Adrenoceptor-mediated activation of liver glycogen phosphorylase: effects of thyroid state

Life Sciences, Vol . 24, pp . 35-42 Printed in the U .S .A .

Pergamon Press

ADßSNOCSPTOR-MEDIATED ACTIVATIOAi OF LIVER GLYCOGEN PHOSPHORYLASE: EFFECTS OF THYROID STATE Harold G . Preiksaitis and George Ruaos Department of Pharmacology S Therapeutics, McGill University, Montreal, Quebec, Canada, H3G lY6 (Received in final form November 16, 1978) SUMMARY The éffects of altered thyroid state on the adrenergic activation of rat liver glycogen phoaphorylase were studied in hepatocytes isolated from normal, thyroidectomized and thyroxine-treated rats . Basal phos In normal rate, phorylase _a activity was similar in the three groups . the order of potency of agoniste was adrenaline > pheaylephrine > isoproterenol, and the effect of adrenaline was blocked by dihydroergocryPtine (105!i) or pheaorybenzamine (105M) but sot by propranolol (10 51i) . These results indicate that in the normal rat activation of thin enzyme occurs via a-adrenoceptors. Thyroxine treatment increased sensitivity to all three agoniats but did not change their relative potencies. Thyroidectomy dramatically altered the pattern of activation by agoniste eo that the order of potency now was isoproterenol > adrenaline > phenylephrine and activation by adrenaline was inActivation of hibited only by propranolol and not by a-antagonists . phosphorylaee was now mediated via ß-adrenocaptors . The effect of thyroidectomy on the adreaocaptor response pattern of the liver develops slowly (more than 2 weeks) and is opposite to the shift from ß to a previously observed is the heart. (Br. J. Pharmacol . 59 :177, These findings indicate that hypothyroidism in rate changes 1977) . activation of liver glycogen phosphorylase from a to e ß- type response and that the direction of thyroid-dependent changes in receptor balance is tissue-dependent . Thyroid hormones can influence the reposes of various tissues to cateAltered cholamines, but the mechanism of these interactions 1e not clear. disposition of catecholamiaes does not appear to play a role is these changes (1,2) . Recently, it has been shows that hypothyroidism increases the a- and decreases the ß-component of the positive isotropic response of rat atria to catecholamiaes, while thyroid hormone treatment has the opposite effect (3) . This finding indicates that in the heart, where a- and ß-receptors mediate the same end response, thyroid hormones can influence the balance between the two. In moat species the activation of liver glycogen phosphorylase by catechol~{spa is mediated by ß-adrenoceptors (4)', whereas in the rat it is an a-receptor mediated effect (5,6) . In the present experiments we have tented the effect of thyroid hormone treatment and thyroidectomy on adrenoceptormediated glycogen phosphorylase activation in rat liver. If adreaocaptor responses is the liver are controlled in the same way as those in the heart, one might ezpect that thyroxine treatment would change liver phoaphorylase activation from an a- to a ß-type response, and that thyroidectomy would enhance sensitivity to a-adrenergic stimulation. Unexpectedly, we find the opposite : 0300-9653/79/0101-003502 .00/0 Copyright (c) 1979 Pergamon Press

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thyroxine treatment dons not significantly alter the adrenoceptor response pattern of the liver, whereas thyroidectomy changes phosphorylase activation from a- to a predominantly -type response . Preliminary accounts of some of these results have been published in abstract form (7,8) . Methode Experiments were done on hepatocytes from normal, hypothyroid and hyperthyroid male Sprague-Dawley rats, weighing 250 to 350 g and fed ad libitum . Hypothyroidism was induced by surgical thyroidectomy, performed 3months before the experiment . Rate were made hyperthyroid by daily iatraperitoneal injections of 1 mg/kg 1-thyroxine for 8 days before the experiment . Changes is thyroid state were confirmed by measuring serum thyroxine levels by radioimmunoassay (Table 1) . Hepatocytea were isolated between 9 :00 and 11 :00 a .m . each day by the method of Berry and Friend (9),with some modifications . Rata were aseathetized with sodium pentobarbital (50 mg/kg) and hgparisized (500 IU/kg) . The vans portae was Caaaulated sad perfused with Ca free Rrebs-Henseleit solution (10 min, 30-35 ml/min) . During this period, the liver was transferred to a per fusion aeration apparatus . After preperfusion, the medium was changed to 100 ml of Rreba-Henseleit buffer containing 4.2 mM Ca 2+ and 0.051 crude collagenase (Sigma type I) and perfusion was continued for 10 min with recycling. The liver was than disrupted by gentle combing and digestion of the collagenaseliver suspension was continued in a shaker bath for as additional 20 min. The perfusion sad all subsequent incubations were carried out at 37°C, under as atmosphere of 5x C02 in 02, and at a pH of 7 .4 . At the end of incubation, the cell suspension was filtered through nylon mesh and cooled oa ice. The filtrate was centrifuged for 2 min at 50 x g, the pellet was washed and centrifuged twice, and resuepended in &rebs-Henseleit buffer containing 2 .5 mM Ca2+ . Thin method routinely yielded cells with > 85Z viability as assessed by exclusion of trypaa blue . To determine phosphorylase acitivity 1.0 ml aliquots of cell suspension were homogenized in as equal volume of ice-cold assay buffer (morpholinoaulphonic acid 100 mM, NaF 300 mM, EDTA 5 .0 mM, dithiothreitol 2.0 mM, final pH 6.5) with a Brinkmann Polytroa. The homogenate was centrifuged at 3,000 x g for 10 min and glycogen phoaphorylase was assayed in duplicate aliquots of the supernatant, by measuring the incorporation of 14C-glucose-l-P into glycogen . Total incubation volume was 0 .1 ml and the reaction was started by the addition of glycogen sad 14 C-glucose-l-P to give a final concentration of 1X and 15 mM, respectively . The assay was rendered specific for phoaphorylase a by including 0 .5 mM caffeine (10) . Incubation (30°C, 30 min) was terminated by spotting 50 pl aliquots of the assay mixture on Whatman 41 filter paper discs, which where further processed(11) and then the retained radioactivity was measured by liquid scintillation counting at as efficiency of 50-60Z . Unite of enzyme activity are naaomoles of 14 C-glucose incorporated into glycogen per min per mg protein. Protein concentration in the 3,000 x g supernatant was determined by the method of Lowry et al . (12) . Three ml aliquots of cell suspension were preincubated for 30 min at 37°C under an atmosphere of 5Z C02 in 02 before the addition of agoniste . Preliminary time-course experiments showed that phoaphorylase a activity was stable after 25 min. Antagonists were added at the start of this preiacubatioa period . Addition of agoniat was followed by a rapid (lees than 1 min) increase in activity which remained at plateau for at least 10 min. For all eaperimeats shown in the present report glycogen phoQphorylase a activity was determined is aliquots of cell suspension taken immediately before and 3 .0 min after the addition of agoniste . Complete concentration-response curves for various

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agoniata we=e produced in each experiment sad adrenaline concentration-response curves were also determined in the presence of the a-receptor antagonists phenoxybenzamine (POB) and dihydroergocryptine (DHEC), and the ß-adrenoceptor antagoaiet propranolol. TABLE 1 The influence of thyroid state on the potency of agoniata in activating xat liver ptioephorylase Normal Serum T4 level (ag/ml)

T4-treated

Thyroidectonized 3 months 2 weeks

50 .3 t 9 .9

98 .3 t 2 .0*

8 .2 f 2 .4*

9 .9 t 2 .1

5.35 t 0 .38 (5) 7 .73 t 0 .11 (13) 6.21 t 0.10 (8)

5 .75 t 0 .34 (5) 8.41 t 0 .39 (8) 6 .58 f 0 .20 (5)

8.77 t 0.25* (4) 7 .96 t 0.27 (5) 5 .54 t 0.15* (3)

6 .02 t 0 .36 (2) 7 .45 t 0 .12 (2) 5.77 t 0 .12 (2)

-0 .86 t 0 .39

-0 .83 t 0.39

+3 .23 t 0.29*

+0 .25 ± 0.25

Agoniet potency (PD2)

dl-isoproterenol 1-adrenaline 1-pheaylephrine Log potency ratio (isoproterenol/ phenylephrine)

Values are means and their standard errors, or half the range where only 2 values were obtained . pD2 is the negative logarithm of the agoniat concentration producing half~maxinal enzyme activation . Number of experiments is shown in pareathesea . * indicates significant difference from corresponding values in normal rata as determined by two-tailed t teat (P < 005) . Results The type of adrenoceptor involved in the activation of phoaphorylase vas deduced from the relative potencies of different agoniata and from the effectiveness of a- and ß-adrenoceptor antagonists in blocking the response to the mixed a-ß agonist, adrenaline . Basal phoaphorylase a activity was not changed by either thyroidectomy or thyroxine treatment. Théorder of potency of agoniata in hepatocytes from normal rats was adrenaline > pheaylephrine > isoproterenol (Table 1) which confirms recent evidence that the response is mediated by a-adrenoceptora (5,6) . Thyroxine-treatment increased the sensitivity to all three agoniata somewhat, but their relative potencies remained unaltered . Hour ever, in hypothyroid rata the potency of ieoproterenol increased, whereas that of phenylephrine decreased significantly, ae also indicated by a reversal of their potency ration (Table 1) . This resulted in an order of potency characteri~tic of ß-adrenoceptora (ieoprotereaol > adrenaline > phenylephrine) . Fig. 1 shows that in normal rata isoproterenol was not only the least potent agoniat, but its efficacy was also significantly less than that of the other two agoniata . In hypothyroid rate, the efficacy of isoproterenol was higher while the efficacy of the other tw agoniata was lower than in normal animale . The change in response pattern in hypothyroid rate developed slowly . Changes in agoniet potencies were minimal in two rats tested at two weeks

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Adrenergic Activation of Glycogen Phosphorylase

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Normal

iZ0 100 8W ô0 ,~ 40 40 e 100

. Thrrold~ctomis~d r~ ..I ."

!f0

,~ ~. A~

. . .. .....

f~!

T~, .:~".

.r r

Effects of dl-isoprotereaol, 1adrenaline and 1-pheaylephrine oa liver glycogen phorphorylase activity . Units of ene activity are naaomoles of glucose incorporated into glycogen per min per mg protein . Each point represents the mean of 3 to 13 experiments . Vertical bare indicate standard errors .

20I

0

1~101~ 10~ 1öy 10'd 10's 10~ Aponbt Conantration (lAÎ

followi thyroidectomy, although serum T4 levels were as low as at 3 months (Table ~. Reversal of these changes by thyroaine-treatment of hypothyroid rats was more rapid. Preliminary experimeata indicated that the relative potency of agonista in calls from hyperhyroid rate treated with 1 mg/kg of thyroaine for 8 days was similar to that in hepatocytes from normal or hyperthyroid rate (8) . The altered order of potency of agoaista is hypo thyroid rate was associated with a compatible change in the effectiveness of a- and ß-adrenoceptor antagonists. Figure 2 illustrates that the increase in phosphorylasa activity produced by adrenaline in normal hepatocytes was substantially inhibited by In cells from DHEC and blocked by POB, but was not affected by propranolol. hypothyroid animals the a-receptor antagoniste did not significantly inhibit phosphorylase activation by adrenaline, whereas propranolol was an affective ß-Receptors are inhibitor, particularly at low adrenaline concentrations, generally more sensitive than a-receptors to stimulation by the same catecholamiae (3,13,14) and the absence of block by propranolol at high adrenaline concentrations may be due to residual a-receptor activity . This possibility is supported bq the finding that combined exposure to propranolol (10-SM) and phenoxybenzamine (10 ~ of hepatocytes from 2 hypertyroid rats inhibited the effect of adrenaline at all concentrations sad the block produced was greater than that with propranolol alone (results not shown) . Discussion The observations presented indicate a change in the adrenoceptor-mediated activation of glycogen phosphorylase from an a-type response in normal rat liver to a predominantly ß-type effect in hepatocytes from hypo thyroid rats, but do not reveal the mechanism of this change, Since the emergence of a ß-

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120 100 SO d0 "0 20

100 80 80 40 20

ô

~oA

W W~ ~de W W AdronaYns

ô

W ,d'8 ut~ ~arg Ws ~d4

Conq~tragon

(AA)

FIG . 2 Inhibition by a- and ß-adreaoceptor antagonists of adrenaline-induced phoaphorylase activation . Adrenaline concentration-response curves were determined in the absence of antagonist (control) or in the presence of dihydroergocryptine (DHEC), pheaorybeazamine (POB) or propranolol (Propr .) . Each point represents the mesa of 2 to 8 eaperiments. Vertical bars indicate standard errors . Antagonists ware added 30 min prior to adrenaline, to give a final concentration of 10-5M. These concentrations are similar to those used by others in isolated liver cells (6,16) . The absence of significant receptor blockade with antagonist concentration below 10-6![ could be partly due to uptake sad metabolism of these compounds by liver cel,la (15,26) . type response pattern, as indicated by the increased potency of isoprotereaol, is accompanied by a decrease in both the potency and efficacy of the a-receptor agonist phenylephrine, it ie clear that a- and ß-recepto activities change in a reciprocal +~g*+++pr. The finding that the effect of 10-~ adrenaline, a concentration close to threshold for both the euthyroid and hypothyroid liver, was blocked only by a-Mockers in the former and only by propranolol in the latter (Fig .2), is also more compatible with a reciprocal change in the two receptors than with an enhanced ß-receptor activity simply masking an unchanged a-activ ity in thyroidectomized rata . Such a mechanism has bees proposed by Guellaen et al . (15) to account for the change in the glycogenolytic response from an toa mixed a-ß patterä in hepatocytea isolated from adrenalectomized rats (16) . The number of binding sites for the radiolabellad S-antagonist dihydro-

a

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alprenolol (DHA) was 23-fold leas than radiolabelled DHEC binding sites, and doubled after adrenalectomy while the latter remained unchanged (15) . The mechanism by which adrenalectomy and thyroidectomy affect liver adrenoceptor responses may be different; doses of cortisone that reversed the effect of adrenalectomy (17) did not influence the effect of thyroidectomy (8) . Moreover it is possible that only a fraction of DHEC binding sites represent physiological a-receptors (18) and that a change in this fraction could have remained undetected (15) . Changes in the balance of cardiac a- and ß-adrenoceptor responses under various conditions have been noted and as interconversion of the two receptors has bees proposed as the underlying mechanism (3, 19, 20) . While the present ezperimenta do not provide direct proof for this hypothesis, it remains a plausible explanation for the altered response pattern observed in hapatocytes from hypothyroid rate . It is of course true that while the receptor activities were changed is an apparently reciprocal manner, this does not preclude the possibility that a- and ß-adrenoceptore change independently . Alternatively, a change in the coupling of receptors to the effector system, or in the sensitivity of the enzymes involved in phosphorylase activation to the coupling signal (e .g . Ca 2+ or cyclic AMP) could also account for the observed changes . Receptor binding studies could help distinguish between changes at or beyond the receptor if the identity of binding sites with functional receptors could be satisfactorily established. Of relevance to the present experiments are studies dealing with the binding of DHA and DHEC to cardiac tissue of rats in different thyroid states . Aa increase in the number of DHA binding sites and a corresponding decrease in DHEC binding aitea has been observed after thyroid hormone treatment of euthyroid (21, 22) or hypothyroid rats (23, 24), and appear to correlate well with The correlation the change in receptor balance for the isotropic response (3) . is less good when the euthyroid and hypothyroid states are compared : the number of both DHA and DHEC binding sites were found to decrease after thyroidectomy (21, 22), whereas a-receptor responses were clearly increased (3) . Correction of thyroid deficient state by T3 treatment further decreased DHEC binding (24) . Hence, the influence of thyroid state on DHEC binding in cardiac tissues ie equivocal, and explanation of the above discrepancies must await further study . The present results confirm as effect of thyroid state on adrenoceptor properties in the rat liver. Most striking, however, is the observation that the charge in response pattern from a- to ß- type was observed in hypothyroidism The direction of this and not after thyroxine treatment as first anticipated. change ie opposite to the shift in the balance of adrenoceptors from ß to a in the myocardium o~ hypothyroid rate (3) or adipose tissue frôm hypothyroid Thus the present findings indicate for the first time human subjects (25) . that the effect of thyroid hormones os the adrenoceptor response pattern ie tissue dependent . We have no ready explanation for why this should be so ; however, the fact that these differences do indeed exist, provides a potentially useful experimental model for further study of the regulation and possible relationships between a- asd ß-adreaoceptors . Ackaowledgements We thank Mre . L. Fallavolita for skilfully performing the thyroidectomiea . This study was supported by the Medical Research Council of Canada . References 1.

R.P . ZIMON, E. V. Flock, 80 808-814 (1967) .

G.M. TYES, S .G . SHEDS and C .A . OWEN, Endocrinology

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2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .

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