentration in the intact thymocyte. These findings reveal that T3 stimulates adenylate cyclase activity in rat thymocyte plasma membrane preparations. With respect to calcium dependence, inhibition by alprenolol, and response to thyronine analogs, this effect has properties similar to those of the increase in cellular cAMP concentration induced by T3 in the intact thymocyte. It can be concluded, therefore, that the effect of T3 to increase 2-deoxyglucose uptake by the rat thymocyte in vitro, a response consequent to an increase in thymocyte cAMP concentration, derives from a stimulatory effect of the hormone on adenylate cyclase itself.

The uptake of 2-deoxy-D-[1-3H]glucose ([1-3H]DOG)by cultured chick embryo heart cells was stimulated by exposure to triiodothyronine (T3). The concentration of free T3 for half maximal stimulation of [1-3H]DOG uptake was 8.7 x 10(-10)M. Nuclear binding studies revealed that the Kd for T3 was 4.2 x 10(-10)M. The molar potencies of other thyroactive compounds in stimulating [1-3H]DOG uptake, relative to L-T3, were: 500% for 3'-isopropyl-3,5-diiodo-L-thyronine, 20% for L-thyroxine (T4), 17% for D-T3, 2.2% for D-T4, 0.6% for 3,5-diiodo-L-thyronine and less than 0.1% for 3',5',3-triiodo-L-thyronine and L-thyronine. These data suggest that cultured chick embryo heart cells may serve in the study of the mode of action of thyroid hormones.

We have previously demonstrated, both in vivo and in vitro, that 3,5,3'-tri-L-iodothyronine (T3) increases the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG) in rat thymocytes by acting at the level of the plasma membrane. In the present study, the effect of hypothyroidism on the basal uptake of 2-DG by rat thymocytes and their response to T3 was examined. Rats were rendered hypothyroid by thyroidectomy at 21 days of age, and experiments performed with 31- and 56-day-old animals. Uptake of 2-DG by thymocytes from hypothyroid rats, both in vivo and in vitro, was greater in 31-day-old animals and less in 56-day-old animals than that in euthyroid rats. In both age-groups, hypothyroidism increased cellular responsiveness to T3, shifting the dose-response curve to the left. Similar results were obtained in experiments in which animals were rendered hypothyroid by KCIO4. Injection of thyroid hormones into rats treated with KCIO4 reversed the effects of hypothyroidism on uptake of [3H]2-DG by thymocytes and their response to T3. From these observations it was concluded that hypothyroidism produces a time-dependent change in basal sugar uptake by rat thymocytes, and increases cellular responsiveness to the effect of T3 at the level of the plasma membrane.

In previous studies we have shown that the thyroid hormone T3 induces a prompt increase in the cAMP concentration of rat thymocytes in vitro. This is followed by and very likely leads to an increase in cellular uptake of the glucose analog 2-deoxyglucose (2-DG). Since these effects of T3 were shown to require the presence of Ca2+ in the suspending medium, it seemed reasonable to determine whether T3 would influence the metabolism of calcium itself. In standard medium containing 1 mM Ca2+ and 45Ca as a tracer, T3 induced a very prompt, dose-related, but transient, increase in thymocyte calcium accumulation. This effect was evident within the first minute after the addition of T3 (including processing time) and is to our knowledge the most rapid effect of T3 yet demonstrated. The effect reached a maximum very shortly thereafter and then abated within a few minutes. Uptake of 45Ca from Ca2+-free medium (5 microM Ca2+ as a contaminant) was also increased by T3, but under these conditions, the increase was sustained for the entire 120-min period of study. At a concentration of 25 microM, lanthanum (La3+) unexpectedly produced a rapid and marked (4- to 6-fold) increase in 45Ca accumulation in the thymocytes. The increase in cellular calcium accumulation produced by La3+ was not associated with any effect on thymocyte cAMP concentration or 2-deoxyglucose (2-DG) uptake. However, La3+ potentiated the response of the cell to T3 in respect to calcium accumulation, cAMP concentration, and 2-DG uptake, shifting the dose-response curve of the latter two effects to the left. In view of the calcium dependence of the T3 effect on thymocyte cAMP concentration and 2-DG uptake, and the effect of La3+ to concomitantly enhance the sensitivity of the cell to the effects of T3 on calcium accumulation and cAMP and 2-DG metabolism, we suggest that the prompt increase in cellular Ca2+ uptake induced by T3 that we demonstrated is causally related to its subsequent effect on cellular cAMP concentration and 2-DG uptake.

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.

Thyroid hormone produces a prompt, plasma membrane-mediated increase in several metabolic functions in the rat thymocyte. These effects of thyroid hormone require calcium and are inhibited by the beta-adrenergic antagonist alprenolol. In the present study, the interrelationship between thyroid hormone and adrenergic agents, and the concept that calcium serves as the first messenger for the rapid action of thyroid hormone in the rat thymocyte are further examined. T3 produced a very rapid and dose-related increase in both 45 calcium accumulation and cytoplasmic free calcium concentration. These effects of T3, like its other calcium-dependent and prompt effects, were inhibited by beta-, but not by alpha-, adrenergic antagonists. Studies with selective beta-adrenergic agents revealed that the inhibitory effect was beta-1 in nature. In addition, beta-adrenergic agonists were found to promote additively T3 action, possibly through their beta-2 activity. Hence, the present study provides further support for the concept that calcium serves as the first messenger in the rapid, plasma membrane-mediated action of thyroid hormone in the rat thymocyte.

The effect of age on the responsiveness of rat thymocytes to 3,5,3'-tri-iodothyronine (T3) was studied. It has been demonstrated previously that the plasma membrane-mediated effect of T3 to increase sugar uptake by rat thymocytes is influenced by age and sex. In both sexes, T3 given in vitro stimulated sugar uptake in cells from animals of 15 days of age, had no effect at 21 days and was again effective at 26 days. In the male, thymocytes from animals of 40 days of age and older were refractory to T3. However, in the female, T3, although less effective than in cells from 26-day-old animals, remained stimulatory in cells from 40- and 60-day-old rats. T3 had no effect in cells from animals of 90 days of age and older. In in-vivo studies in which female rats of 26, 60 and 90 days of age were first injected with T3 and 1 h later with [3H]2-deoxyglucose, the responsiveness of thymocytes to T3 also declined progressively with advancing age; T3 was most effective in cells from 26-day-old animals, less stimulatory in 60-day-old and essentially without effect in cells from 90-day-old animals. From these observations we have concluded that in both male and female rats the responsiveness of thymocytes to T3 declines progressively with age, and that this decline occurs at an earlier age in cells obtained from males.

The present experiments were designed to explore the mechanism whereby 3,5,3'-triiodothyronine (T3) stimulates the uptake of 2-deoxy-D-glucose (2-DG) in rat thymocytes in vitro. Addition of T3 evoked a transient, dose-related increase in cellular cyclic (c) AMP concentrations, evident within 5 min. followed soon by an increase in 2-DG uptake. The effects of T3 on both cAMP concentration and 2-DG uptake were dependent upon the presence of extracellular calcium. Epinephrine also induced a sequential increase in thymocyte cAMP concentration and 2-DG uptake. These responses were more prompt than those to T3, but were calcium independent. As with their combined effects on 2-DG uptake, T3 and epinephrine produced synergistic or additive effects on cellular cAMP concentration. Dibutyryl cAMP also stimulated 2-DG uptake, an effect that was more prompt than that of epinephrine, and like that of epinephrine, was calcium independent. Prior or simultaneous addition of L-alprenolol (10 microM), which, we have previously shown, blocks the effect of both T3 and epinephrine on 2-DG uptake, also blocked the increase in thymocyte cAMP concentration induced by these agents. In contrast, L-alprenolol failed to block the increase in 2-DG uptake produced by dibutyryl cAMP. On the basis of these observations we suggest that T3 increases 2-DC uptake in the rat thymocyte by increasing the cellular concentration of cAMP, which then acts to enhance sugar transport. The increase in 2-DC uptake induced by epinephrine is also mediated by an increase in cAMP concentration. The greater response of cellular cAMP concentration to T3 and epinephrine when added together than to either agent added alone may explain their synergistic action to increase 2-DG uptake. We suggest that these actions of T3 and epinephrine are both initiated at the level of the plasma membrane.

The effect of 3,5,3'-triiodo-L-thyronine (T3; 10(-8)M) on sugar transport was studied in cultured chick embryo heart cells. T3 did not increase the uptake of L-glucose, a sugar which enters the cell by simple diffusion. The effect of T3 on the kinetic parameters of the uptake of both 3-0-methyl-D-glucose (3-OMET) and 2-deoxy-D-glucose (2-DOG) was studied after 6 and 24 h exposure to T3. T3 had no effect on the Km for either sugar. T3 increased the Vmax for the first 6 h or exposure for 3-OMET, a sugar which is only transported, and increased the Vmax after 6 and 24 h for 2-DOG, a sugar both transported and phosphorylated. T3 also increased the relative phosphorylation of 2-DOG within the cells in the period between 6 and 24 h. The data show a biphasic effect of T3 on sugar accumulation, an early effect on transport along and a later effect on phosphorylation as well.

We have previously demonstrated that T3 increases adenylate cyclase activity in preparations of plasma membranes from rat thymocytes. On the basis of this and other evidence, we have postulated that the increased cAMP concentration and consequent increase in 2-deoxyglucose (dGlc) uptake that T3 induces in the intact thymocyte is the consequence of a similar stimulation of adenylate cyclase activity. To obtain further evidence to this point, we have now conducted experiments with MDL 12330A [N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride], a compound that inhibits adenylate cyclase activity in several other tissues. In thymocyte plasma membrane preparations, MDL 12330A induced a concentration-dependent inhibition of both basal enzyme activity (activity in the absence of hormone) and the increase in activity induced by T3 and epinephrine. In the intact thymocyte, MDL 12330A greatly limited the marked increase in cellular cAMP concentration induced by maximally effective concentrations of the phosphodiesterase inhibitor 3'-isobutyl-1'-methylxanthine. This indicates that MDL 12330A inhibits adenylate cyclase activity in the intact thymocyte as it does in thymocyte plasma membrane preparations. Further, in intact thymocytes incubated with MDL 12330A, there occurred small but significant decreases in basal cAMP concentration and dGlc uptake, and the T3-induced enhancement of these functions was reduced or abolished. These data provide additional evidence that the increase in dGlc uptake in rat thymocytes that T3 induces is linked to an antecedent increase in cellular cAMP concentration, and that the latter results from a T3-induced enhancement of adenylate cyclase activity.

The present studies were undertaken to explore further the mechanism by which T3 increases adenylate cyclase activity and the uptake of the sugar analog 2-deoxyglucose (2-DG) in freshly isolated rat thymocytes. In studies of cells preloaded with the fluorescent probe quin-2, whose fluorescence intensity increases linearly with increases in cytoplasmic free calcium concentration [( Ca2+]i), we have now demonstrated that T3 increases [Ca2+]i in thymocytes suspended in buffer containing 1 mM Ca2+. This effect was extremely prompt, becoming evident much less than 1 min after the addition of T3 and reaching maximal values in about 5-8 min. The subsequent time course of the T3 effect was obscured by an increase in fluorescence intensity in control thymocytes not exposed to T3, beginning after about 8 min of incubation. However, the T3 effect, after reaching its peak, appeared to remain stable for about 5 min and then to decline, abating completely in 18-30 min. No effect of T3 on [Ca2+]i was observed when thymocytes were suspended in Ca2+-free medium. The effect of T3 on [Ca2+]i was concentration dependent, and as with its actions on thymocyte adenylate cyclase activity, cAMP concentration and 2-DG uptake, the lowest effective concentration of T3 was 1 nM. Among several thyronine analogs studied, L-T3 was the most potent, followed in decreasing order of potency by L-T4, D-T3, 3,5-diiodo-3'-isopropyl-L-thyronine, and D-T4. rT3, 3,5-diiodo-L-thyronine, and D,L-thyronine were without effect. l-Alprenolol alone (10 microM) produced a modest increase in thymocyte [Ca2+]i, but, as it does with the effect of T3 on cellular cAMP concentration and 2-DG uptake, it markedly inhibited or abolished the stimulatory effect of T3 on [Ca2+]i. From these observations we conclude that T3 initiates the increase in thymocyte [Ca2+]i by enhancing the influx of extracellular calcium, though the possibility that it also releases calcium from an intracellular calcium pool cannot be excluded. Since the effects of T3 on thymocyte adenylate cyclase activity, cAMP concentration, and 2-DG uptake occur subsequent to these effects on calcium metabolism and require the presence of Ca2+ in the extracellular fluid, we suggest that an increase in [Ca2+]i, due at least partly to an influx of extracellular calcium, is the initiating event in these plasma membrane-mediated responses of the rat thymocyte to T3.

We have previously demonstrated that in the rat thymocyte T3 produces a prompt, extranuclear effect to increase cellular cAMP concentration and 2-deoxyglucose (2-DG) uptake. We have also demonstrated the presence of specific receptors for T3 in the rat thymocyte plasma membrane. From these and other observations we have suggested that T3 initiates these actions by binding to its receptors on the surface of this cell. To test this hypothesis further we have now examined whether a decrease in the binding of T3 to the thymocyte plasma membrane, induced by mild trypsinization, attenuates the effect of T3 on thymocyte cAMP concentration and 2-DG uptake. Mild trypsin treatment of rat thymocytes in cell suspension did not cause appreciable nonspecific damage to the cells, since it did not change the ability of the cells to exclude either trypan blue or mannitol, a marker of extracellular water. Further, treatment with trypsin did not alter the ability of thymocytes to bind concanavalin A. It did produce, however, a dose-related decrease in the binding of T3 to its plasma membrane binding sites owing to a decrease in their number, but not their affinity. This was associated with a progressive decrease in the T3-induced increase in both 2-DG uptake and cAMP concentration. Thus, low concentrations of trypsin had no significant effect on 2-DG uptake in the absence of T3, but decreased significantly the increase in sugar uptake induced by T3, whereas a higher concentration of trypsin (5 mg/ml) decreased basal 2-DG uptake by only 23%, but abolished the response to T3 altogether. T3, epinephrine, and trypsin alone induced dose-related increases in cellular cAMP concentration, the response to trypsin exceeding that to T3 or epinephrine. Trypsinization also resulted in a progressive decrease in the response of cellular cAMP to both hormones, so that at a trypsin concentration of 5 mg/ml no further increase was produced by either hormone. The possibility that a maximum activation of adenylate cyclase by trypsin could explain these results was rendered unlikely by experiments with prostaglandin E1 (PGE1). The increase in cellular cAMP concentration induced by this agent was vastly greater than that produced by trypsin alone. Although the response to PGE1 was greatly inhibited by trypsin, cellular cAMP concentration in thymocytes incubated with trypsin (5 mg/ml) plus PGE1 was much higher than that in cells treated with trypsin alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of the influence of age and sex on the concentrations of total thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3) in serum and on the free T4 and free T3 indices, were conducted in Sprague-Dawley rats of the CD strain varying in age between 10 days and 12 months. Both sex- and age-related differences were found. In all age-groups studied, serum T4 concentrations were higher in the male than in the female, whereas serum T3 concentrations were higher in the female. In both sexes, concentrations of T4 and T3 in serum reached a peak early in life, between the first and second month of age, and declined thereafter. In addition, in both sexes the intensity of thyroid hormone binding, as judged from values of the in-vitro uptake of T3, did not change with age, suggesting that free T4 and T3 concentrations in the serum display the same sex differences and age-related changes as do the concentrations of total T4 and T3. It remains to be determined whether these sex- and age-related alterations in serum thyroid hormone concentration are expressed in differences in the activity of various thyroid hormone-dependent processes.

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.

We have previously demonstrated that T3 increases adenylate cyclase activity in rat thymocyte plasma membranes by a mechanism that is calcium dependent. In the present studies we have examined whether calmodulin participates in this response to T3. Initial experiments provided evidence that calmodulin is involved in regulating the activity of the guanyl nucleotide-dependent adenylate cyclase in this tissue. Thus, compared with findings in freshly prepared membranes, dialysis of thymocyte plasma membrane preparations for 20 h at 4 C decreased their calmodulin concentrations from an initial value of approximately 600 ng/100 micrograms protein by about 50% and decreased adenylate cyclase activity by approximately 80%. Although storage of the same preparations under comparable conditions resulted in no change in calmodulin content, it caused an approximate 30% decrease in adenylate cyclase activity. Addition of calmodulin had no effect on adenylate cyclase activity in fresh or stored membrane preparations, but produced a dose-dependent increase in enzyme activity in preparations that had been dialyzed. Further, when added to freshly isolated membranes, three calmodulin antagonists, trifluoperazine, calmidazolium, and calmodulin antibodies, all produced a concentration-dependent inhibition of adenylate cyclase activity, and this was completely reversed in all cases by the addition of high concentrations of exogenous calmodulin. The stimulation of guanyl nucleotide-dependent adenylate cyclase activity that T3 induced in fresh membrane preparations was present in membranes that had been stored, but was absent in those that had been dialyzed. In addition, the response to T3 in fresh membranes was inhibited or abolished by all three calmodulin antagonists. Both in dialyzed membranes and in the presence of antagonists, the response to T3 was restored by the addition of exogenous calmodulin. We conclude that calmodulin influences the activity of the guanyl nucleotide-dependent adenylate cyclase in rat thymocytes and ultimately mediates the stimulation of enzyme activity that T3 produces. Such mediation very likely explains the calcium-dependent nature of the stimulatory effect of T3 on thymocyte adenylate cyclase activity.

3,5,3'-Triiodothyronine (T3) produced a rapid and transient increase in 45Ca uptake and cytoplasmic free calcium concentration in rat thymocytes, which is the most rapid effect of T3 in this system. This effect was manifested in cells suspended in medium containing 1 mM calcium. The T3 effect on 45Ca uptake was evident at 15-30 s, reached maximum at 30-60 s, and returned to control values at 5 min. The T3 effect on cytoplasmic free calcium concentration was seen after 30 s, reached maximum at 7 min, and returned to control values after 24 min. In cells suspended in Ca2+-free medium, T3 produced a similar rapid increase in 45Ca uptake, which was sustained for at least 60 min, but T3 failed to change cytoplasmic free calcium concentration. Alprenolol (10 microM) blocked the stimulatory effects of T3 on these two functions in a similar fashion. From these results, I suggest that in rat thymocytes T3 influences cellular calcium economy through a biphasic mechanism in which T3 first increases calcium uptake which, in turn, is followed by a release of calcium from intracellular pool(s), resulting in a further increase in cytoplasmic free calcium concentration and the activation of Ca2+ -regulated systems. Moreover, the present study provides further support for the postulate that in the rat thymocyte calcium serves as the first messenger for the plasma membrane-mediated stimulatory effects of T3 on several metabolic functions.

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.

T3 produced a prompt and biphasic change in the uptake of 2-deoxy-D-glucose (dGlc) by atria, ventricles, diaphragm, and fat in the rat in vivo. At the lower physiological doses T3 produced a dose-related increase in tissue dGlc uptake; the lowest effective concentration was 5-10 ng/100 g BW, and maximal effect of about 75-100% increase above control values was seen at a T3 dose of 25 (diaphragm) or 100 ng. At pharmacological doses of 5 and 50 micrograms/100 g BW T3 inhibited dGlc uptake by about 50%. Evaluation of the physiological related stimulatory effect of T3 on dGlc uptake in the four tissues revealed that it was independent of new protein synthesis, because it was not inhibited by cycloheximide which blocked [3H]leucine incorporation by more than 95%, and that it was thyroid hormone specific, as judged from the order of potency of several T3 analogs: 3'-isopropyl-L-T2 greater than or equal to L-T3 greater than L-T4 = D-T3 greater than D-T4; L-rT3, 3,5-L-T2, and DL-thyronine were without effect. Additional studies demonstrated that in the four tissues employed T3 acted to promote sugar uptake by increasing the activity of the sugar transport system located at the plasma membrane. The present study together with previous in vitro and in vivo studies, in the same and other tissues, provide a strong evidence in support of a physiological role for the action of thyroid hormone at the level of the plasma membrane to increase cellular sugar uptake.

We have previously demonstrated that 3,5,3'-tri-iodo-L-thyronine (T3) produces a very rapid and transient increase in calcium uptake and cytoplasmic free calcium concentration in the rat thymocyte, and have postulated that Ca2+-ATPase may contribute to the overall effect of T3 on cellular calcium metabolism. In the present study, we show that in the rat thymocyte, T3 increased plasma membrane Ca2+-ATPase activity. This effect of T3 was very rapid, seen at 30 s after the addition of the hormone, and was concentration-related, evident at a physiological concentration as low as 1 pM. Evaluation of the effect of several thyronine analogues on Ca2+-ATPase activity revealed the following order of potency: D-T3 greater than or equal to 3'-isopropyl-L-T2 = L-T3 = L-T4 = D-T4 greater than L-rT3 greater than 3,5-L-T2 greater than DL-thyronine. Studies with the calmodulin antagonist trifluoperazine demonstrated that thymocyte Ca2+-ATPase activity and its stimulation by T3 are influenced by calmodulin. Other studies showed that several adrenergic agents, agonists and antagonists, had no effect on thymocyte Ca2+-ATPase activity and its stimulation by T3. From these and previous observations, we would suggest that in the rat thymocyte, the T3-induced increase in Ca2+-ATPase activity, which enhances the expulsion of calcium from the cell, plays a role in the diminution and transiency of the stimulatory effect of T3 on thymocyte calcium metabolism.

In previous studies we have demonstrated that 3,5,3'-triiodothyronine (T3) in vitro produces a prompt increase in the uptake of the sugar analogue 2-deoxyglucose (2-DG) by freshly isolated rat thymocytes. This effect is prompt, being evident at 20 min after addition of T3, is independent of new protein synthesis, and can be elicited by physiologic concentrations of the hormone. In the present studies, we have sought to determine whether physiologic doses of T3 are capable of inducing an increase in 2-DG uptake in the thymocytes of the living animal. Therefore, 26-28-d-old female rats were injected with increasing doses of i.v. T3, followed 60 min later by 3H-labeled 2-DG. 30 min later, animals were killed, thymocytes were isolated, and their 3H content determined. Uptake of [3H]2-DG was increased by T3 in a dose-dependent manner. The lowest effective dose was 10 ng/100 g of body weight (30% above control) and the maximally effective dose 1 microgram/100 g of body weight (116% above control). The effect of T3 was independent of new protein synthesis in that it was not blocked by a dose of cycloheximide that inhibited the incorporation of [3H]leucine into thymocyte protein by 92-95%. Comparable studies with various thyronine analogues revealed the following rank order of potency: L-T3 greater than L-3,5,3'5'-tetraiodothyronine (L-T4) greater than D-T3 greater than or equal to D-T4 greater than L-3,3'5'-triiodothyronine greater than 3'-isopropyl-3,5-L-diiodothyronine (T2) = 3,5-L-T2. DL-thyronine was without effect. These studies indicate that T3 in physiologic doses acts in vivo to increase the uptake of sugar by rat thymocytes by a mechanism that is extranuclear in origin, in that it is independent of new protein synthesis. The findings support the conclusion that the previously demonstrated effects of T3 on thymocyte sugar uptake in vitro, which seem clearly to be mediated at the level of the plasma membrane, have physiologic relevance.

The uptake of 2-deoxy-D-[1-3H]glucose ([1-3H]DOG) by cultured chick embryo heart cells was stimulated by L-triiodothyronine (T3). This stimulation began with the exposure to T3, rose linearly for about 8 h and reached a plateau around 24h. Actinomycin D, puromycin and cyclohexmide had no effect on the stimulated sugar uptake during the first 6h. These agents prevented the further increase in the rate of [1-3H]DOG uptake in T3-treated cultures that occurred between 6 and 24h. In the presence of hydroxyurea the uptake of [1-3H]DOG in both control and T3-treated cells was unchanged. These results suggest that some of the effects of T3 may not require new synthesis of either RNA, proteins or DNA.