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Mechanisms of altered beta-adrenergic responsiveness in the hyperthyroid and hypothyroid turkey erythrocyte
Life Sciences, Vol. 30, pp. 663-673 Printed in the U.S.A.
Pergamon Press
MECHANISMS OF ALTERED BETA-ADRENERGICRESPONSIVENESSIN THE HYPERTHYROIDAND HYPOTHYROIDTURKEY ERYTHROCYTE John P. Bilezikian and John N. Loeb Departments of Medicine and Pharmacology,College of Physicians and Surgeons, Columbia University,New York, N.Y. 10032
SUMMARY
Studies on the relationshipbetween thyroid hormone and the beta-adrenergiccatecholamineshave been carried out in the turkey erythrocyte. Conditions of thyroid hormone excess and deficiency were examined with respect to their effects on the beta receptor itself, as well as to their effects on associatedbiochemical and physiologicalindices of beta receptor function, including agoniststimulated adenylate cyclase activity, cellular cyclic AMP generation, and catecholamine-induced stimulationof potassium ion influx. Erythrocytes obtained frcanhypothyroidturkeys shmed a marked (~50%) reduction in beta receptor number without any change in receptor affinity for agonists or antagonists. Catecholamine-sensitive adenylate cyclase activity and cellular cyclic AMF levels were similarly potasreduced. The sensitivity of these cells to agonist-stimulated sium influx was significantlydecreased,but maximal agonist-stimulated transport rate was unchanged. Analysis of the quantitative relaticmshipbetween beta receptor number, agonist concentration, and level of catecholamine-stimulated potassium influx indicates that, at any given absolute level of receptor occupancy,the level of agonist-stimulatedpotassium influx is identical in hypothyroid and normal erythrocytes,and that the diminishedphysiologicalsensitivity of the hypothyroidcell is attributablein its entirety to a reduction in beta receptor number per -se. The results obtained in the hyperthyroidturkey erythrocytewere strikinglydifferent. Here, beta receptor number, binding affinity cyclase for agonists and antagonists,catecholamine-sensitive,adenylate activity, and maximal cyclic AMF levels were all unchanged. In contrast, maximal agonist-stimulatedpotassium ion transport was markedly reduced, while the concentraticmof isoproterenolrequired for halfmaximal stimulationwas only slightly increased. Analysis of the relationshipbetween beta receptor number, agonist concentration,and catecholamine-stimulated potassium influx rate indicates that, at all absolute levels of beta receptor occupancy,the stimulationof monovalent cation influx is markedly blunted in the hyperthyroidcell. In contrast to the findings in the hypothyroid cell, where decreased physiologic sensitivityto catecholaminesis directly attributable to a reduction in beta receptor number, the primary abnormalityresponsible for diminished catecholamineresponsivenessin the hyperthyroid cell would appear to be located at a point "distal" to the beta receptor itself.
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INTRODUCTION It has become apparent from many years of clinical observation that betaadrenergic tone is increased in hy-perthyroidism and decreased in hypothyroidNumerous experimental models to study these observations have focused ism (1). upon catecholamine-sensitive adenylate cyclase and phosphorylase activities, as well as upon direct physiological concomitants of catecholamine actions (2, has been directed at the beta-adrenergic receptor 3) * More recently , attention itself, which is the initial site of interaction between beta agonists and the With the development of highly specific radioligands that bind target cell. to this receptor, it has become possible, in a variety of tissues, to test the possibility that thyroid hormone-induced alterations in catecholamine responConsistent with siveness are associated with changes at the receptor level. this hypothesis, some investigators have shown that beta receptors are increased in hyperthyroidism and decreased in hypothyroidism (4-14). However, in other experimental models, beta receptor number has not been found to be influIrrespective of the actual results enced by thyroid hormone (7,8,13,15-18). have been limited to an relating to beta receptor number, many of these studies examination of the beta receptor only and have not included an analysis of biochemical or physiological consequences of hormone-receptor interaction (412,14,16). Hence, in many experimental models of hyperthyroidism and hypothyroidism, it has remained uncertain whether a change, or lack of change, in beta receptor number can be related to alterations in cellular functions regulated by the catecholamines. In a series of investigations extending over the past several years, we have examined the effects of hyperthyroidism and hypothyroidism in the domestic turkey (13,19), whose erythrocytes not only contain a well-characterized betaadrenergic receptor (20) and a catecholamine-sensitive adenylate cyclase system (21), but additionally exhibit a physiological response to beta agonists in namely a striking and specific enhancement of monovalent cation trans-vitro, By virtue of these properties the turkey erythrocyte has become a port (22). useful model in which the known sequence of events initiated by binding of catecholamines to the beta receptor can be evaluated with regard to the changes induced by thyrotoxicosis or hypothyroidism. Alterations at the receptor level can be quantitatively studied in relation to changes in corresponding biochemical and physiological indices to which receptor occupancy is linked. The approach made possible by this model reveals a fascinating but rather complex relationship between thyroid hormone and the catecholamines. METHODS All methods have been previously published in detail. These include the following procedures: maintenance of turkeys (13,23), preparation of washed erythrocytes and a hormone-responsive membrane fraction (13), assay for adenylate cyclase activity (24), ]125I]iodohydroxybenzylpindolol ([125I]IHYP) and assays (25,26), measurement of cellular cyclic AMP (27), ~~J-Jl;;-Jl;;;,;i;~;~ K+ influx rate in intact cells (26), and measurement of intracellular sodium and potassium ion concentrations (28). RESULTS Establishment
of hyperthyroidism
and hypothyroidism
Hyperthyroidism was induced by the addition of L-thyroxine (3 ug/ml) to the drinking water. The daily intake of L-thyroxine per turkey was approximately 600-900 Pg. Hypothyroidism was established either by the use of a lowiodine diet and the addition of 0.5% sodium perchlorate to the drinking water,
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or by the intravenous administration of an ablative dose (lo-15 mCi) of 13lI-. Confirmation of thyroid hormone excess or deficiency was made by clinical observation as well as by actual measurement of circulating levels of thyroid hormone. Plasma thyroxine concentrations in hyperthyroid and hypothyroid birds were 10.0 i 1.4 ug/dl and 0.34 t 0.04 vg/dl, respectively, both significantly (~~0.05) different from normal (0.57 ? 0.08 ug/dl). Basal body temperature in in hyperthyroid and hypothyroid birds was 41.0 f O.lZ’C and 39.8 i O.lO”C, respectively, both significantly (p
of
[125I]IHyP
to intact
turkey
erythrocytes
Beta receptors were detected by the binding of [1251]IHYP, a radioligand which has been shown previously in turkey erythrocytes to bind to sites displaying the characteristics of beta-adrenergic receptors (20). In erythrocytes from hyperthyroid and hypothyroid turkeys, beta receptors were found to be identical to those in control erythrocytes with respect to binding affinity for the antagonist hydroxybenzylpindolol (Kd= 40 PM) and for the agonist isoproterenol (Kd = 0.15 uM; Table I). By Scatchard analysis (29)) however, hypothyroid turkey erythrocytes displayed a 50% reduction in beta receptor number as compared to normal cells. In contrast, beta receptor number was normal in hyperthyroid turkey erythrocytes . TABLE I
Binding
of
[125I]IHYp Normal,
to Erythrocytes and Hypothyroid Hyperthyroid
40 pM
Kd for' [1251]1Hyp Kd
for
Receptor
(-) -isoproterenol sites
per
cell
0.15 260
uM
from Hyperthyroid, Turkeys Normal
40 pM 0.15 260
JJM
Hypothyroid
40 pM 0.15
uM
130
Binding experiments were performed as previously described (13). Equilibrium binding constants (Kd) and binding capacities were determined by Only receptor number in hypothyroid cells the method of Scat chard (29). was found to be significantly different from normal. Adenylate
cyclase
activity
Erythrocytes from hypothyroid turkeys also demonstrated significant reducthe membrane-bound enzyme that meditions in activities of adenylate cyclase, As shown in Table II, there were uniform ates the beta-adrenergic response. and fluoride-stimulatable adeisoproterenol-stimulatable, decreases in basal,
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In contrast, nylate cyclase activities in cells from the hypothyroid turkeys. the data for adenylate cyclase activity in hyperthyroid erythrocytes were inNeither hyperthyroid nor hypodistinguishable from those for normal cells. thyroid erythrocytes showed any change in the sensitivity of adenylate cyclase to stimulation by isoproterenol or to inhibition by the beta-adrenergic inhibitor, propranolol. TABLE II
Adenylate
Cyclase
Activity
Normal,
in Hemolysates
and Hypothyroid
pmol cyclic
+ Isoproterenol + Fluoride
(0 5 mM)
(8 mM)
Turkeys
AMP generated
Hyperthyroid
Basal
from Hyperthyroid,
per
10’
Normal
9.9
? 1.6
10.5 1 2.2
51.9
+ 5.6
45.4
156 f
19
-c 5.7
152 + 19
cell
equivalents
Hypothyroid
4.9 + 1.6* 29.4
+ 5.1*
115 -+ 22*
Adenylate cyclase activity was determined as previously described (13). Values indicate means f 1 SEM. All values in hypothyroid erythrocytes (asterisks) were significantly (~~0.01) lower than normal, including those measured in the presence of GTP or its analogue, guanylyl 5’-imidophosphate. No significant differences were observed between normal and hyperthyroid erythrocytes (p>O.OS). Cyclic
AMP content
In hypothyroid turkeys, cellular cyclic AMP levels in intact erythrocytes after exposure to maximally stimulatory concentrations of isoproterenol were reduced by SO%, a finding consistent with the results obtained for beta receptor number and for adenylate cyclase activity in broken-cell preparations. Also consistent with observations made on beta receptor number and on adenylate cyclase activity was the finding that erythrocytes from hyperthyroid turkeys generated the same maximal cyclic AMP levels in response to high concentrations of isoproterenol as did euthyroid erythrocytes. However, at very low isoproterenol concentrations (S-50 nM), hyperthyroid turkey erythrocytes - in marked contrast TO their behavior at higher concentrations - accumulated substantially more cyclic AMP than did normal erythrocytes (Table III). It appears, therefore, that, within the physiological range, the concentration-response relationship for the hyperthyroid turkey erythrocyte is characterized by an enhanced responsiveness of cyclic AMP levels to catecholamines. Comparison of active mal, and hypothyroid
potassium turkeys
influx
in erythrocytes
from hyperthyroid,
nor-
As shown in Table IV, the rates of potassium influx both in the presence and absence of 1 mM ouabain were similar in all three groups of red cells. In addition, sensitivity of Na,K-ATPase-mediated active potassium influx to inhi-
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with half-maximal inhibition occurring at apbition by ouabain was similar, proximately 4 PM ouabain for all groups of cells at an external potassium ion Moreover, at this potassium concentration, Scatchard concentration of 10 ti. analysis (29) of [3H]ouabain binding in hyperthyroid and hypothyroid cells indicated that both binding affinity and binding capacity were the same as in normal cells. TABLE III
Accumulation
of Cellular
Hyperthyroid,
Cyclic
Normal,
AMP in Erythrocytes
and Hypothyroid pm01 cyclic
Hyperthyroid
50 f
Basal
Turkeys AMP per Normal
15
25 + 12 169 ?r 25
+ (-) -isoproterenol
(10 nM)
281 -+ 50
+ (-) -isoproterenol
(50 uM)
2100 + 50
from
2200 f
log
cells Hypothyroid
300
18 + 7 63 + 13 1200 f
150
Cellular cyclic AMP levels were determined as previously described (13). Values indicate the means* 1 SEM of three separate experiments for basal, for submaximal (10 nM), and for maximal (50 uM) isoproterenol-stimulatable levels of cyclic AMP. Physiological responsiveness of hyperthyroid, cytes to isoproterenol: Quantitative relation tassium influx to occupancy of beta-adrenergic
normal, and hypothyroid of isoproterenol-stimulated receptors
erythropo-
Despite the absence of any intrinsic alterations in the ouabain-inhibitable, active transport system for potassium ion in erythrocytes from hypothythere were significant deviations from northyroid and hyperthyroid turkeys, mal in the catecholamine-responsive transport system for monovalent cations. sensitivity of isoproterenol-induced potassium inIn the hypothyroid cells, flux was markedly reduced, with an increase in the concentration of agonist required for maximal stimulation from 3.920.4 nM to 9.2t1.2 nM (pO.l]). In hyperthyroid cells, on the other hand, the results The magnitude of maximal isoproterenol-stimulated were strikingly different. potassium influx was markedly reduced in hyperthyroid erythrocytes from 3423 meq/l cells/hr to 22 * 2 meq/l cells/hr (p
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TABLE IV Inhibitionof Potassium Influx by Ouabain in Erythrocytesfrom Hyperthyroid,Normal, and HypothyroidTurkeys
Potassium Influx Rate (meq/l cells/hr)
Basal + Ouabain (1 mM)
Hyperthyroid
Normal
Hypothyroid
7.3 + 0.3
8.7 r 0.7
11.9*
1.1 + 0.1
2.4 c 0.3
1.9*
Potassium influx rate was determined as previously described (19). The degree of inhibition of basal potassium influx by ouabain was similar for all three groups of erythrocytes. Values indicate means + 1 SEM except for those marked by an asterisk, which instead are from a single experiment employing pooled blood from three birds. The external potassium ion concentrationwas 10 mM.
, Hyoerthymd
t-1 - I SOPROTERENOL
I
1
1
-7
-6
CONCENTRATION
( log M 1
FIG. 1 Sensitivityof erythrocytesfrom hyperthyroid,normal, and hypothyroid turkeys to stimulationof potassium influx by isoproterenol. Erythrocytes from hyperthyroid (O---O), normal (O-O), or hypothyroid (A---A) turkeys were incubated with a range of (-)-isoproterenolconcentrations and potassium influx rate determined. Points indicate means +SEM for n = 13 (hyperthyroid),n= 14 (normal),or n= 12 (hypothyroid)experiments. Arrows indicate values for half-maximalstimulation [K,) based upon the means of all Ka's within each individual group.
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for half-maximal stimulation of potassium transport (23). In hypothyroid cells - because total beta receptor number is reduced by 50% in the absence of any change in receptor affinity - twice the concentration of isoproterenol is required, at the lower end of the concentration-response curve where occupancy is linear with agonist concentration, to result in a given absolute level of receptor occupancy (Figure 2). At any given level of receptor occupancy, however, the absolute increment in potassium transport induced by beta agonist is identical in the hypothyroid erythrocyte to that in the normal cell (Figure 3). The quantitative relationship between beta receptor occupancy and potassium ion transport is quite different in the hyperthyroid erythrocyte. In this instance, beta receptor number and agonist binding affinity are both unchanged from their respective values in normal cells, indicating that a given low concentration of isoproterenol gives rise to the same absolute level of receptor occupancy as for normal cells (Figure 2). Despite the apparent “normality” of this relationship, however, the magnitude of catecholamine-stimulated potassium transport in the hyperthyroid cell is markedly reduced at all levels of receptor occupancy (Figure 3).
4 (-I-
0
ISOPROTERENOL
12 CONCENTRATION
16 Mvll
FIG. 2 Quantitative relation between beta receptor occupancy and isoproterenol concentration in hyperthyroid, normal, and hypothyroid turkey erythrocytes. The absolute number of occupied beta receptors is shown in relation to a range of very low isoproterenol concentrations (l-16 nM) for hyperthyroid (0), normal (a), and hypothyroid (A) turkey erythrocytes. The number of beta receptors occupied at specified concentrations of isoproterenol is seen to be the same for normal and hyperthyroid erythrocytes but reduced by approximately 50% in hypothyroid cells. Receptor occupancy was calculated as previously described (19).
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OCCUPIED RECEPTORS PER CELL
FIG. 3 Isoproterenol-stimulated potassium influx as a function of recepturkey erythrotor occupancy in hyperthyroid, normal, and hypothyroid Isoproterenol-stimulated potassium influx is plotted against cytes. absolute level of receptor occupancy for hyperthyroid (O---O), normal (O-O), and hypothyroid (A---A) cells. The increment in potassium influx resulting per occupied receptor is seen to be the same for normal and hypothyroid erythrocytes, but to be reduced by approximately 50% in hyperthyroid cells. DISCUSSION The results of this investigation permit several conclusions to be drawn in regard to the relationship between thyroid hormone, the catecholamines, and monovalent cation transport systems in erythrocytes from chronically hyperthyroid and hypothyroid turkeys. In contrast to many other cells in which thyroid hormone has been shown to alter the number or activity of sodium pump units (30-31), ouabain-sensitive Na,K-ATPase effector units are unchanged either in number or in activity in both hyperthyroid and hypothyroid turkey erythrocytes. Basal potassium influx rates, ouabain sensitivity, ouabain binding affinity and binding capacity, and intracellular sodiiun and potassium concentrations are all virtually identical in normal, hyperthyroid, and hypothyroid erythrocytes. In contrast to these findings, a different transport system - one that is resistant to ouabain but responsive to stimulation by the beta-adrenergic catecholamines - is shown here to be profoundly influenced by states of thyroid hormone excess or deficiency. Recent evidence suggests that this latter system represents a (Na++K+) co-transport pathway that is sensitive to inhibition by furosemide (32,33) .
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In the hypothyroid turkey erythrocyte, beta receptor number is markedly reduced, an observation that is consistent with findings in a number of other model systems (5-7,9,13,12). In contrast to many former studies, however, which were restricted to an analysis of the beta receptor-adenylate cyclase complex only, it has been possible in the present system to assign a direct role for the reduction in beta receptor number in the mediation of the associated change in physiologic sensitivity to the catecholamines. At low agonist concentrations, where the physiological stimulation of ion transport is observed, and where receptor occupancy is very nearly linear with agonist concentration, the reduced number of qualitatively normal receptors in the hypothyroid cell leads to a requirement for a reciprocally higher agonist concentration to result in a given absolute level of receptor occupancy. Maximal stimulatable potassium influx is unchanged from that in normal cells, and each agonist-occupied beta receptor in the hypothyroid cell gives rise to the same absolute increment in ion flux rate as in the euthyroid cell. It is apparent, that the observed reduction in beta receptor number per se can actherefore, count entirely for the diminished sensitivity of the hypothyroid tuzey erythrocyte to agonist-stimulated potassium influx. In the hyperthyroid cell, however, the results are more complex and are not simply the converse of those observed in the hypothyroid state. Despite the absence of any change in beta receptor characteristics (either number or affinity for agonists or antagonists), or in maximal stimulatable cyclic AMP levels, the hyperthyroid turkey erythrocyte exhibits a striking reduction in the magnitude of catecholamine-stimulatable potassium influx. The reduction relative to the level of stimulation in the normal cell is evident and proportionately identical at all concentrations of agonist, leading to the conclusion that the degree of stimulation of potassium ion transport resulting from each occupied receptor is markedly reduced. Thus, in contrast to the situation in the hypothyroid turkey erythrocyte, the blunting of the physiologic response at any given concentration of agonist is attributable, not to a change in beta receptor number, but rather to modifications in intracellular events “distal” to Given the fact that cyclic AMP generation at low the beta receptor itself. isoproterenol-concentrations is actually somewhat increased over that in normal cells (13). it would appear either that the increase in cyclic AMP in some specific intracellular-compartment responsible for the stimulation of monovalent cation transport is blunted at a given absolute level of receptor occupancy (despite a heightened increase in total cellular cyclic AMP), or that the post-receptor defect in the hyperthyroid turkey erythrocyte is distal not only to the beta receptor itself but also to the generation of cyclic AMP. as well as from those in many other labIt is evident from our studies, oratories, that the relationship between thyroid,hormone and functions served When viewed solely in the context of beta by the catecholamines is complex. receptors, hypothyroidism has been found to be associated with an increase (34, 35)) a decrease (5-7,9,11-13)) or no change (15,36) in beta receptor number. in hyperthyroidism, beta receptors have been reported to be inSimilarly, creased (4,6,10,X,14), decreased (37), or unchanged (7,8,13,15,16). Even when such data are analyzed in terms of associated physiologic responses, it is apparent that there is great variability from cell to cell and from organ to Both the hyperthyroid and hypothyroid states can each be associated organ. with a heightening or blunting of beta receptor-mediated responsiveness to catecholamines, which, in turn, can be observed either with or without changes It is increasingly clear that the overall enin beta receptor number per se. hancement of beta-adrenergic
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isolated organ. The turkey erythrocytehas proven to be a useful model by virtue of its well-characterizedbeta-adrenergicreceptor and adenylate cyclase system, as well as its characteristicand readily quantifiablephysiological response to catecholamines. To what extent the biochemical and physiological mechanisms elucidated in this avian model system will prove applicable to other systems as well, and to what extent still other mechanisms will be found to play a role in alterationsof catecholamineresponsivenesscharacteristicof altered thyroid states, must await the results of future studies.
ACKNOWLEDGMENTS These studies were supported in part by grants HL-20859, HL-12738, HD-05506, and TI-AM-07271 from the National Institutes of Health. J.P.B. is the recipient of Research Career DevelopmentAward HL-00383.
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J.P. BILEZIKIAN, J.N. LOEB, and D.E. GAMMON, Poult. Sci. 59 628-634 (1980) Y. SALOMON, C. LONDOS, and M. RODBELL, Anal. Biochem. 58 al-548 (1974). H. FURUKAWA, J.P. BILEZIKIAN, and J.N. LOEB, Biochim. Eophys. Acta 598 345-356 (1980). H. FURUKAWA, J.P. BILEZIKIAN, and J.N. LOEB, J. Gen. Physiol. -76 499-516 (1980). A.L. STEINER, C.W. PARKER, and D.M. KIPNIS, J. Biol. Chem. -247 1106-1113 (1972). J.R. SACHS and L.G. WELT, J. Clin. Invest. 46 65-76 (1967). G. SCATCHARD, Ann. N.Y. Acad. Sci. 51 660-6B (1949). T.J. SMITH and I.S. EDELMAN, Fed. PGc. 38 2150-2153 (1979). M.H. LIN and T. AKERA, J. Biol. Chem. 25T723-726 (1978). W.F. SCHMIDT III and T.J. M&ANUS, J. ??%. Physiol. 70 81-97 (1977). H.C. PALFREY, P.W. FEIT, and P. GREENGARD, Am. J. PhFiol. -238 C139-Cl48 (1980). C.C. MALBON, J. Biol. Chem. 255 8692-8699 (1980). C.C. MALBON, S.-Y. LI, and Jx FAIN, J. Biol. Chem. 253 8820-8825 (1978). J.N. FAIN, Life Sci. 28 1745-1754 (1981). c.c. MALBON and M.L. ?XEENBERG, J. Clin. Invest., in press.
EDITED GENERAL DISCUSSION Paper of Bilezikian
Dratman asked what the step distal to adenyl cyclase is. Bilezikian indicated there are many steps distal to beta-receptor but was uncertain what they are. He is certain that it is not simply receptor binding and induction of CAMP. McCarron brought up the question whether the turkey's receptor binding can be modified by altering dietary intake, i.e. high sodium or potassium, but this had not been studied. Ridgway then inquired whether it is known if sodium and potassium shifts result in changes in intracellular calcium since increases in intracellular Ca++ might result in the low renin levels seen in hypothyroidism. Bilezikian replied that there are no changes in Ca fluxes when one exposes normal turkey erythrocytes to beta-adrenergic agents. Werner brought up the increase in intracellular sodium in hyperthyroidism in man and whether Bilezikian had seen this in his turkeys. The reply was not in this model, but it is true in man. Rosenqvist noted he had observed in the rat that there is less calcium in mitochondria isolated from hypothyroid hepatocytes than from normal hepatocytes.
Pergamon Press
MECHANISMS OF ALTERED BETA-ADRENERGICRESPONSIVENESSIN THE HYPERTHYROIDAND HYPOTHYROIDTURKEY ERYTHROCYTE John P. Bilezikian and John N. Loeb Departments of Medicine and Pharmacology,College of Physicians and Surgeons, Columbia University,New York, N.Y. 10032
SUMMARY
Studies on the relationshipbetween thyroid hormone and the beta-adrenergiccatecholamineshave been carried out in the turkey erythrocyte. Conditions of thyroid hormone excess and deficiency were examined with respect to their effects on the beta receptor itself, as well as to their effects on associatedbiochemical and physiologicalindices of beta receptor function, including agoniststimulated adenylate cyclase activity, cellular cyclic AMP generation, and catecholamine-induced stimulationof potassium ion influx. Erythrocytes obtained frcanhypothyroidturkeys shmed a marked (~50%) reduction in beta receptor number without any change in receptor affinity for agonists or antagonists. Catecholamine-sensitive adenylate cyclase activity and cellular cyclic AMF levels were similarly potasreduced. The sensitivity of these cells to agonist-stimulated sium influx was significantlydecreased,but maximal agonist-stimulated transport rate was unchanged. Analysis of the quantitative relaticmshipbetween beta receptor number, agonist concentration, and level of catecholamine-stimulated potassium influx indicates that, at any given absolute level of receptor occupancy,the level of agonist-stimulatedpotassium influx is identical in hypothyroid and normal erythrocytes,and that the diminishedphysiologicalsensitivity of the hypothyroidcell is attributablein its entirety to a reduction in beta receptor number per -se. The results obtained in the hyperthyroidturkey erythrocytewere strikinglydifferent. Here, beta receptor number, binding affinity cyclase for agonists and antagonists,catecholamine-sensitive,adenylate activity, and maximal cyclic AMF levels were all unchanged. In contrast, maximal agonist-stimulatedpotassium ion transport was markedly reduced, while the concentraticmof isoproterenolrequired for halfmaximal stimulationwas only slightly increased. Analysis of the relationshipbetween beta receptor number, agonist concentration,and catecholamine-stimulated potassium influx rate indicates that, at all absolute levels of beta receptor occupancy,the stimulationof monovalent cation influx is markedly blunted in the hyperthyroidcell. In contrast to the findings in the hypothyroid cell, where decreased physiologic sensitivityto catecholaminesis directly attributable to a reduction in beta receptor number, the primary abnormalityresponsible for diminished catecholamineresponsivenessin the hyperthyroid cell would appear to be located at a point "distal" to the beta receptor itself.
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INTRODUCTION It has become apparent from many years of clinical observation that betaadrenergic tone is increased in hy-perthyroidism and decreased in hypothyroidNumerous experimental models to study these observations have focused ism (1). upon catecholamine-sensitive adenylate cyclase and phosphorylase activities, as well as upon direct physiological concomitants of catecholamine actions (2, has been directed at the beta-adrenergic receptor 3) * More recently , attention itself, which is the initial site of interaction between beta agonists and the With the development of highly specific radioligands that bind target cell. to this receptor, it has become possible, in a variety of tissues, to test the possibility that thyroid hormone-induced alterations in catecholamine responConsistent with siveness are associated with changes at the receptor level. this hypothesis, some investigators have shown that beta receptors are increased in hyperthyroidism and decreased in hypothyroidism (4-14). However, in other experimental models, beta receptor number has not been found to be influIrrespective of the actual results enced by thyroid hormone (7,8,13,15-18). have been limited to an relating to beta receptor number, many of these studies examination of the beta receptor only and have not included an analysis of biochemical or physiological consequences of hormone-receptor interaction (412,14,16). Hence, in many experimental models of hyperthyroidism and hypothyroidism, it has remained uncertain whether a change, or lack of change, in beta receptor number can be related to alterations in cellular functions regulated by the catecholamines. In a series of investigations extending over the past several years, we have examined the effects of hyperthyroidism and hypothyroidism in the domestic turkey (13,19), whose erythrocytes not only contain a well-characterized betaadrenergic receptor (20) and a catecholamine-sensitive adenylate cyclase system (21), but additionally exhibit a physiological response to beta agonists in namely a striking and specific enhancement of monovalent cation trans-vitro, By virtue of these properties the turkey erythrocyte has become a port (22). useful model in which the known sequence of events initiated by binding of catecholamines to the beta receptor can be evaluated with regard to the changes induced by thyrotoxicosis or hypothyroidism. Alterations at the receptor level can be quantitatively studied in relation to changes in corresponding biochemical and physiological indices to which receptor occupancy is linked. The approach made possible by this model reveals a fascinating but rather complex relationship between thyroid hormone and the catecholamines. METHODS All methods have been previously published in detail. These include the following procedures: maintenance of turkeys (13,23), preparation of washed erythrocytes and a hormone-responsive membrane fraction (13), assay for adenylate cyclase activity (24), ]125I]iodohydroxybenzylpindolol ([125I]IHYP) and assays (25,26), measurement of cellular cyclic AMP (27), ~~J-Jl;;-Jl;;;,;i;~;~ K+ influx rate in intact cells (26), and measurement of intracellular sodium and potassium ion concentrations (28). RESULTS Establishment
of hyperthyroidism
and hypothyroidism
Hyperthyroidism was induced by the addition of L-thyroxine (3 ug/ml) to the drinking water. The daily intake of L-thyroxine per turkey was approximately 600-900 Pg. Hypothyroidism was established either by the use of a lowiodine diet and the addition of 0.5% sodium perchlorate to the drinking water,
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or by the intravenous administration of an ablative dose (lo-15 mCi) of 13lI-. Confirmation of thyroid hormone excess or deficiency was made by clinical observation as well as by actual measurement of circulating levels of thyroid hormone. Plasma thyroxine concentrations in hyperthyroid and hypothyroid birds were 10.0 i 1.4 ug/dl and 0.34 t 0.04 vg/dl, respectively, both significantly (~~0.05) different from normal (0.57 ? 0.08 ug/dl). Basal body temperature in in hyperthyroid and hypothyroid birds was 41.0 f O.lZ’C and 39.8 i O.lO”C, respectively, both significantly (p
of
[125I]IHyP
to intact
turkey
erythrocytes
Beta receptors were detected by the binding of [1251]IHYP, a radioligand which has been shown previously in turkey erythrocytes to bind to sites displaying the characteristics of beta-adrenergic receptors (20). In erythrocytes from hyperthyroid and hypothyroid turkeys, beta receptors were found to be identical to those in control erythrocytes with respect to binding affinity for the antagonist hydroxybenzylpindolol (Kd= 40 PM) and for the agonist isoproterenol (Kd = 0.15 uM; Table I). By Scatchard analysis (29)) however, hypothyroid turkey erythrocytes displayed a 50% reduction in beta receptor number as compared to normal cells. In contrast, beta receptor number was normal in hyperthyroid turkey erythrocytes . TABLE I
Binding
of
[125I]IHYp Normal,
to Erythrocytes and Hypothyroid Hyperthyroid
40 pM
Kd for' [1251]1Hyp Kd
for
Receptor
(-) -isoproterenol sites
per
cell
0.15 260
uM
from Hyperthyroid, Turkeys Normal
40 pM 0.15 260
JJM
Hypothyroid
40 pM 0.15
uM
130
Binding experiments were performed as previously described (13). Equilibrium binding constants (Kd) and binding capacities were determined by Only receptor number in hypothyroid cells the method of Scat chard (29). was found to be significantly different from normal. Adenylate
cyclase
activity
Erythrocytes from hypothyroid turkeys also demonstrated significant reducthe membrane-bound enzyme that meditions in activities of adenylate cyclase, As shown in Table II, there were uniform ates the beta-adrenergic response. and fluoride-stimulatable adeisoproterenol-stimulatable, decreases in basal,
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In contrast, nylate cyclase activities in cells from the hypothyroid turkeys. the data for adenylate cyclase activity in hyperthyroid erythrocytes were inNeither hyperthyroid nor hypodistinguishable from those for normal cells. thyroid erythrocytes showed any change in the sensitivity of adenylate cyclase to stimulation by isoproterenol or to inhibition by the beta-adrenergic inhibitor, propranolol. TABLE II
Adenylate
Cyclase
Activity
Normal,
in Hemolysates
and Hypothyroid
pmol cyclic
+ Isoproterenol + Fluoride
(0 5 mM)
(8 mM)
Turkeys
AMP generated
Hyperthyroid
Basal
from Hyperthyroid,
per
10’
Normal
9.9
? 1.6
10.5 1 2.2
51.9
+ 5.6
45.4
156 f
19
-c 5.7
152 + 19
cell
equivalents
Hypothyroid
4.9 + 1.6* 29.4
+ 5.1*
115 -+ 22*
Adenylate cyclase activity was determined as previously described (13). Values indicate means f 1 SEM. All values in hypothyroid erythrocytes (asterisks) were significantly (~~0.01) lower than normal, including those measured in the presence of GTP or its analogue, guanylyl 5’-imidophosphate. No significant differences were observed between normal and hyperthyroid erythrocytes (p>O.OS). Cyclic
AMP content
In hypothyroid turkeys, cellular cyclic AMP levels in intact erythrocytes after exposure to maximally stimulatory concentrations of isoproterenol were reduced by SO%, a finding consistent with the results obtained for beta receptor number and for adenylate cyclase activity in broken-cell preparations. Also consistent with observations made on beta receptor number and on adenylate cyclase activity was the finding that erythrocytes from hyperthyroid turkeys generated the same maximal cyclic AMP levels in response to high concentrations of isoproterenol as did euthyroid erythrocytes. However, at very low isoproterenol concentrations (S-50 nM), hyperthyroid turkey erythrocytes - in marked contrast TO their behavior at higher concentrations - accumulated substantially more cyclic AMP than did normal erythrocytes (Table III). It appears, therefore, that, within the physiological range, the concentration-response relationship for the hyperthyroid turkey erythrocyte is characterized by an enhanced responsiveness of cyclic AMP levels to catecholamines. Comparison of active mal, and hypothyroid
potassium turkeys
influx
in erythrocytes
from hyperthyroid,
nor-
As shown in Table IV, the rates of potassium influx both in the presence and absence of 1 mM ouabain were similar in all three groups of red cells. In addition, sensitivity of Na,K-ATPase-mediated active potassium influx to inhi-
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with half-maximal inhibition occurring at apbition by ouabain was similar, proximately 4 PM ouabain for all groups of cells at an external potassium ion Moreover, at this potassium concentration, Scatchard concentration of 10 ti. analysis (29) of [3H]ouabain binding in hyperthyroid and hypothyroid cells indicated that both binding affinity and binding capacity were the same as in normal cells. TABLE III
Accumulation
of Cellular
Hyperthyroid,
Cyclic
Normal,
AMP in Erythrocytes
and Hypothyroid pm01 cyclic
Hyperthyroid
50 f
Basal
Turkeys AMP per Normal
15
25 + 12 169 ?r 25
+ (-) -isoproterenol
(10 nM)
281 -+ 50
+ (-) -isoproterenol
(50 uM)
2100 + 50
from
2200 f
log
cells Hypothyroid
300
18 + 7 63 + 13 1200 f
150
Cellular cyclic AMP levels were determined as previously described (13). Values indicate the means* 1 SEM of three separate experiments for basal, for submaximal (10 nM), and for maximal (50 uM) isoproterenol-stimulatable levels of cyclic AMP. Physiological responsiveness of hyperthyroid, cytes to isoproterenol: Quantitative relation tassium influx to occupancy of beta-adrenergic
normal, and hypothyroid of isoproterenol-stimulated receptors
erythropo-
Despite the absence of any intrinsic alterations in the ouabain-inhibitable, active transport system for potassium ion in erythrocytes from hypothythere were significant deviations from northyroid and hyperthyroid turkeys, mal in the catecholamine-responsive transport system for monovalent cations. sensitivity of isoproterenol-induced potassium inIn the hypothyroid cells, flux was markedly reduced, with an increase in the concentration of agonist required for maximal stimulation from 3.920.4 nM to 9.2t1.2 nM (p
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TABLE IV Inhibitionof Potassium Influx by Ouabain in Erythrocytesfrom Hyperthyroid,Normal, and HypothyroidTurkeys
Potassium Influx Rate (meq/l cells/hr)
Basal + Ouabain (1 mM)
Hyperthyroid
Normal
Hypothyroid
7.3 + 0.3
8.7 r 0.7
11.9*
1.1 + 0.1
2.4 c 0.3
1.9*
Potassium influx rate was determined as previously described (19). The degree of inhibition of basal potassium influx by ouabain was similar for all three groups of erythrocytes. Values indicate means + 1 SEM except for those marked by an asterisk, which instead are from a single experiment employing pooled blood from three birds. The external potassium ion concentrationwas 10 mM.
, Hyoerthymd
t-1 - I SOPROTERENOL
I
1
1
-7
-6
CONCENTRATION
( log M 1
FIG. 1 Sensitivityof erythrocytesfrom hyperthyroid,normal, and hypothyroid turkeys to stimulationof potassium influx by isoproterenol. Erythrocytes from hyperthyroid (O---O), normal (O-O), or hypothyroid (A---A) turkeys were incubated with a range of (-)-isoproterenolconcentrations and potassium influx rate determined. Points indicate means +SEM for n = 13 (hyperthyroid),n= 14 (normal),or n= 12 (hypothyroid)experiments. Arrows indicate values for half-maximalstimulation [K,) based upon the means of all Ka's within each individual group.
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for half-maximal stimulation of potassium transport (23). In hypothyroid cells - because total beta receptor number is reduced by 50% in the absence of any change in receptor affinity - twice the concentration of isoproterenol is required, at the lower end of the concentration-response curve where occupancy is linear with agonist concentration, to result in a given absolute level of receptor occupancy (Figure 2). At any given level of receptor occupancy, however, the absolute increment in potassium transport induced by beta agonist is identical in the hypothyroid erythrocyte to that in the normal cell (Figure 3). The quantitative relationship between beta receptor occupancy and potassium ion transport is quite different in the hyperthyroid erythrocyte. In this instance, beta receptor number and agonist binding affinity are both unchanged from their respective values in normal cells, indicating that a given low concentration of isoproterenol gives rise to the same absolute level of receptor occupancy as for normal cells (Figure 2). Despite the apparent “normality” of this relationship, however, the magnitude of catecholamine-stimulated potassium transport in the hyperthyroid cell is markedly reduced at all levels of receptor occupancy (Figure 3).
4 (-I-
0
ISOPROTERENOL
12 CONCENTRATION
16 Mvll
FIG. 2 Quantitative relation between beta receptor occupancy and isoproterenol concentration in hyperthyroid, normal, and hypothyroid turkey erythrocytes. The absolute number of occupied beta receptors is shown in relation to a range of very low isoproterenol concentrations (l-16 nM) for hyperthyroid (0), normal (a), and hypothyroid (A) turkey erythrocytes. The number of beta receptors occupied at specified concentrations of isoproterenol is seen to be the same for normal and hyperthyroid erythrocytes but reduced by approximately 50% in hypothyroid cells. Receptor occupancy was calculated as previously described (19).
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OCCUPIED RECEPTORS PER CELL
FIG. 3 Isoproterenol-stimulated potassium influx as a function of recepturkey erythrotor occupancy in hyperthyroid, normal, and hypothyroid Isoproterenol-stimulated potassium influx is plotted against cytes. absolute level of receptor occupancy for hyperthyroid (O---O), normal (O-O), and hypothyroid (A---A) cells. The increment in potassium influx resulting per occupied receptor is seen to be the same for normal and hypothyroid erythrocytes, but to be reduced by approximately 50% in hyperthyroid cells. DISCUSSION The results of this investigation permit several conclusions to be drawn in regard to the relationship between thyroid hormone, the catecholamines, and monovalent cation transport systems in erythrocytes from chronically hyperthyroid and hypothyroid turkeys. In contrast to many other cells in which thyroid hormone has been shown to alter the number or activity of sodium pump units (30-31), ouabain-sensitive Na,K-ATPase effector units are unchanged either in number or in activity in both hyperthyroid and hypothyroid turkey erythrocytes. Basal potassium influx rates, ouabain sensitivity, ouabain binding affinity and binding capacity, and intracellular sodiiun and potassium concentrations are all virtually identical in normal, hyperthyroid, and hypothyroid erythrocytes. In contrast to these findings, a different transport system - one that is resistant to ouabain but responsive to stimulation by the beta-adrenergic catecholamines - is shown here to be profoundly influenced by states of thyroid hormone excess or deficiency. Recent evidence suggests that this latter system represents a (Na++K+) co-transport pathway that is sensitive to inhibition by furosemide (32,33) .
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In the hypothyroid turkey erythrocyte, beta receptor number is markedly reduced, an observation that is consistent with findings in a number of other model systems (5-7,9,13,12). In contrast to many former studies, however, which were restricted to an analysis of the beta receptor-adenylate cyclase complex only, it has been possible in the present system to assign a direct role for the reduction in beta receptor number in the mediation of the associated change in physiologic sensitivity to the catecholamines. At low agonist concentrations, where the physiological stimulation of ion transport is observed, and where receptor occupancy is very nearly linear with agonist concentration, the reduced number of qualitatively normal receptors in the hypothyroid cell leads to a requirement for a reciprocally higher agonist concentration to result in a given absolute level of receptor occupancy. Maximal stimulatable potassium influx is unchanged from that in normal cells, and each agonist-occupied beta receptor in the hypothyroid cell gives rise to the same absolute increment in ion flux rate as in the euthyroid cell. It is apparent, that the observed reduction in beta receptor number per se can actherefore, count entirely for the diminished sensitivity of the hypothyroid tuzey erythrocyte to agonist-stimulated potassium influx. In the hyperthyroid cell, however, the results are more complex and are not simply the converse of those observed in the hypothyroid state. Despite the absence of any change in beta receptor characteristics (either number or affinity for agonists or antagonists), or in maximal stimulatable cyclic AMP levels, the hyperthyroid turkey erythrocyte exhibits a striking reduction in the magnitude of catecholamine-stimulatable potassium influx. The reduction relative to the level of stimulation in the normal cell is evident and proportionately identical at all concentrations of agonist, leading to the conclusion that the degree of stimulation of potassium ion transport resulting from each occupied receptor is markedly reduced. Thus, in contrast to the situation in the hypothyroid turkey erythrocyte, the blunting of the physiologic response at any given concentration of agonist is attributable, not to a change in beta receptor number, but rather to modifications in intracellular events “distal” to Given the fact that cyclic AMP generation at low the beta receptor itself. isoproterenol-concentrations is actually somewhat increased over that in normal cells (13). it would appear either that the increase in cyclic AMP in some specific intracellular-compartment responsible for the stimulation of monovalent cation transport is blunted at a given absolute level of receptor occupancy (despite a heightened increase in total cellular cyclic AMP), or that the post-receptor defect in the hyperthyroid turkey erythrocyte is distal not only to the beta receptor itself but also to the generation of cyclic AMP. as well as from those in many other labIt is evident from our studies, oratories, that the relationship between thyroid,hormone and functions served When viewed solely in the context of beta by the catecholamines is complex. receptors, hypothyroidism has been found to be associated with an increase (34, 35)) a decrease (5-7,9,11-13)) or no change (15,36) in beta receptor number. in hyperthyroidism, beta receptors have been reported to be inSimilarly, creased (4,6,10,X,14), decreased (37), or unchanged (7,8,13,15,16). Even when such data are analyzed in terms of associated physiologic responses, it is apparent that there is great variability from cell to cell and from organ to Both the hyperthyroid and hypothyroid states can each be associated organ. with a heightening or blunting of beta receptor-mediated responsiveness to catecholamines, which, in turn, can be observed either with or without changes It is increasingly clear that the overall enin beta receptor number per se. hancement of beta-adrenergic
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isolated organ. The turkey erythrocytehas proven to be a useful model by virtue of its well-characterizedbeta-adrenergicreceptor and adenylate cyclase system, as well as its characteristicand readily quantifiablephysiological response to catecholamines. To what extent the biochemical and physiological mechanisms elucidated in this avian model system will prove applicable to other systems as well, and to what extent still other mechanisms will be found to play a role in alterationsof catecholamineresponsivenesscharacteristicof altered thyroid states, must await the results of future studies.
ACKNOWLEDGMENTS These studies were supported in part by grants HL-20859, HL-12738, HD-05506, and TI-AM-07271 from the National Institutes of Health. J.P.B. is the recipient of Research Career DevelopmentAward HL-00383.
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EDITED GENERAL DISCUSSION Paper of Bilezikian
Dratman asked what the step distal to adenyl cyclase is. Bilezikian indicated there are many steps distal to beta-receptor but was uncertain what they are. He is certain that it is not simply receptor binding and induction of CAMP. McCarron brought up the question whether the turkey's receptor binding can be modified by altering dietary intake, i.e. high sodium or potassium, but this had not been studied. Ridgway then inquired whether it is known if sodium and potassium shifts result in changes in intracellular calcium since increases in intracellular Ca++ might result in the low renin levels seen in hypothyroidism. Bilezikian replied that there are no changes in Ca fluxes when one exposes normal turkey erythrocytes to beta-adrenergic agents. Werner brought up the increase in intracellular sodium in hyperthyroidism in man and whether Bilezikian had seen this in his turkeys. The reply was not in this model, but it is true in man. Rosenqvist noted he had observed in the rat that there is less calcium in mitochondria isolated from hypothyroid hepatocytes than from normal hepatocytes.