0013-7227/78/0132-0480$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 2 Printed in U.S.A.
Studies on Autoregulation of Prolactin Secretion from Perifused Rat Pituitary Glands in the Basal and Thyrotropin-Releasing Hormone-Stimulated States* BRUCE L. MALTZ, MAIRE T. BUCKMAN,f AND GLENN T. PEAKE WITH EXPERT TECHNICAL ASSISTANCE OF JOSEPHINE MORRIS
Medicine and Research Services, Veterans Administration Hospital, and the Department of Medicine, The University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 ABSTRACT. In order to evaluate autoregulation of PRL secretion at the pituitary gland, spontaneous and TRH-stimulated PRL secretion was determined in an isolated anterior pituitary (AP) perifusion system utilizing normal and chronically hyperprolactinemic (MtTW 15 tumor-bearing) male rat pituitaries. Decreased AP weight in chronically hyperprolactinemic rats [5.7 ± 0.1 (SE) VS. 7.2 ± 0.2 mg in controls, P < 0.001] was associated with lower mean basal PRL secretion compared to controls (9.9 ± 1.8 vs. 20.6 ± 2.7 ng/min/AP, P < 0.01). PRL secretion from normal AP was not altered by the addition of ovine PRL (oPRL) at 10 or 100 ng/ml (19.9 ± 3.8 and 19.2 ± 2.9 ng/min/AP, respectively). TRHstimulated PRL secretion from normal AP in the absence of oPRL was 133 ± 7% of baseline (designated as
100%) and this did not differ from the response observed in the presence of 10 or 100 ng/ml oPRL (134 ± 9 and 133 ± 1%, respectively). Basal PRL secretion from tumor rat AP was likewise unaffected by the presence of medium oPRL (9.9 ± 1.8 in the absence of oPRL and 10.0 ± 1.9 ng/min/AP in the presence of 100 ng/ml oPRL). TRH-stimulated PRL secretion from tumor rat AP was 149 ± 10% of baseline in the absence of medium oPRL and 140 ± 7% in the presence of 100 ng/ml oPRL (P > 0.10). The data suggest that autoregulation of PRL secretion exists, and this regulation may not be mediated directly at the pituitary gland but rather at higher, possibly hypothalamic, centers. (Endocrinology 103: 480, 1978)
A
UTOREGULATION of pituitary PRL quently reported. These in vitro experiments secretion has been amply demonstrated using static incubation systems have given in the rat. Decreased pituitary PRL stores conflicting results, with one report indicating have been found in rats rendered hyperprolac- that addition of oPRL to the medium detinemic by 1) injection with transplantable creased PRL release (7) and another report PRL-secreting pituitary tumors (1-3); 2) demonstrating no effect of increased medium transplantation of normal rat pituitaries under PRL on release from incubated pituitaries (8). the kidney capsule (4); and 3) administration In the experiments reported here, a continof ovine PRL (oPRL) (4). The hypothalamus uous anterior pituitary (AP) perifusion system (3, 5) and the pituitary gland (6) have been was utilized. The advantages of this system suggested as sites of PRL autoregulation. In include: 1) the continuous exchange of mevivo experiments have not allowed definitive dium which probably more closely approxiseparation of hypothalamic vs. pituitary influ- mates physiological conditions; and 2) assessences on PRL autoregulation (1-4). In con- ment of changes in minute-to-minute rates of trast, in vitro experiments using isolated pi- hormone release which may reveal transient tuitary glands to evaluate feedback regulation alterations in hormone secretion which might directly at the pituitary gland have been infre- otherwise be obscured in the usual static incubation system. As alterations in basal hormone secretion may be less discriminatory Received August 8, 1977. than those of stimulated hormone secretion Address requests for reprints to: Dr. Maire T. Buckman, Research Service (151), Veterans Administration and as stimulated hormone secretion has not Hospital, 2100 Ridgecrest Drive S.E., Albuquerque, New been previously studied, the effects of physiMexico 87131. ological concentrations of medium oPRL on * This work was supported by NIH Grants HD-05794- TRH-provoked PRL secretion were investi05 and RR-00997-02. gated. PRL responsiveness was studied in pit Recipient of VA Clinical Investigator Award. 480 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 06:32 For personal use only. No other uses without permission. . All rights reserved.
AUTOREGULATION OF PRL SECRETION
tuitaries obtained from normal and from chronically hyperprolactinemic MtTW 15 tu* mor-bearing male rats. Materials and Methods Animals
y
Male Wistar-Furth rats were purchased from Sprague-Dawley, Madison, WI. Fourteen animals were inoculated in the sc tissue of the hind leg with the PRL- and GH-secreting transplantable pituitary tumor MtTW 15, as previously described (9). Animals were housed in individual cages in a temperature-controlled (70 ± 1 F) and artificially lighted room with an automatic light-dark cycle (lights on from 0600-1800 h). Standard rat chow and tap water were available ad libitum. Perifusion protocol
481
either no PRL or 10 or 100 ng/ml oPRL (NIH-PS11; biological potency estimate, 26 IU/mg). After a 1-h stabilization period, during which time hormone secretion tended to be erratic, 2-min aliquots were collected over a 30-35-min period. After this basal period, 100 ng/ml TRH (Abbott lot 844-8901) was added to the perifusate. Mean hormone secretion was determined over the subsequent 30-45-min period. In order to assure pituitary tissue viability and continued hormonal responsiveness after approximately 2.5 h of perifusion, the response to a conventional GH secretogogue, aminophylline, was determined during the initial six experiments utilizing normal pituitary glands. After a 30-min postTRH period, 2.5 mg/ml aminophylline ([theophylline2] • ethylenediamine, Sigma Chemical Co.) were added to the perfusing buffer. In each instance, GH secretion increased by at least 2.5-fold above baseline (range, 2.5- to 10-fold above the mean basal levels). Thus, after several hours of perifusion, GH responsiveness remained intact, indicating a viable pituitary gland.
Pituitaries were obtained from animals without anesthesia by rapid decapitation by guillotine and blood was collected from the trunk vessels. After decapitation, the pituitary fossa was quickly ex- RIAs posed, the posterior pituitary was dissected free Rat PRL (rPRL) (11) and rat GH (rGH) (12) and discarded, and the anterior pituitary was care- were assayed as previously described. All determifully removed, weighed on a Roller Smith precision nations were done in duplicate and all samples from balance, and placed in a perifusion chamber. Each a single study were assayed in the same assay. chamber contained a single whole anterior pituitary oPRL at concentrations up to 1000 ng/ml failed to gland. cross-react in the rPRL assay. Thus, addition of 10 The perifusion system utilized was a modification or 100 ng/ml oPRL to the perfusate did not interof the method described by Carlson et al. (10). fere with assay of rPRL released into the medium. Modified Gey and Gey buffer contained 111 mM NaCl, 3.7 mM KC1, 0.79 mM Na2 HPO4 7H2O, 0.22 Statistics mM KH2PO4, 27 mM NaHCO3, 0.28 mM Statistics using the paired two-tailed Student's t MgSO4 7H2O, 1.03 mM MgCl2 6H2O, 2.64 mM CaCl2, 1 mg/ml glucose, and 1 g/100 ml bovine test were performed to compare mean basal and serum albumin (Sigma, Cohn fraction V). The TRH-stimulated PRL secretion in the same pituibuffer was gased with a mixture of humidified 95% tary gland. All other statistical calculations were O2-5% CO2 at 37 C. Utilizing a Harvard peristaltic performed using the unpaired two-tailed Student's pump set at 0.5 ml/min, the buffer was pumped t test. through Tygon S50 TL tubing, with an id of 1.6 mm (Norton Plastics and Synthetics Division), Results which was connected to the perifusion chamber. The chamber consisted of a 13-mm steel Millipore Serum PRL concentration filter holder outfitted with a steel grid and an 8-jnm Mean serum PRL concentration was 5 ± 1 membrane filter (Sartorius Divisions, Brinkmann Instruments). Media, tubing, and chambers were ng/ml (SE) for normal male rats and 19,177 ± submerged in a constant 37 C water bath. Effluent 4,449 ng/ml for MtTW 15 tumor-bearing rats. from the perifusion chambers was collected on an automatic fraction collector at 2-min intervals. Pituitary weights These fractions were frozen at -20 C until assayed. Pituitary atrophy in the MtTW 15 tumor To evaluate the effect of the presence of PRL in the perifusing medium on endogenous PRL secre- group vs. control group was evident by a sigtion, the modified Gey and Gey buffer contained nificant decrease in both absolute pituitary
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MALTZ, BUCKMAN, AND PEAKE
482
weight (5.7 ± 0.1 vs. 7.2 ± 0.2 mg, P < 0.001) and pituitary weight per 100 g BW (1.8 ± 0.08 vs. 2.9 ±0.04, P< 0.001). Representative patterns of spontaneous and TRH-stimulated PRL secretion (Fig. 1) Basal PRL secretion from normal and tumor rat pituitaries in the absence and presence of 10 and 100 ng/ml oPRL was fairly constant in some pituitaries and pulsatile in others. After addition of TRH to the perifusing medium, a pulsatile pattern of PRL secretion was frequently seen and this was not significantly altered by addition of oPRL. Patterns of PRL secretion were highly variable and the frequency and magnitude of the pulsations did not seem to be consistently altered by the addition of oPRL. Basal PRL secretion (Table 1) Basal PRL secretion was significantly de-
Kndo • 1978 Vol 103 • No 2
creased in tumor pituitary glands compared to controls in the absence and presence of oPRL. Addition of oPRL to the medium failed to alter PRL secretion from either normal or tumor pituitary glands. TRH-stimulated PRL secretion from normal rat pituitaries (Fig. 2) Pituitary PRL secretion in response to TRH was variable and an occasional pituitary gland (8.5%) failed to respond altogether. In the absence of oPRL, perifusion with TRH resulted in a mean increase in PRL of 133 ± 7% of baseline (P < 0.005 compared to baseline secretion). With the addition of 10 ng/ml oPRL to the perifusate, TRH produced a similar mean increase of 134 ± 9% (P < 0.01). When 100 ng/ml oPRL was added to the medium, TRH produced a mean rise to 133 ± 7% (P < 0.001). TRH-stimulated PRL secretion in the presence of 100 ng/ml oPRL
, , t
FIG. 1. Representative patterns of individual normal and MtTW 15 tumor pituitary PRL secretion in the basal and TRH-stimulated states in the presence and absence of medium oPRL. The arrows indicate time of TRH addition to the medium. The ordinate represents medium rPRL and the abscissa represents the tube number. Each tube represents a 2-min collection.
II 13 15 17 19 21 23 25 27 29 31
II 13 15 17 19 21 23 25 27 29 31
TUBE
• • , • t • • • , 13 15 17 19 21 23 25 27 29 31
NUMBER
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483
AUTOREGULATION OF PRL SECRETION
TABLE 1. Basal and TRH-stimulated incremental change in PRL secretion in normal and MtTW 15 tumor-bearing rat pituitaries in the absence of medium oPRL and in the presence of 10 and 100 ng/ml oPRL.
Medium oPRL (ng/ml) 0 10 100
Basal PRL secretion (ng/min/AP)
TRH-stimulated PRL secretion APRL (ng/min/AP)
Normal 20.6 ± 2.7 (11) 19.9 ± 3.8 (11) 19.2 ± 2.9 (11)
APRL (ng/min/mg AP)
Tumor 9.9 ± 1.8" (7) 10.0 ± 1.9'' (7)
Normal
Tumor
Normal
Tumor
5.8 ± 1.4 5.6 ± 1.6 5.5 ± 1.0
4.9 ± 1.2
0.9 ± 0.2 0.9 ± 0.3 0.8 ± 0.2
0.9 ± 0.2
4.1 ± 1.3
0.8 ± 0.2
Values are expressed as .r ± SE. Parentheses indicate number of pituitaries in each group. " P < 0.01 compared to normal. '' P < 0.05 compared to normal.
MtT WI5
BASE TRH 10ng/ml oPRL
BASE " TRH IOOng/ml oPRL
FIG. 2. TRH-stimulated PRL secretion of individual conBASE TRH lOOng/ml o PRL o o PRL trol rat pituitaries in the presence of 0, 10, and 100 ng oPRL/ml perifusing medium. Baseline secretion is desig- FIG. 3. TRH-stimulated PRL secretion of individual nated as 100%. The horizontal bars represent the mean MtTW 15 tumor-bearing rat pituitaries in the presence of stimulated responses. 0 and 100 ng oPRL/ml perifusing medium. Baseline secretion is designated as 100%. The horizontal bars repredid not differ significantly from that observed sent the mean stimulated responses in the two groups.
in the absence of oPRL (P > 0.10) or with addition of 10 ng/ml oPRL (P > 0.10). Thus, TRH-induced PRL secretion from normal pituitary glands was not affected by the presence of 10 or 100 ng/ml oPRL in the medium. TRH-stimulated PRL secretion from MtTW 15 tumor rat pituitaries (Fig. 3) In the absence of oPRL, addition of 100 ng/ml TRH to the perifusing medium resulted in a mean increase in medium PRL of 149 ± 10% of baseline secretion (P < 0.005). Compared to normal rat pituitaries, mean TRHstimulated PRL secretion from tumor rat pituitaries, expressed as a percentage of baseline, was greater, but the difference was not significant (133 ± 7 vs. 149 ± 10%, P > 0.10). Addition of 100 ng oPRL to the perifusate was associated with a mean PRL increase of 140 ± 7% of baseline secretion (P < 0.02). The difference in TRH-induced PRL secretion in tumor rat pituitaries not exposed to oPRL
compared to those exposed to 100 ng/ml oPRL was not significantly different (P > 0.10). In the presence of 100 ng/ml oPRL, addition of TRH resulted in a slightly greater mean PRL response, expressed as a percentage of baseline, in pituitaries obtained from tumor rats than those from normal rats (140 ± 7% vs. 133 ± 7%, P > 0.10). Incremental (Table 1)
TRH-induced
PRL response
In normal rat pituitary glands, the mean incremental rise in PRL secretion after TRH administration was unaffected by the presence of 10 and 100 ng/ml oPRL. Likewise, addition of 100 ng/ml oPRL to medium perifusing tumor rat pituitaries failed to result in a significant change in the TRH-induced incremental PRL rise. Although tumor rat pituitaries seemed to have a somewhat diminished TRHstimulated incremental PRL response com-
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MALTZ, BUCKMAN, AND PEAKE
484
pared to controls, both in the absence of oPRL and in the presence of 100 ng/ml oPRL, these differences were not statistically significant (P > 0.10). To evaluate whether the smaller size of the pituitaries from the tumor-bearing rats affected the incremental rise in PRL after TRH administration, the incremental response was expressed in milligrams of AP weight (Table 1, last two columns). When expressed in this manner, the incremental changes were identical in normal and tumor rat pituitaries. This suggests that the functional PRL reserve elicited by TRH from each milligram of tissue was comparable in normal and tumor rat pituitary glands. Discussion The hypothalamus and the anterior pituitary are two logical sites at which PRL may feed back to regulate its own secretion. Experimental evidence has implicated both the hypothalamus and the pituitary as the site of this autoregulation. Hypothalamic PRL release-inhibiting factor (PIF) content has been reported to be increased in MtTW 15 and MtTW 5 tumor-bearing rats (3). In contrast to this observation, MacLeod and Abad (13) reported preliminary evidence that rats with hormone-secreting pituitary tumors do not exhibit altered releasing or inhibiting factors in extracts of their hypothalamic tissues. The pituitary itself as a locus for feedback regulation is suggested by the studies of Nicoll et al. (7) who reported that addition of oPRL to an in vitro pituitary incubation system resulted in decreased release of PRL during the first hour over a 4-h incubation period; there was, however, no significant overall difference compared to controls. Voogt and Ganong (8) could demonstrate no difference in cumulative release of PRL from male pituitaries incubated continuously or with replacement of medium every 30 min, suggesting that the accumulated PRL released failed to alter subsequent PRL secretion. Addition of oPRL at 0.1-100.0 jug/3 ml medium for 2 h also had no effect on PRL release. These authors concluded that the pituitary gland was not a site of PRL feedback regulation.
Kndo V«l 10.) , N o ;
The present studies also suggest that PRL secretion is not autoregulated by a direct effect at the pituitary gland. Basal PRL secretion from normal rat pituitaries was not affected by the presence of physiological concentrations of PRL (10 or 100 ng/ml medium). Likewise, 100 ng/ml oPRL did not alter basal secretion from MtTW 15 tumor-bearing rat pituitaries. Thus, basal secretion was not affected in either normal or chronically hyperprolactinemic rat pituitary glands by the presence of oPRL in the perifusate. As stimulated hormone secretion may, in some instances, provide a more sensitive indicator of alterations in secretory response, the direct pituitary PRL secretogogue, TRH, was added to the perifusate. TRH was found to exert a modest stimulatory effect on PRL secretion from normal and chronically hyperprolactinemic pituitaries. Although both basal and TRH-induced PRL secretion per pituitary gland was decreased in the hyperprolactinemic state, the mean incremental rise per mg pituitary tissue was similar from hyperprolactinemic and control pituitary glands. This observation suggests that TRH-releasable PRL stores per mg pituitary tissue were similar in hyperprolactinemic and control pituitary glands. Despite the fact that in vitro de novo synthesis of PRL per mg pituitary tissue has been shown to be decreased in animals bearing PRL-secreting tumors (13, 14), the readily releasable pool of stored PRL per mg tissue in our study was not diminished by exposure of the pituitary to the chronically hyperprolactinemic state. The addition of oPRL to the perifusing medium did not further alter TRH-stimulated PRL release from either normal or MtTW 15 tumor rat pituitaries. Thus, the TRH-releasable pool of PRL was not affected by either prior chronic exposure to hyperprolactinemia or continued exposure to more physiological concentrations of PRL in the perfusate. In the present study, rats bearing the MtTW 15 PRL- and GH-secreting ectopic pituitary tumor had significantly lower pituitary weight compared to normal controls when expressed either in milligrams alone or in relation to body weight. This observation is in
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AUTOREGULATION OF PRL SECRETION agreement with those by MacLeod etal. (1, 2) who reported pituitary atrophy in rats bearing several PRL-secreting ectopic tumors. Chen et al. (3) likewise reported decreased AP weight in ovariectomized and adrenalecto•mized MtTW 5 and MtTW 15 tumor-bearing animals compared to controls. As all ectopic tumors studied secreted other hormones in addition to PRL, PRL alone could not be definitively implicated in the observed AP atrophy. To delineate further the role of hyperprolactinemia in pituitary atrophy, Sinha and Tucker (4) induced isolated hyperprolactinemia by injecting oPRL or by transplantation of normal pituitaries from donor animals under the kidney capsule of recipient animals. The resultant hyperprolactinemia in these experiments was associated with decreased pituitary weight. The latter studies directly implicate PRL in mediating the observed pituitary atrophy. The pituitary atrophy observed in hyperprolactinemic animals has been shown to be associated with decreased pituitary PRL concentration in ectopic PRL-secreting tumor-bearing rats (1-3), in oPRLtreated rats (4), and in rats carrying several normal pituitary transplants (4). The studies presented here further demonstrate that spontaneous PRL secretion is also decreased in chronically hyperprolactinemic rats compared to controls. Thus, chronic hyperprolactinemia is associated with pituitary atrophy, decreased pituitary PRL concentration, and, in the present study, decreased PRL secretion. We conclude from these studies that autoregulation of PRL secretion does occur, as demonstrated by pituitary atrophy and decreased spontaneous PRL secretion from rat pituitaries exposed to chronic hyperprolactinemia. However, this regulation does not occur at the level of the pituitary gland and may involve a hormone pool distinct from the TRH-releasable PRL pool.
Acknowledgment We thank Dr. Albert F. Parlow, Dr. Alfred E. Wilhelmi, and the National Pituitary Agency, NIAMDD, for giving us the oPRL and the materials for the rPRL and GH
485
RIAs. The TRH used in these studies was a gift from Abbott Laboratories, North Chicago, IL. We acknowledge Pat Dodd for typing the manuscript.
References 1. MacLeod, R. M., M. C. Smith, and G. W. DeWitt, Hormonal properties of transplanted pituitary tumor and their relation to the pituitary gland, Endocrinology 79: 1149, 1966. 2. MacLeod, R. M., G. W. DeWitt, and M. C. Smith, Suppression of pituitary gland hormone content by pituitary tumor hormones, Endocrinology 82: 889, 1968. 3. Chen, C. L., H. Minaguchi, and J. Meites, Effects of transplanted pituitary tumors on host pituitary prolactin secretion, Proc Soc Exp Biol Med 126: 317, 1967. 4. Sinha, Y. N., and H. A. Tucker, Pituitary prolactin content and mammary development after chronic administration of prolactin, Proc Soc Exp Biol Med 128: 84, 1968. 5. Meites, J., and J. A. Clemens, Hypothalamic control of prolactin secretion, Vitam Horm 30: 165, 1972. 6. Spies, H. G., and M. T. Clegg, Pituitary as a possible site of prolactin feedback in autoregulation, Neuroendocrinology 8: 205, 1971. 7. Nicoll, C. S., Aspects of the neural control of prolactin secretion, In Ganong, W. F., and L. Martini (eds.), Frontiers in Neuroendocrinology, Oxford University Press, New York, 1971, p. 291. 8. Voogt, J. L., and W. F. Ganong, In vitro evidence against the anterior pituitary as a site of negative feedback of prolactin, Proc Soc Exp Biol Med 147: 795, 1974. 9. Peake, G. T., I. K. Mariz, and W. H. Daughaday, Radioimmunoassay of growth hormone in rats bearing somatotropin producing tumors, Endocrinology 83: 714, 1968. 10. Carlson, H. E., I. K. Mariz, and W. H. Daughaday, Thyrotropin-releasing hormone stimulation and somatostatin inhibition of growth hormone secretion from perfused rat adenohypophyses, Endocrinology 94: 1709, 1974. 11. Birge, C. A., L. S. Jacobs, C. T. Hammer, and W. H. Daughaday, Catecholamine inhibition of prolactin secretion by isolated rat adenohypophyses, Endocrinology 86: 120, 1970. 12. Peake, G. T., A. L. Steiner, and W. H. Daughaday, Guanosine 3'5' cyclic monophosphate is a potent pituitary growth hormone secretogogue, Endocrinology 90: 212, 1972. 13. MacLeod, R. M., and A. Abad, On the control of prolactin and growth hormone synthesis in rat pituitary glands, Endocrinology 83: 799, 1968. 14. MacLeod, R. M., and J. E. Lehmeyer, Restoration of prolactin synthesis and release by the administration of monoaminergic blocking agents to pituitary tumorbearing rats, Cancer Res 34: 345, 1974.
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Vol. 103, No. 2 Printed in U.S.A.
Studies on Autoregulation of Prolactin Secretion from Perifused Rat Pituitary Glands in the Basal and Thyrotropin-Releasing Hormone-Stimulated States* BRUCE L. MALTZ, MAIRE T. BUCKMAN,f AND GLENN T. PEAKE WITH EXPERT TECHNICAL ASSISTANCE OF JOSEPHINE MORRIS
Medicine and Research Services, Veterans Administration Hospital, and the Department of Medicine, The University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 ABSTRACT. In order to evaluate autoregulation of PRL secretion at the pituitary gland, spontaneous and TRH-stimulated PRL secretion was determined in an isolated anterior pituitary (AP) perifusion system utilizing normal and chronically hyperprolactinemic (MtTW 15 tumor-bearing) male rat pituitaries. Decreased AP weight in chronically hyperprolactinemic rats [5.7 ± 0.1 (SE) VS. 7.2 ± 0.2 mg in controls, P < 0.001] was associated with lower mean basal PRL secretion compared to controls (9.9 ± 1.8 vs. 20.6 ± 2.7 ng/min/AP, P < 0.01). PRL secretion from normal AP was not altered by the addition of ovine PRL (oPRL) at 10 or 100 ng/ml (19.9 ± 3.8 and 19.2 ± 2.9 ng/min/AP, respectively). TRHstimulated PRL secretion from normal AP in the absence of oPRL was 133 ± 7% of baseline (designated as
100%) and this did not differ from the response observed in the presence of 10 or 100 ng/ml oPRL (134 ± 9 and 133 ± 1%, respectively). Basal PRL secretion from tumor rat AP was likewise unaffected by the presence of medium oPRL (9.9 ± 1.8 in the absence of oPRL and 10.0 ± 1.9 ng/min/AP in the presence of 100 ng/ml oPRL). TRH-stimulated PRL secretion from tumor rat AP was 149 ± 10% of baseline in the absence of medium oPRL and 140 ± 7% in the presence of 100 ng/ml oPRL (P > 0.10). The data suggest that autoregulation of PRL secretion exists, and this regulation may not be mediated directly at the pituitary gland but rather at higher, possibly hypothalamic, centers. (Endocrinology 103: 480, 1978)
A
UTOREGULATION of pituitary PRL quently reported. These in vitro experiments secretion has been amply demonstrated using static incubation systems have given in the rat. Decreased pituitary PRL stores conflicting results, with one report indicating have been found in rats rendered hyperprolac- that addition of oPRL to the medium detinemic by 1) injection with transplantable creased PRL release (7) and another report PRL-secreting pituitary tumors (1-3); 2) demonstrating no effect of increased medium transplantation of normal rat pituitaries under PRL on release from incubated pituitaries (8). the kidney capsule (4); and 3) administration In the experiments reported here, a continof ovine PRL (oPRL) (4). The hypothalamus uous anterior pituitary (AP) perifusion system (3, 5) and the pituitary gland (6) have been was utilized. The advantages of this system suggested as sites of PRL autoregulation. In include: 1) the continuous exchange of mevivo experiments have not allowed definitive dium which probably more closely approxiseparation of hypothalamic vs. pituitary influ- mates physiological conditions; and 2) assessences on PRL autoregulation (1-4). In con- ment of changes in minute-to-minute rates of trast, in vitro experiments using isolated pi- hormone release which may reveal transient tuitary glands to evaluate feedback regulation alterations in hormone secretion which might directly at the pituitary gland have been infre- otherwise be obscured in the usual static incubation system. As alterations in basal hormone secretion may be less discriminatory Received August 8, 1977. than those of stimulated hormone secretion Address requests for reprints to: Dr. Maire T. Buckman, Research Service (151), Veterans Administration and as stimulated hormone secretion has not Hospital, 2100 Ridgecrest Drive S.E., Albuquerque, New been previously studied, the effects of physiMexico 87131. ological concentrations of medium oPRL on * This work was supported by NIH Grants HD-05794- TRH-provoked PRL secretion were investi05 and RR-00997-02. gated. PRL responsiveness was studied in pit Recipient of VA Clinical Investigator Award. 480 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 06:32 For personal use only. No other uses without permission. . All rights reserved.
AUTOREGULATION OF PRL SECRETION
tuitaries obtained from normal and from chronically hyperprolactinemic MtTW 15 tu* mor-bearing male rats. Materials and Methods Animals
y
Male Wistar-Furth rats were purchased from Sprague-Dawley, Madison, WI. Fourteen animals were inoculated in the sc tissue of the hind leg with the PRL- and GH-secreting transplantable pituitary tumor MtTW 15, as previously described (9). Animals were housed in individual cages in a temperature-controlled (70 ± 1 F) and artificially lighted room with an automatic light-dark cycle (lights on from 0600-1800 h). Standard rat chow and tap water were available ad libitum. Perifusion protocol
481
either no PRL or 10 or 100 ng/ml oPRL (NIH-PS11; biological potency estimate, 26 IU/mg). After a 1-h stabilization period, during which time hormone secretion tended to be erratic, 2-min aliquots were collected over a 30-35-min period. After this basal period, 100 ng/ml TRH (Abbott lot 844-8901) was added to the perifusate. Mean hormone secretion was determined over the subsequent 30-45-min period. In order to assure pituitary tissue viability and continued hormonal responsiveness after approximately 2.5 h of perifusion, the response to a conventional GH secretogogue, aminophylline, was determined during the initial six experiments utilizing normal pituitary glands. After a 30-min postTRH period, 2.5 mg/ml aminophylline ([theophylline2] • ethylenediamine, Sigma Chemical Co.) were added to the perfusing buffer. In each instance, GH secretion increased by at least 2.5-fold above baseline (range, 2.5- to 10-fold above the mean basal levels). Thus, after several hours of perifusion, GH responsiveness remained intact, indicating a viable pituitary gland.
Pituitaries were obtained from animals without anesthesia by rapid decapitation by guillotine and blood was collected from the trunk vessels. After decapitation, the pituitary fossa was quickly ex- RIAs posed, the posterior pituitary was dissected free Rat PRL (rPRL) (11) and rat GH (rGH) (12) and discarded, and the anterior pituitary was care- were assayed as previously described. All determifully removed, weighed on a Roller Smith precision nations were done in duplicate and all samples from balance, and placed in a perifusion chamber. Each a single study were assayed in the same assay. chamber contained a single whole anterior pituitary oPRL at concentrations up to 1000 ng/ml failed to gland. cross-react in the rPRL assay. Thus, addition of 10 The perifusion system utilized was a modification or 100 ng/ml oPRL to the perfusate did not interof the method described by Carlson et al. (10). fere with assay of rPRL released into the medium. Modified Gey and Gey buffer contained 111 mM NaCl, 3.7 mM KC1, 0.79 mM Na2 HPO4 7H2O, 0.22 Statistics mM KH2PO4, 27 mM NaHCO3, 0.28 mM Statistics using the paired two-tailed Student's t MgSO4 7H2O, 1.03 mM MgCl2 6H2O, 2.64 mM CaCl2, 1 mg/ml glucose, and 1 g/100 ml bovine test were performed to compare mean basal and serum albumin (Sigma, Cohn fraction V). The TRH-stimulated PRL secretion in the same pituibuffer was gased with a mixture of humidified 95% tary gland. All other statistical calculations were O2-5% CO2 at 37 C. Utilizing a Harvard peristaltic performed using the unpaired two-tailed Student's pump set at 0.5 ml/min, the buffer was pumped t test. through Tygon S50 TL tubing, with an id of 1.6 mm (Norton Plastics and Synthetics Division), Results which was connected to the perifusion chamber. The chamber consisted of a 13-mm steel Millipore Serum PRL concentration filter holder outfitted with a steel grid and an 8-jnm Mean serum PRL concentration was 5 ± 1 membrane filter (Sartorius Divisions, Brinkmann Instruments). Media, tubing, and chambers were ng/ml (SE) for normal male rats and 19,177 ± submerged in a constant 37 C water bath. Effluent 4,449 ng/ml for MtTW 15 tumor-bearing rats. from the perifusion chambers was collected on an automatic fraction collector at 2-min intervals. Pituitary weights These fractions were frozen at -20 C until assayed. Pituitary atrophy in the MtTW 15 tumor To evaluate the effect of the presence of PRL in the perifusing medium on endogenous PRL secre- group vs. control group was evident by a sigtion, the modified Gey and Gey buffer contained nificant decrease in both absolute pituitary
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MALTZ, BUCKMAN, AND PEAKE
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weight (5.7 ± 0.1 vs. 7.2 ± 0.2 mg, P < 0.001) and pituitary weight per 100 g BW (1.8 ± 0.08 vs. 2.9 ±0.04, P< 0.001). Representative patterns of spontaneous and TRH-stimulated PRL secretion (Fig. 1) Basal PRL secretion from normal and tumor rat pituitaries in the absence and presence of 10 and 100 ng/ml oPRL was fairly constant in some pituitaries and pulsatile in others. After addition of TRH to the perifusing medium, a pulsatile pattern of PRL secretion was frequently seen and this was not significantly altered by addition of oPRL. Patterns of PRL secretion were highly variable and the frequency and magnitude of the pulsations did not seem to be consistently altered by the addition of oPRL. Basal PRL secretion (Table 1) Basal PRL secretion was significantly de-
Kndo • 1978 Vol 103 • No 2
creased in tumor pituitary glands compared to controls in the absence and presence of oPRL. Addition of oPRL to the medium failed to alter PRL secretion from either normal or tumor pituitary glands. TRH-stimulated PRL secretion from normal rat pituitaries (Fig. 2) Pituitary PRL secretion in response to TRH was variable and an occasional pituitary gland (8.5%) failed to respond altogether. In the absence of oPRL, perifusion with TRH resulted in a mean increase in PRL of 133 ± 7% of baseline (P < 0.005 compared to baseline secretion). With the addition of 10 ng/ml oPRL to the perifusate, TRH produced a similar mean increase of 134 ± 9% (P < 0.01). When 100 ng/ml oPRL was added to the medium, TRH produced a mean rise to 133 ± 7% (P < 0.001). TRH-stimulated PRL secretion in the presence of 100 ng/ml oPRL
, , t
FIG. 1. Representative patterns of individual normal and MtTW 15 tumor pituitary PRL secretion in the basal and TRH-stimulated states in the presence and absence of medium oPRL. The arrows indicate time of TRH addition to the medium. The ordinate represents medium rPRL and the abscissa represents the tube number. Each tube represents a 2-min collection.
II 13 15 17 19 21 23 25 27 29 31
II 13 15 17 19 21 23 25 27 29 31
TUBE
• • , • t • • • , 13 15 17 19 21 23 25 27 29 31
NUMBER
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483
AUTOREGULATION OF PRL SECRETION
TABLE 1. Basal and TRH-stimulated incremental change in PRL secretion in normal and MtTW 15 tumor-bearing rat pituitaries in the absence of medium oPRL and in the presence of 10 and 100 ng/ml oPRL.
Medium oPRL (ng/ml) 0 10 100
Basal PRL secretion (ng/min/AP)
TRH-stimulated PRL secretion APRL (ng/min/AP)
Normal 20.6 ± 2.7 (11) 19.9 ± 3.8 (11) 19.2 ± 2.9 (11)
APRL (ng/min/mg AP)
Tumor 9.9 ± 1.8" (7) 10.0 ± 1.9'' (7)
Normal
Tumor
Normal
Tumor
5.8 ± 1.4 5.6 ± 1.6 5.5 ± 1.0
4.9 ± 1.2
0.9 ± 0.2 0.9 ± 0.3 0.8 ± 0.2
0.9 ± 0.2
4.1 ± 1.3
0.8 ± 0.2
Values are expressed as .r ± SE. Parentheses indicate number of pituitaries in each group. " P < 0.01 compared to normal. '' P < 0.05 compared to normal.
MtT WI5
BASE TRH 10ng/ml oPRL
BASE " TRH IOOng/ml oPRL
FIG. 2. TRH-stimulated PRL secretion of individual conBASE TRH lOOng/ml o PRL o o PRL trol rat pituitaries in the presence of 0, 10, and 100 ng oPRL/ml perifusing medium. Baseline secretion is desig- FIG. 3. TRH-stimulated PRL secretion of individual nated as 100%. The horizontal bars represent the mean MtTW 15 tumor-bearing rat pituitaries in the presence of stimulated responses. 0 and 100 ng oPRL/ml perifusing medium. Baseline secretion is designated as 100%. The horizontal bars repredid not differ significantly from that observed sent the mean stimulated responses in the two groups.
in the absence of oPRL (P > 0.10) or with addition of 10 ng/ml oPRL (P > 0.10). Thus, TRH-induced PRL secretion from normal pituitary glands was not affected by the presence of 10 or 100 ng/ml oPRL in the medium. TRH-stimulated PRL secretion from MtTW 15 tumor rat pituitaries (Fig. 3) In the absence of oPRL, addition of 100 ng/ml TRH to the perifusing medium resulted in a mean increase in medium PRL of 149 ± 10% of baseline secretion (P < 0.005). Compared to normal rat pituitaries, mean TRHstimulated PRL secretion from tumor rat pituitaries, expressed as a percentage of baseline, was greater, but the difference was not significant (133 ± 7 vs. 149 ± 10%, P > 0.10). Addition of 100 ng oPRL to the perifusate was associated with a mean PRL increase of 140 ± 7% of baseline secretion (P < 0.02). The difference in TRH-induced PRL secretion in tumor rat pituitaries not exposed to oPRL
compared to those exposed to 100 ng/ml oPRL was not significantly different (P > 0.10). In the presence of 100 ng/ml oPRL, addition of TRH resulted in a slightly greater mean PRL response, expressed as a percentage of baseline, in pituitaries obtained from tumor rats than those from normal rats (140 ± 7% vs. 133 ± 7%, P > 0.10). Incremental (Table 1)
TRH-induced
PRL response
In normal rat pituitary glands, the mean incremental rise in PRL secretion after TRH administration was unaffected by the presence of 10 and 100 ng/ml oPRL. Likewise, addition of 100 ng/ml oPRL to medium perifusing tumor rat pituitaries failed to result in a significant change in the TRH-induced incremental PRL rise. Although tumor rat pituitaries seemed to have a somewhat diminished TRHstimulated incremental PRL response com-
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MALTZ, BUCKMAN, AND PEAKE
484
pared to controls, both in the absence of oPRL and in the presence of 100 ng/ml oPRL, these differences were not statistically significant (P > 0.10). To evaluate whether the smaller size of the pituitaries from the tumor-bearing rats affected the incremental rise in PRL after TRH administration, the incremental response was expressed in milligrams of AP weight (Table 1, last two columns). When expressed in this manner, the incremental changes were identical in normal and tumor rat pituitaries. This suggests that the functional PRL reserve elicited by TRH from each milligram of tissue was comparable in normal and tumor rat pituitary glands. Discussion The hypothalamus and the anterior pituitary are two logical sites at which PRL may feed back to regulate its own secretion. Experimental evidence has implicated both the hypothalamus and the pituitary as the site of this autoregulation. Hypothalamic PRL release-inhibiting factor (PIF) content has been reported to be increased in MtTW 15 and MtTW 5 tumor-bearing rats (3). In contrast to this observation, MacLeod and Abad (13) reported preliminary evidence that rats with hormone-secreting pituitary tumors do not exhibit altered releasing or inhibiting factors in extracts of their hypothalamic tissues. The pituitary itself as a locus for feedback regulation is suggested by the studies of Nicoll et al. (7) who reported that addition of oPRL to an in vitro pituitary incubation system resulted in decreased release of PRL during the first hour over a 4-h incubation period; there was, however, no significant overall difference compared to controls. Voogt and Ganong (8) could demonstrate no difference in cumulative release of PRL from male pituitaries incubated continuously or with replacement of medium every 30 min, suggesting that the accumulated PRL released failed to alter subsequent PRL secretion. Addition of oPRL at 0.1-100.0 jug/3 ml medium for 2 h also had no effect on PRL release. These authors concluded that the pituitary gland was not a site of PRL feedback regulation.
Kndo V«l 10.) , N o ;
The present studies also suggest that PRL secretion is not autoregulated by a direct effect at the pituitary gland. Basal PRL secretion from normal rat pituitaries was not affected by the presence of physiological concentrations of PRL (10 or 100 ng/ml medium). Likewise, 100 ng/ml oPRL did not alter basal secretion from MtTW 15 tumor-bearing rat pituitaries. Thus, basal secretion was not affected in either normal or chronically hyperprolactinemic rat pituitary glands by the presence of oPRL in the perifusate. As stimulated hormone secretion may, in some instances, provide a more sensitive indicator of alterations in secretory response, the direct pituitary PRL secretogogue, TRH, was added to the perifusate. TRH was found to exert a modest stimulatory effect on PRL secretion from normal and chronically hyperprolactinemic pituitaries. Although both basal and TRH-induced PRL secretion per pituitary gland was decreased in the hyperprolactinemic state, the mean incremental rise per mg pituitary tissue was similar from hyperprolactinemic and control pituitary glands. This observation suggests that TRH-releasable PRL stores per mg pituitary tissue were similar in hyperprolactinemic and control pituitary glands. Despite the fact that in vitro de novo synthesis of PRL per mg pituitary tissue has been shown to be decreased in animals bearing PRL-secreting tumors (13, 14), the readily releasable pool of stored PRL per mg tissue in our study was not diminished by exposure of the pituitary to the chronically hyperprolactinemic state. The addition of oPRL to the perifusing medium did not further alter TRH-stimulated PRL release from either normal or MtTW 15 tumor rat pituitaries. Thus, the TRH-releasable pool of PRL was not affected by either prior chronic exposure to hyperprolactinemia or continued exposure to more physiological concentrations of PRL in the perfusate. In the present study, rats bearing the MtTW 15 PRL- and GH-secreting ectopic pituitary tumor had significantly lower pituitary weight compared to normal controls when expressed either in milligrams alone or in relation to body weight. This observation is in
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AUTOREGULATION OF PRL SECRETION agreement with those by MacLeod etal. (1, 2) who reported pituitary atrophy in rats bearing several PRL-secreting ectopic tumors. Chen et al. (3) likewise reported decreased AP weight in ovariectomized and adrenalecto•mized MtTW 5 and MtTW 15 tumor-bearing animals compared to controls. As all ectopic tumors studied secreted other hormones in addition to PRL, PRL alone could not be definitively implicated in the observed AP atrophy. To delineate further the role of hyperprolactinemia in pituitary atrophy, Sinha and Tucker (4) induced isolated hyperprolactinemia by injecting oPRL or by transplantation of normal pituitaries from donor animals under the kidney capsule of recipient animals. The resultant hyperprolactinemia in these experiments was associated with decreased pituitary weight. The latter studies directly implicate PRL in mediating the observed pituitary atrophy. The pituitary atrophy observed in hyperprolactinemic animals has been shown to be associated with decreased pituitary PRL concentration in ectopic PRL-secreting tumor-bearing rats (1-3), in oPRLtreated rats (4), and in rats carrying several normal pituitary transplants (4). The studies presented here further demonstrate that spontaneous PRL secretion is also decreased in chronically hyperprolactinemic rats compared to controls. Thus, chronic hyperprolactinemia is associated with pituitary atrophy, decreased pituitary PRL concentration, and, in the present study, decreased PRL secretion. We conclude from these studies that autoregulation of PRL secretion does occur, as demonstrated by pituitary atrophy and decreased spontaneous PRL secretion from rat pituitaries exposed to chronic hyperprolactinemia. However, this regulation does not occur at the level of the pituitary gland and may involve a hormone pool distinct from the TRH-releasable PRL pool.
Acknowledgment We thank Dr. Albert F. Parlow, Dr. Alfred E. Wilhelmi, and the National Pituitary Agency, NIAMDD, for giving us the oPRL and the materials for the rPRL and GH
485
RIAs. The TRH used in these studies was a gift from Abbott Laboratories, North Chicago, IL. We acknowledge Pat Dodd for typing the manuscript.
References 1. MacLeod, R. M., M. C. Smith, and G. W. DeWitt, Hormonal properties of transplanted pituitary tumor and their relation to the pituitary gland, Endocrinology 79: 1149, 1966. 2. MacLeod, R. M., G. W. DeWitt, and M. C. Smith, Suppression of pituitary gland hormone content by pituitary tumor hormones, Endocrinology 82: 889, 1968. 3. Chen, C. L., H. Minaguchi, and J. Meites, Effects of transplanted pituitary tumors on host pituitary prolactin secretion, Proc Soc Exp Biol Med 126: 317, 1967. 4. Sinha, Y. N., and H. A. Tucker, Pituitary prolactin content and mammary development after chronic administration of prolactin, Proc Soc Exp Biol Med 128: 84, 1968. 5. Meites, J., and J. A. Clemens, Hypothalamic control of prolactin secretion, Vitam Horm 30: 165, 1972. 6. Spies, H. G., and M. T. Clegg, Pituitary as a possible site of prolactin feedback in autoregulation, Neuroendocrinology 8: 205, 1971. 7. Nicoll, C. S., Aspects of the neural control of prolactin secretion, In Ganong, W. F., and L. Martini (eds.), Frontiers in Neuroendocrinology, Oxford University Press, New York, 1971, p. 291. 8. Voogt, J. L., and W. F. Ganong, In vitro evidence against the anterior pituitary as a site of negative feedback of prolactin, Proc Soc Exp Biol Med 147: 795, 1974. 9. Peake, G. T., I. K. Mariz, and W. H. Daughaday, Radioimmunoassay of growth hormone in rats bearing somatotropin producing tumors, Endocrinology 83: 714, 1968. 10. Carlson, H. E., I. K. Mariz, and W. H. Daughaday, Thyrotropin-releasing hormone stimulation and somatostatin inhibition of growth hormone secretion from perfused rat adenohypophyses, Endocrinology 94: 1709, 1974. 11. Birge, C. A., L. S. Jacobs, C. T. Hammer, and W. H. Daughaday, Catecholamine inhibition of prolactin secretion by isolated rat adenohypophyses, Endocrinology 86: 120, 1970. 12. Peake, G. T., A. L. Steiner, and W. H. Daughaday, Guanosine 3'5' cyclic monophosphate is a potent pituitary growth hormone secretogogue, Endocrinology 90: 212, 1972. 13. MacLeod, R. M., and A. Abad, On the control of prolactin and growth hormone synthesis in rat pituitary glands, Endocrinology 83: 799, 1968. 14. MacLeod, R. M., and J. E. Lehmeyer, Restoration of prolactin synthesis and release by the administration of monoaminergic blocking agents to pituitary tumorbearing rats, Cancer Res 34: 345, 1974.
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