Effect of thyrotropin-releasing hormone and bromoergocriptine on growth hormone and prolactin secretion in perfused pituitary adenoma tissues of acromegaly.

0021-972X/78/4706-1251$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society

Vol. 47, No. 6 Printed in U.S.A.

Effect of Thyrotropin-Releasing Hormone and Bromoergocriptine on Growth Hormone and Prolactin Secretion in Perfused Pituitary Adenoma Tissues of Acromegaly MIYUKI ISHIBASHI* AND TOHRU YAMAJI Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan ABSTRACT. To determine the site of action of TRH and 2-brom-a-ergocriptine (CB154) on pituitary hormone release in acromegalic patients, the effect of these substances on GH and PRL secretion was examined in perfused pituitary adenoma tissues obtained at surgery from subjects with acromegaly. Relatively stable baseline secretion levels of GH and PRL were followed by an abrupt and marked discharge of the hormones after TRH infusion in all of the experiments. The pattern of GH response was essentially the same as that of PRL. Moreover, a dose-response relationship was obtained between the TRH concentrations infused and the magnitude of GH and PRL responses. The infusion of CB154, on the other hand, inhibited

A

BERRANT or paradoxical GH responses L to various stimuli are frequently observed in acromegalic patients. Dompaminergic agonists, such as L-dopa (1), 2-brom-aergocriptine (CB154) (2), and dopamine (3) have been shown to inhibit GH as well as PRL release in some patients with acromegaly. Administration of TRH (4, 5) or LRH (6-8), on the other hand, may elevate the circulating levels of GH in this pathological condition. Of interest in this regard is that there exists a close correlation between the response to CB154 and that to TRH (9, 10). In patients who secrete GH in response to TRH, CB154 usually depresses their serum GH concentrations. The mechanism by which TRH and dopaminergic drugs affect GH secretion, however, remains unknown. They may act at the hypothalamus or, alternatively, may influence GH release by a direct action on the pituitary. Received April 25, 1978. * To whom requests for reprints should be addressed.

both GH and PRL secretion in three experiments performed on different adenoma tissues. This effect of CB154 was prompt and lasted for a long period even after the infusion was discontinued. When TRH was perfused concomitantly with CB154, the stimulatory effect of TRH on GH release was maintained, while TRH-induced PRL secretion was completely blocked. The results suggest that both TRH and CB154 possess a direct action on pituitary adenoma cells of acromegaly and that aberrant GH responses to TRH and dopaminergic agonists in acromegalic patients may be explained by the altered cellular membrane receptors of the adenoma of these subjects. (J Clin Endocrinol Metab 47: 1251, 1978)

The present study was undertaken 1) to determine the site of action of TRH and CB154, and 2) to clarify the interaction of these two substances by examining their effect on GH and PRL secretion from pituitary adenoma tissue of acromegalic patients using an in vitro perfusion technique. Materials and Methods Pituitary adenoma Pituitary adenoma tissue was obtained at surgery from seven patients with active acromegaly. Light microscopy revealed that all of the pituitary adenomas were eosinophilic. Acromegalic patients These seven patients consisted of three men and four women, aged between 19-48 yr. Their preoperative clinical data are summarized in Table 1. They exhibited physical features of acromegaly, elevated baseline serum GH concentrations, and failure of the serum GH levels to suppress during an oral glucose tolerance test. Intravenous injection of 500 /ig TRH elicited both GH and PRL release

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TABLE 1. Laboratory findings of the seven acromegalic patients Response to TRH (500 ng iv) Case

Age

no.

(yr)

1 2 3 4 5 6 7

33 19 31 31 34

35 48

GH (ng/ml) M F F M M F F

598 57 62 146 220 60 80

PRL (ng/ml) 30 100 11

GHa

PRL°

212 775 200

152 390 290 211 153 341 375

8.5

1151

15 9.4 9.1

3125

285 428

Response to CB154 (5 mg orally) GH*

PRL6

8.6 49

6.8 24

25 29

21 49

Pituitary adenoma tissue used in this study (mg) 50 103 27 75 68 25 70

° Peak GH nd PRL concentrations were expressed in terms of a percentage of the control levels. * The lowest GH and PRL concentrations observed after CB154 administration were expressed in terms of percentage of the control levels.

in all of the subjects. Two patients (patients 1 and 2) showed elevated basal PRL concentrations with an exaggerated (patient 2) or normal (patient 1) PRL response to TRH, while baseline PRL levels as well as TRH-induced PRL release were normal or subnormal in the remaining five patients. Injection of 100 jug LRH had no effect on serum GH or PRL concentrations in any of the subjects. In four patients studied, administration of a single dose (5 mg orally) of CB154 invariably suppressed their serum GH and PRL levels to less than 50% of the baseline values. Perfusion studies The perfusion procedures employed in this study were modifications of those originally described by Lacy et al. (11). Surgically removed, fresh pituitary adenoma tissue, weighing 25-103 mg, was minced with scissors in isotonic saline and then placed en bloc on a Millipore filter (SMWP 02500, Millipore Co., MA). The filter was fitted in a Millipore filtration chamber (Swinnex-25), which was submerged in a water bath at 37 C. The tissue was perfused with Krebs-Ringer-bicarbonate buffer containing 7.72 mM calcium, 0.5% human serum albumin, and 0.2% glucose at a constant flow rate of 0.5 ml/min by means of a peristaltic pump. The buffer was freshly prepared in each experiment and equilibrated with a gaseous phase of 95% O2 and 5% CO2 throughout the period of perfusion. The pH of this buffer ranged from 7.35-7.40. Test substances were dissolved in the fresh gassed buffer and introduced into the chamber via a side-arm constant infusion pump. Theflowpressure was continuously monitored and flow rate, during the infusion of test materials, was adjusted to the same 0.5 ml/min as in the control perfusion. Perfusate was collected every 5 min (2.5 ml) on a

fraction collector. Samples were stored at -20 C until analyzed. CB154 was a gift from Dr. von Orelli (Sandoz, Basel, Switzerland). Synthetic TRH and LRH were purchased from Tanabe Pharmaceutical Co. (Osaka, Japan). RIAs GH and PRL concentrations in each fraction were estimated by RIAs, as previously described in detail (9, 12). The materials for RIAs were kindly provided by the NIAMDD and the National Pituitary Agency. Statistical analysis Significance of changes in hormone concentrations after TRH and CB154 infusion were evaluated by analysis of variance.

Results Baseline secretion of GH and PRL Perfusion of adenoma tissue with the medium resulted in a relatively stable baseline secretion of GH and PRL (Figs. 1-3). PRL secretion was observed in most experiments using pituitary adenoma obtained from acromegalic patients with normoprolactinemia (patients 3-7) as well as from those with hyperprolactinemia (patients 1 and 2), though its secretion rate was far less compared with that of GH. Infusion of synthetic LRH at a concentration of 0.2 or 1 jug/ml for 10 min had no effect on GH or PRL release, as was the case in vivo (Figs. 1 and 2).


TRH AND BROMOERGOCRIPTINE IN ACROMEGALY Effect of TRH on GH and PRL secretion Effect of TRH at a concentration of 1 jug/ml for 10 min was examined in three adenoma tissues (Fig. 1). In all of the experiments, a definite increase in both GH and PRL levels was obtained. The peak GH and PRL values after TRH infusion are significantly greater than the mean + 3 SD of basal hormone concentrations (six preinfusion values) in each

200 180 160 140 120 100 80 60 40

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experiment (P < 0.01). The pattern of GH response was essentially the same as that of PRL. When the adenoma tissue from hyperprolactinemic patients (patients 1 and 2) was utilized, the PRL response was usually more pronounced. The response appeared as early as 5 min after the initiation of the TRH infusion. GH and PRL levels peaked at 10-30 min and then gradually declined to the baseline at 25-45 min. The peak GH concentrations ranged from 470-1030% of the control, while the maximum rate of PRL secretion was 205-1540% of the baseline. Q, Synthetic TRH infusion for 10 min at vary^3

2000

20 0

1500

1000

80 70

500

60

^

50

™

noi

40 z 30 «

ex:

10 °" o t!

•g 1 —. = uj

° 2000

M 1500 o *

1000

t

4000 3000 2000 20

40

60

80 100 120 140 160 180 200 220 *

TIME

IN MINUTES

1000 0

FIG. 1. GH and PRL responses to LRH or TRH infusion in three separate experiments using different pituitary adenoma tissues. LRH and TRH were infused at a concentration of 1 ng/ml for 10 min. Each experiment was started at 0 time.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 TIME

IN

MINUTES

FIG. 3. Effect of CB154 (10 jug/ml) on basal as well as TRH-indused GH and PRL secretion from three pituitary adenoma tissues. TRH at a concentration of 1 jug/ml was infused for 10 min. Each experiment was started at 0 time.

FIG. 2. GH and PRL responses of pituitary adenoma tissue to different doses of TRH. LRH at a concentration of 0.2 /ig/ml and TRH ranging from 10 ng/ml to 1 jug/ml were infused for 10 min. The experiment was started at 0 time.

80

100

TIME

120

140

160

240

MINUTES



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ing concentrations from 10 ng/ml to 1 jug/ml promptly produced a significant increase {P < 0.01) in effluent GH levels (Fig. 2). A doseresponse relationship between the concentrations of TRH and the GH responses was demonstrated. Similarly, though quantitatively less, the tissue secreted PRL in response to TRH. This effect was clear at a higher dose of TRH. Effect of CB154 on GH and PRL secretion Perfusion of CB154 at a concentration of 10 jug/ml precipitously decreased the basal secretion of GH and PRL from adenoma tissue (Fig. 3). This effect was already apparent by 10 min after CB154 infusion. The GH and PRL concentrations during and after CB154 infusion are significantly lower than the mean — 3 SD of the baseline levels (six preinfusion values) in each experiment (P < 0.01). The secretory rate of GH was depressed to 17-35% of the control levels. When the infusion was discontinued, the suppressive action of CB154 lasted as long as 80 min. In two experiments, TRH was infused at a concentration of 1 jug/ml over a period of 10 min concomitantly with CB154 (Fig. 3). A significant increase (P < 0.01) in the GH secretion rate was observed, although the baseline GH levels were greatly reduced. However, TRH-induced PRL release was completely blocked in all of the experiments. Similarly, infusion of TRH resulted in a marked GH secretion in the period after the cessation of CB154 perfusion. No stimulatory effect of TRH on PRL secretion was again demonstrable on this occasion. Discussion Administration of TRH has been shown to elicit GH release in several pathological conditions, including acromegaly (4, 5, 13-16). Whether this effect may be mediated by the hypothalamus or, alternatively, may be attributed to altered cellular membrane receptors of the GH-producing pituitary adenomas was not determined. The present study clearly demonstrates that TRH possesses a direct action on pituitary adenoma tissue of acro-

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megalic patients to induce GH release. One may argue that the elevated GH levels after TRH infusion observed in this study were merely due to fluctuations in basal GH secretory rate. However, this possibility is highly unlikely, since a fairly constant hormone secretion was obtained in the control period and the effect of TRH appeared promptly after the initiation of infusion, which is consistent with an in vivo finding. Moreover, a doseresponse relationship between TRH concentrations and GH responses was established (Fig. 2). The finding that LRH had no effect on GH secretion, as was the case in vivo, also supports this view. PRL secretion was demonstrated in pituitary adenoma tissues from five acromegalic patients with normoprolactinemia as well as those from two hyperprolactinemic subjects. One possibility is that adenoma tissues were contaminated with adjacent normal pituitary lactotrophs. However, this is a less probable explanation, based on the facts that the tissues were obtained at transsphenoidal selective removal of adenomas and histological examination performed in randomly selected tissue pieces revealed that all of them possessed characteristics of eosinophilic adenoma. Relevant in this context is an immunohistochemical observation that identified PRL-producing cells in adenomas obtained from acromegalic patients whose serum PRL levels were not elevated (17,18). Coupled with the present finding that TRH induces PRL release in a similar manner as that of GH, it was concluded that PRL-secreting cells exist in most eosinophilic adenoma tissues of acromegalics, which possess cellular membrane receptors to TRH. This may explain an exaggerated PRL response to TRH in some acromegalic subjects with hyperprolactinemia. CB154 has been shown to suppress the circulating levels of PRL in men (19) as well as in experimental animals (20). This drug is also effective in lowering serum GH concentrations in many acromegalic patients, and its use in the medical management of this disease has been proposed (2, 9, 10, 21, 22). No consensus is available at present regarding the site of action of CB154 in acromegalics. While it is


TRH AND BROMOERGOCRIPTINE IN ACROMEGALY

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generally believed that dopaminergic drugs However, the time course of GH response to act on the central nervous system, a direct TRH in acromegalic patients is completely effect of CB154 has been suggested (3, 23). different from that observed in these condiOur study clearly shows that CB154 has a tions, which suggests a divergent mode of acdirect action on pituitary adenoma tissue to tion of TRH in acromegaly and in other pathinhibit both GH and PRL secretion. This ef- ological states without pituitary tumor. fect was prompt and continued for a long Acknowledgments period even after the infusion was discontinThe authors are indebted to Professor K. Kosaka, The ued. When T R H was infused concomitantly Third Department of Internal Medicine, for his advice with CB154, the stimulatory effect of T R H on and continuous encouragement throughout the study; to GH release was not overcome. Similarly, T R H Assistant Professor K. Hirakawa and his colleagues, Deresulted in a rise in GH levels when T R H was partment of Neurosurgery, for providing us with pituitary followed by CB154 infusion. In sharp contrast, adenoma tissues obtained at surgery; and to Dr. H. Sando, TRH-mediated PRL secretion was completely The Third Department of Internal Medicine, for his helpblocked in both situations. These results are ful suggestions on the perfusion system. They are also grateful to Dr. W. von Orelli (Sandoz, Ltd., Basel, Switzconsonant with those obtained in vivo (9). erland) for the gift of CB154. Taken together with the finding alluded to above that T R H exerts its effect directly on References pituitary adenoma, the result led us to the 1. LIUZZI, A., P. G. CHIODINI, L. BOTALLA, G. CREMASCOLI, AND F. SILVESTRINI, Inhibitory effect of L-dopa on GH release in conclusion that there exist two different recepacromegalic patients, J Clin Endocrinol Metab 35: 941,1972. tors to T R H and CB154 in GH-producing cells 2. LIUZZI, A., P. G. CHIODINI, L. BOTALLA, G. CREMASCOLI, E. E. MULLER, AND F. SILVESTRINI, Decreased plasma growth of adenoma tissue from acromegalic subjects hormone (GH) levels in acromegalics following CB154 (2-Bror, alternatively, T R H and CB154 share a a-ergocryptine) administration, J Clin Endocrinol Metab 38: 910, 1974. common site of action on a cellular membrane 3. CAMANNI, F., F. MASSARA, L. BELFORTE, A. ROSATELLO, AND receptor on adenoma tissues and a smaller G. M. MOLINATTI, Effect of dopamine on plasma growth hormone and prolactin levels in normal and acromegalic subdose of CB154 employed in this study failed jects, J Clin Endocrinol Metab 44: 465, 1977. to block TRH-induced GH release. In light of 4. IRIE, M., AND T. TSUSHIMA, Increase of serum growth hormone concentration following thyrotropin-releasing hormone the finding that GH inhibitory action of injection in patients with acromegaly and gigantism, J Clin CB154 is demonstrable only in patients who Endocrinol Metab 35: 97, 1972. secrete GH in response to TRH, the latter 5. SCHALCH, D. S., D. GONZALEZ-BARCENA, A. J. KASTIN, A. V. SCHALLY, AND L. A. LEE, Abnormalities in the release of TSH view might be more likely. The receptor of in response to thyrotropin-releasing hormone (TRH) in patients with disorders of the pituitary, hypothalamus and basal PRL-secreting cells to T R H , on the other ganglia, J Clin Endocrinol Metab 35: 609, 1972. hand, may be identical to the site of action of 6. RUBIN, A. L., S. R. LEVIN, R. I. BERNSTEIN, J. B. TYRRELL, C. NOACCO, AND P. H. 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Tenth Annual Meeting of the European Thyroid Association The 10th Annual Meeting of the European Thyroid Association will be held in Newcastle upon Tyne, July 2-6, 1979. The preliminary program, abstract forms, and registration forms are available from: Dr. W. M. G. Tunbridge (ETA 10) Ward 2 Office Newcastle General Hospital Newcastle upon Tyne NE4 6BE United Kingdom Registration fee for non-members is £45. The deadline for submission of abstracts is February 24, 1979, and the registration deadline is March 31, 1979.


Growth hormone and prolactin secretion of pituitary adenoma.

Induction of growth hormone and prolactin secretion by luteinizing hormone-releasing hormone and its blockade by bromoergocriptine in acromegalic patients.

Effect of bromoergocriptine on TRH-induced growth hormone and prolactin release in acromegalic patients.

Effect of starvation on pituitary growth hormone cells and blood growth hormone and prolactin levels in the rat.

Effects of alcohol on growth hormone secretion in acromegaly.

Effect of galanin on the growth hormone response to growth hormone-releasing hormone in acromegaly.

Follicle stimulating hormone-secreting pituitary adenoma: inappropriate secretion and effect of pulsatile luteinizing hormone releasing hormone analogue (buserelin) administration.

Effect of pyridoxine on pituitary release of growth hormone and prolactin in childhood and adolescence.

Growth hormone release inhibiting hormone: actions on thyrotrophin and prolactin secretion after thyrotrophinreleasing hormone.

The effect of bombesin and related peptides on prolactin and growth hormone secretion in the rat.

The dynamics of growth hormone and prolactin secretion in acromegalic patients with "mixed" pituitary tumours.

Effect of sodium valproate on the secretion of prolactin, cortisol and growth hormone in migraine patients.

Metformin inhibits proliferation and growth hormone secretion of GH3 pituitary adenoma cells.

Effects of hypothalamic dopamine on growth hormone-releasing hormone-induced growth hormone secretion and thyrotropin-releasing hormone-induced prolactin secretion in goats.

Pulsatile growth hormone secretion in patients with acromegaly and normal men: the effects of growth hormone-releasing hormone infusion.

The effect of "binge" ethanol exposure on growth hormone and prolactin gene expression and secretion.

Effect of chicken hypothalamus on prolactin and growth hormone secretion in male chickens.

Effect of somatostatin on abnormal growth hormone and prolactin secretion in patients with the carcinoid syndrome.

Effect of naloxone or levallorphan on serum prolactin concentrations and apomorphine-induced growth hormone secretion.

Ultrastructural localization of prolactin, growth hormone and luteinizing hormone by immunocytochemical techniques in the bovine pituitary.

Growth hormone and prolactin secretion in cultured somatomammotroph cells.

A case of pituitary adenoma producing both growth hormone (GH) and adrenocorticotropic hormone (ACTH).

The effect of varying prolactin levels on pituitary luteinizing hormone and follicle-stimulating hormone response to gonadotropin-releasing hormone.

Effects of prolonged treatment with diltiazem on pituitary secretion of luteinizing hormone, follicle-stimulating hormone, thyrotropin and prolactin.