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The effects of estradiol on pituitary responsiveness to dopamine in vitro: A comparison of ovariectomized fischer 344 and Holtzman rats
Life Sciences, Vol. 50, pp. 235-243 Printed in the USA
Pergamon Press
THE EFFECTS OF ESTRADIOL ON PITUITARY RESPONSIVENESS TO DOPAMINE IN VITRO: A COMPARISON OF OVARIECTOMIZED FISCHER 344 AND HOLTZMAN RATS 1 David Lawson and Patrick Parker Department of Physiology Wayne State University School of Medicine Detroit, MI 48201 (Received in final form November 13, 1991)
Summary The objective of this study was to determine if the effectiveness of dopamine as an inhibitor of prolactin is altered by estradiol in strains of rats which show marked differences in estrogen-induced pituitary hyperplasia. Groups of Fischer 344 and Holtzman Sprague-Dawley rats were ovariectomized and implanted with Silastic capsules of estradiol. Rats were sacrificed by rapid decapitation following a brief period of ether anesthesia at 2, 4, 6, 8 weeks (F-344) or at 2 and 8 weeks (Holtzman) of estradiol treatment. The pituitary was removed and cut into fragments which were either snap frozen for initial prolactin content measurements or incubated for 60 min in the presence or absence of dopamine (i x 10-6M). Prolactin was measured in the plasma, in sonicates of the pituitary and in the incubation medium by double antibody radioirmmunoassay. Pituitary weight and plasma levels of prolactin were significantly less in Holtzman rats compared to Fischer 344 females at 2 or 8 weeks of estradiol treatment but pituitary concentrations of prolactin were not different between the two strains. Pituitary fragments from Fischer 344 rats studied at 2 and 4 weeks of estradiol treatment did not respond to the removal of dopamine in vitro whereas pituitary fragments from Holtzman rats obtained at 2 weeks of estradiol treatment did release significantly more prolactin in the absence than in the presence of dopamine. Pituitary fragments taken from Fischer 344 rats at 6 and 8 weeks were responsive to dopamine whereas pituitary tissue from Holtzman rats was not responsive at 8 weeks. The data indicate that temporal differences in responsiveness to the inhibitory effects of dopamine occur in strains which are susceptible or resistant to the formation of pituitary tumors following prolonged estradiol treatment. It has long been recognized that Fischer 344 (F-344) rats are more prone to develop estrogen-induced pituitary hyperplasia than rats of the SpragueDawley strain. Wiklund, Wertz and Gorski (i) showed that pituitary weight, DNA content, and prolactin synthesis were all higher in F-344 rats than in Holtzman Sprague-Dawley rats over an 8-week period of DES treatment and that these changes were due, at least in part, to factors within the pituitary
iSupported by NIH grant RR 08167-12. 0024-3205/92 $5.00 + .00
Copyright (c) 1991 Pergamon Press plc
236
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since pituitaries from F-344 donors, but not those from Holtzman donors, respond to DES treatment when transplanted into common recipients. Although it appears that lactotrophs of F-344 rats are more sensitive to one or more of the many actions of estradiol than are those of Sprague-Dawley rats, it has not been established which of these actions are responsible for the differences between strains. One action of estrogen is to interfere with the inhibitory action of dopamine on prolactin release (2-9). Part of this effect of estrogen in vivo is as a result of indirect (via prolactin) inhibition of the tuberinfundibular dopaminergic system which has been observed following long-term estrogen treatment (I0). However, short-term estrogen treatment has been shown to increase the concentration of dopamine arriving at the pituitary in the portal blood (6,11). In addition, changes in the level of dopamine in the portal blood do not explain the apparent local pituitary control of hyperplasia reported by Wiklund et al. (i). Estrogen has also been shown to eliminate low affinity pituitary dopamine receptors (9) and to decrease total dopamine receptor number (12). However, DiPaolo et al. (13) reported no change in dopamine receptor binding following estrogen treatment. In a recent review (14), Enjalbert proposed mechanisms by which estrogen may affect the responsiveness to dopamine at a point(s) after receptor binding. In fact, Nansel et al. (7) have reported intracellular effects (increase of iysosomal enzymes) of dopamine which may be reduced by estrogen. The purpose of the present study was to determine if the differences in estrogen-induced pituitary hyperplasia and prolactin secretion between F-344 and Holtzman rats are associated with differences in the in vitro responsiveness to dopamine at various periods of treatment with estradiol in vivo. Materials Animals Mature female F-344 and Holtzman Dawley, Indianapolis, IN) were housed lighting (lights on 0600 to 2000 hrs), (50%) with food and tap water provided and 7 days later given a subcutaneous 10-15 mg/implant).
and Methods
Sprague-Dawley rats (Harlan Spraguetwo per cage in a room with controlled temperature (23C) and relative humidity ad lib. Each rat was ovariectomized Silastic implant of estradiol-17B (E2,
Experimental Procedures Six rats of each strain were anesthetized with ether for approximately one minute and then decapitated between 1200 and 1230 hrs at 2, 4, 6, and 8 weeks (F-344) or 2 and 8 weeks of E2 treatment (Holtzman) and the anterior pituitaries and blood were collected. Plasma obtained from the trunk blood was stored at -20C until assayed for prolactin. The anterior pituitary was cut into fragments (2-5 mg in size). Two fragments from each rat were immediately snap frozen on dry ice as non-incubated controls and the remaining fragments were placed into wells of 24-well tissue culture plates containing one ml of alpha-modified Minimal Essential Medium (aMEM) buffered with 21 mM NaHCO3 and containing 0.1% bovine serum albumin (BSA), 0.1% ascorbic acid, and 10-6M dopamine. The plates were incubated at 37C in a humidified atmosphere of 95% 02-5% CO 2 for 60 min. The pituitary fragments were then transferred to other plates the wells of which contained c~MEM, 0.1% BSA, 0.1% ascorbic acid, 21 mM NaHCO3 with or without 10-6M dopamine and the incubation was continued for a second 60 min period. The incubated pituitary fragments were then collected and snap frozen on dry ice. The medium from both incubation periods was collected and frozen at -20C until assayed for prolactin. The frozen pituitary fragments were weighed and then sonicated for i0 seconds in one ml of assay buffer (phosphate-buffered saline, PBS) containing 1% Triton X-100
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and 0.1% bovine serum albumin. The sonicates were allowed to stand at room temperature for one hour and then they were diluted 1:20 in assay buffer and stored at -20C until assayed for prolactin. Prolactin Radioimmunoassay Plasma, pituitary sonicates and incubation medium were assayed for prolactin by double antibody radioimmunoassay as described previously (15). Following appropriate pre-assay dilution, these samples were assayed in two dilutions in duplicate using NIAMDD-RP-I (ii IU/mg) as standard. The concentrations of prolactin were expressed as either ng/ml or ~g/mg wet tissue weight. In addition, the prolactin released into the incubation medium during the second hour was expressed as % of prolactin available for release. Prolactin available for release was calculated by subtracting the amount of prolactin released into incubation medium during the first hour from the average prolactin content of the non-incubated pituitary fragments from the same rat. Statistical Analysis One way ANOVA and Tukey's hsd multiple comparison test were used to compare means across times of estradiol treatment. The comparison of the effects of dopamine vs. no dopamine and the differences between strains at each period of estradiol treatment was done using an F-test. Significant differences were assessed at P<0.05. Results Total pituitary weight, plasma levels of prolactin and pituitary prolactin content in both F-344 and Holtzman rats from 2 to 8 weeks of estradiol treatment are shown in Figure IA, B and C respectively. Significant increases in pituitary weight and plasma levels of prolactin were observed in the F-344 rats over the duration of estradiol treatment, but pituitary prolactin content significantly decreased over the same period. Holtzman rats showed a significantly smaller increase in pituitary weight over the 8 week period of estradiol treatment compared to F-344 rats. Plasma levels of prolactin increased significantly over this period in Holtzman rats but the level at 8 weeks was significantly lower than plasma levels in F-344 rats. Pituitary content of prolactin in the Holtzman strain was not significantly different from the F-344 rats at 2 or 8 weeks of estradiol treatment and a decrease across the duration of steroid treatment was also observed. The release of prolactin from pituitary fragments into medium in the presence or absence of dopamine (expressed as ~g/mg AP) is shown in Figure 2. At 2 weeks of estradiol treatment pituitaries of Holtzman rats released significantly more prolactin in the absence than in the presence of dopamine, but a similar difference was not present at 8 weeks of treatment (Figure 2A). Prolactin release in the presence of dopamine was not different between weeks 2 and 8 in Holtzman rats. Prolactin release from pituitary fragments of F-344 rats was not significantly increased by the removal of dopamine at 2 or 4 weeks but at 6 and 8 weeks of estradiol treatment dopamine withdrawal significantly increased prolactin release (Figure 2B). The data in Figure 2B also show that dopamine was more effective in inhibiting prolactin release at 6 and 8 weeks of estradiol treatment than at 2 or 4 weeks in F-344 rats. When in vitro prolactin release was expressed as a % of that available for release and hence corrected for the decrease in pituitary content seen with increased duration of estrogen treatment, the same qualitative differences in responsiveness to dopamine were observed in both strains at
238
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Vol.
50, No.
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P I T U I T A R Y WEIGHT, P L A S M A A N D P I T U I T A R Y P R O L A C T I N IN O V A R I E C T O M I Z E D F I S C H E R 344 (F-344) A N D H O L T Z M A N S P R A G U E - D A W L E Y (HO) R A T S F O L L O W I N G E S T R A D I O L TREATMENT. A. T o t a l p i t u i t a r y weight, B. P l a s m a levels of p r o l a c t i n , C. Pituitary prolactin concentration. +Bars r e p r e s e n t m e a n ± S.E.M. M e a n s w i t h d i f f e r e n t letters (a, b, a, S) w i t h i n strains are s t a t i s t i c a l l y d i f f e r e n t P<0.05 (n=6-12).
3, 1992
Vol. 50, NO. 3, 1992
Lactotroph Responsiveness After Estrogen
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FIG. 2 EFFECT OF DOPAMINE ON PROLACTIN RELEASE IN VITRO FROM PITUITARY EXPLANTS OF OVARIECTOMIZED HOLTZMAN (A) AND FISCHER 344 (B) RATS AT VARIOUS TIMES OF ESTRADIOL TREATMENT. Bars represent mean ± S.E.M. (n=6). Means within strains with different letters (a,b) are significantly different. *,*** +DA vs. -DA at each estrogen treatment period (P<0.05 or P<0.001).
239
240
Lactotroph Responsiveness After Estrogen
Vol. 50, No. 3, 1992
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FIG. 3 EFFECT OF DOPAMINE ON PROLACTIN RELEASE IN VITRO FROM PITUITARY EXPLANTS OF OVARIECTOMIZED HOLTZMAN (A) OR FISCHER 344 (B) RATS A T VARIOUS TIMES OF ESTRADIOL TREATMENT. Data calculated from that shown in Figure 2. Pituitary content available for release was pituitary content of non-incubated tissue minus prolactin released during first hour of incubation. Statistical comparisons are as shown in Figure 2.
Vol. 50, No. 3, 1992
Lactotroph Responsiveness After Estrogen
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each period of estrogen treatment examined (Figure 3A and B). However, the differences across weeks of estrogen treatment were less dramatic than seen in Figure 2. In fact, there were no significant differences in prolactin release in the presence of dopamine across the 8 weeks of estrogen treatment in the F-344 rats (Figure 3B). Discussion The results of the present study indicate that F-344 and Holtzman rats are different with respect to the temporal changes in pituitary weight, plasma concentrations of prolactin and in vitro responsiveness to dopamine during an 8 week period of estradiol treatment. On the other hand, both strains showed similar changes in pituitary concentration of prolactin over the period. It is clear from the current study that F-344 rats show a more marked pituitary hyperplasia to estradiol than do Holtzman rats. This confirms the observations of Wiklund et al. ( I ) . Rats of the F-344 strain also released significantly more prolactin into the plasma than did Holtzman females. The large difference in plasma prolactin between strains extends the previous report of Stone et al. (16). Since the pituitary concentrations of prolactin were not significantly different between the strains, the large difference in plasma levels of prolactin could have been due in part to the larger mass of the F-344 pituitaries. However, the large difference in plasma levels could have also been due to a greater decrease in hypophysial portal blood dopamine or a greater reduction in the inhibitory effects of dopamine in F-344 than in Holtzman rats. Dopamine did not inhibit in vitro prolactin release in F-344 rats at 2 and 4 weeks of estradiol treatment whereas the inhibitory effects were restored by 6 and 8 weeks of estrogen treatment. These observations suggest that the long-lasting hyperprolactinemia in this strain cannot be completely explained by a sustained reduction in the effects of dopamine on the lactotroph. One must be cautious about extrapolating results obtained in vitro to the in vivo condition. However, we do have unpublished data which show that bromocryptine does not significantly inhibit prolactin release in vivo in F344 rats treated with DES for 1 week but is effective at 4 weeks of DES treatment. Perhaps the long-lasting hyp~rprolactinemia seen in vivo is due to a reduced concentration of dopamine arriving at the lactotroph or to an increase in prolactin releasing hormones from the hypothalamus or stimulatory paracrine or autocrine factors from the pituitary itself. The local production of stimulatory factors within the pituitary could explain the results of Wiklund et al. (I) that pituitary transplants from F-344 rats, but not those from Holtzman rats, become hyperplastic in common recipients treated with estrogen. However, there is also strong evidence that dopamine reaching the lactotroph is reduced by long term estrogen treatment either by decreased release from tuberoinfundibular neurons (i0) or by the dilution of portal blood by systemic blood carried to the pituitary by non-portal vessels formed by arteriogenesis (17). The pituitaries of the F-344 rats in the current study began to show gross vascular changes at 6 weeks of estradiol treatment suggesting that arteriogenesis may play a role in the later stages of the hyperplasia and hyperprolactinemia noted in this strain. That pituitaries of Holtzman rats ultimately showed a decreased responsiveness to the inhibitory effects of dopamine in vitro indicates that Holtzman and F-344 rats do respond similarly to estradiol treatment albeit over a different time course. Early reports indicated that Sprague-Dawley rats do develop pituitary hyperplasia if estrogen treatment is continued for a sufficiently long period (18). Clearly, the strains showed marked temporal differences in responsiveness to dopamine but it is not clear what mechanisms
242
Lactotroph Responsiveness After Estrogen
account for the differences. to address these mechanisms.
Vol. 50, No. 3, 1992
Ongoing studies in this laboratory will attempt
One possible factor that may influence the observed temporal changes in the responsiveness to dopamine is the level of prolactin in the intracellular pool that can be inhibited by dopamine. Mena and his coworkers (19) have shown that newly synthesized prolactin is strongly inhibited by dopamine whereas the prolactin that has been stored in the lactotroph for more than one hour is not as susceptible to dopamine inhibition. In the F-344 rat, it is possible that late in estrogen treatment much of the intracellular prolactin was newly synthesized and strongly inhibited by dopamine whereas in the early periods of estradiol treatment a greater proportion of the intracellular prolactin was in the stored component and not so easily inhibited by dopamine. On the other hand, in the Holtzman rats more pituitary prolactin may have been in the newly synthesized, dopamine responsive pool, early in estradiol treatment, but as the treatment was continued a greater proportion of stored prolactin may have been present. In summary, the data obtained in the current study show that there is an association between the loss of pituitary responsiveness to dopamine and the susceptibility of rat strains to show pituitary hyperplasia in response to DES treatment. Acknowledgements The authors wish to thank Drs. Raiti and Parlow of the National Pituitary Agency and the NIDDK for rat prolactin used for standards and for iodination. We also wish to thank Dr. Richard Gala for the use of equipment to do the radioiranunoassay. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17.
J. WIKLUND, N. WERTZ and J. GORSKI, Endocrinology 109 1700-1707 (1981). V. RAYMOND, M. BEAULIEU, F. LABRIE and J. BOISSIER, Science 200 11731175 (1978). S. JAQUES and R.R. GALA, Acta Endocrinol. 92 437-447 (1979). L. FERLAND, F. LABRIE, C. EUVRARD and J-P. RAYNAUD, Mol. Cell. Endocrinol. 14 199-204 (1979). B. WEST and P.S. DANNIES, Endocrinology 106 1108-1113 (1980). G.A. GUDELSKY, D.D. NANSEL and J.C. PORTER, Endocrinology 108 440-444 (1981). D.D. NANSEL, G.A. GUDELSKY, M.J. REYMOND and J.C. PORTER, Endocrinology 108 903-907 (1981). V. GIGUERE, H. MEUNIER, R. VEILLEUX and F. LABRIE, Endocrinology 111 857-862 (1982). D. BRESSION, A.M. BRANDI, P. PAGESY, M. LE DAFNIET, M. MARTINET, S. BRAILLY, M. MICHARD and F. PEILLON, Endocrinology 116 1905-1911 (1985). K.T. DEMAREST, G.D. RIEGLE and K.E. MOORE, Neuroendo. 39 193-200 (1984). N.S. PILOTTE, D.R. BURT and C.E. BARRACLOUGH, Endocrinology 114 23062311 (1984). M.L. HEIMAN and N. BEN-JONATHAN, Endocrinology Iii 1057-1060 (1982). T. DIPAOLO, R. CARMICHAEL, F. LABRIE and J-P. RAYNAUD, Mol. Cell. Endocrinol. 16 99-112 (1979). A. ENJALBERT, Horm. Res. 31 6-12 (1989). E.Y.H. KUO and R.R. GALA, Biochim. Biophys. Acta 264 462-471 (1972). J.P. STONE, S. HOLTZMAN and C.J. SHELLABARGER, Cancer Res. 39 773-778 (1979). J. SCHECTER, N. AHMAD and R. WEINER, Amer. J. Anat. 179 315-323 (1987).
Vol. 50, No. 3, 1992
18. 19.
Lactotroph Responsiveness After Estrogen
C.W. WELSCH, T. JENKINS, Y. AMENOMORI and J. MEITES, Experientia 27 1350-1352 (1971). F. MENA, C. CLAPP, D. AGUAYO, M.T. MORALES, C.E. GROSVENOR and G. MARTINEZ DE LA ESCALERA, Endocrinology 125 1814-1820 (1989).
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Pergamon Press
THE EFFECTS OF ESTRADIOL ON PITUITARY RESPONSIVENESS TO DOPAMINE IN VITRO: A COMPARISON OF OVARIECTOMIZED FISCHER 344 AND HOLTZMAN RATS 1 David Lawson and Patrick Parker Department of Physiology Wayne State University School of Medicine Detroit, MI 48201 (Received in final form November 13, 1991)
Summary The objective of this study was to determine if the effectiveness of dopamine as an inhibitor of prolactin is altered by estradiol in strains of rats which show marked differences in estrogen-induced pituitary hyperplasia. Groups of Fischer 344 and Holtzman Sprague-Dawley rats were ovariectomized and implanted with Silastic capsules of estradiol. Rats were sacrificed by rapid decapitation following a brief period of ether anesthesia at 2, 4, 6, 8 weeks (F-344) or at 2 and 8 weeks (Holtzman) of estradiol treatment. The pituitary was removed and cut into fragments which were either snap frozen for initial prolactin content measurements or incubated for 60 min in the presence or absence of dopamine (i x 10-6M). Prolactin was measured in the plasma, in sonicates of the pituitary and in the incubation medium by double antibody radioirmmunoassay. Pituitary weight and plasma levels of prolactin were significantly less in Holtzman rats compared to Fischer 344 females at 2 or 8 weeks of estradiol treatment but pituitary concentrations of prolactin were not different between the two strains. Pituitary fragments from Fischer 344 rats studied at 2 and 4 weeks of estradiol treatment did not respond to the removal of dopamine in vitro whereas pituitary fragments from Holtzman rats obtained at 2 weeks of estradiol treatment did release significantly more prolactin in the absence than in the presence of dopamine. Pituitary fragments taken from Fischer 344 rats at 6 and 8 weeks were responsive to dopamine whereas pituitary tissue from Holtzman rats was not responsive at 8 weeks. The data indicate that temporal differences in responsiveness to the inhibitory effects of dopamine occur in strains which are susceptible or resistant to the formation of pituitary tumors following prolonged estradiol treatment. It has long been recognized that Fischer 344 (F-344) rats are more prone to develop estrogen-induced pituitary hyperplasia than rats of the SpragueDawley strain. Wiklund, Wertz and Gorski (i) showed that pituitary weight, DNA content, and prolactin synthesis were all higher in F-344 rats than in Holtzman Sprague-Dawley rats over an 8-week period of DES treatment and that these changes were due, at least in part, to factors within the pituitary
iSupported by NIH grant RR 08167-12. 0024-3205/92 $5.00 + .00
Copyright (c) 1991 Pergamon Press plc
236
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After Estrogen
Vol. 50, No. 3, 1992
since pituitaries from F-344 donors, but not those from Holtzman donors, respond to DES treatment when transplanted into common recipients. Although it appears that lactotrophs of F-344 rats are more sensitive to one or more of the many actions of estradiol than are those of Sprague-Dawley rats, it has not been established which of these actions are responsible for the differences between strains. One action of estrogen is to interfere with the inhibitory action of dopamine on prolactin release (2-9). Part of this effect of estrogen in vivo is as a result of indirect (via prolactin) inhibition of the tuberinfundibular dopaminergic system which has been observed following long-term estrogen treatment (I0). However, short-term estrogen treatment has been shown to increase the concentration of dopamine arriving at the pituitary in the portal blood (6,11). In addition, changes in the level of dopamine in the portal blood do not explain the apparent local pituitary control of hyperplasia reported by Wiklund et al. (i). Estrogen has also been shown to eliminate low affinity pituitary dopamine receptors (9) and to decrease total dopamine receptor number (12). However, DiPaolo et al. (13) reported no change in dopamine receptor binding following estrogen treatment. In a recent review (14), Enjalbert proposed mechanisms by which estrogen may affect the responsiveness to dopamine at a point(s) after receptor binding. In fact, Nansel et al. (7) have reported intracellular effects (increase of iysosomal enzymes) of dopamine which may be reduced by estrogen. The purpose of the present study was to determine if the differences in estrogen-induced pituitary hyperplasia and prolactin secretion between F-344 and Holtzman rats are associated with differences in the in vitro responsiveness to dopamine at various periods of treatment with estradiol in vivo. Materials Animals Mature female F-344 and Holtzman Dawley, Indianapolis, IN) were housed lighting (lights on 0600 to 2000 hrs), (50%) with food and tap water provided and 7 days later given a subcutaneous 10-15 mg/implant).
and Methods
Sprague-Dawley rats (Harlan Spraguetwo per cage in a room with controlled temperature (23C) and relative humidity ad lib. Each rat was ovariectomized Silastic implant of estradiol-17B (E2,
Experimental Procedures Six rats of each strain were anesthetized with ether for approximately one minute and then decapitated between 1200 and 1230 hrs at 2, 4, 6, and 8 weeks (F-344) or 2 and 8 weeks of E2 treatment (Holtzman) and the anterior pituitaries and blood were collected. Plasma obtained from the trunk blood was stored at -20C until assayed for prolactin. The anterior pituitary was cut into fragments (2-5 mg in size). Two fragments from each rat were immediately snap frozen on dry ice as non-incubated controls and the remaining fragments were placed into wells of 24-well tissue culture plates containing one ml of alpha-modified Minimal Essential Medium (aMEM) buffered with 21 mM NaHCO3 and containing 0.1% bovine serum albumin (BSA), 0.1% ascorbic acid, and 10-6M dopamine. The plates were incubated at 37C in a humidified atmosphere of 95% 02-5% CO 2 for 60 min. The pituitary fragments were then transferred to other plates the wells of which contained c~MEM, 0.1% BSA, 0.1% ascorbic acid, 21 mM NaHCO3 with or without 10-6M dopamine and the incubation was continued for a second 60 min period. The incubated pituitary fragments were then collected and snap frozen on dry ice. The medium from both incubation periods was collected and frozen at -20C until assayed for prolactin. The frozen pituitary fragments were weighed and then sonicated for i0 seconds in one ml of assay buffer (phosphate-buffered saline, PBS) containing 1% Triton X-100
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and 0.1% bovine serum albumin. The sonicates were allowed to stand at room temperature for one hour and then they were diluted 1:20 in assay buffer and stored at -20C until assayed for prolactin. Prolactin Radioimmunoassay Plasma, pituitary sonicates and incubation medium were assayed for prolactin by double antibody radioimmunoassay as described previously (15). Following appropriate pre-assay dilution, these samples were assayed in two dilutions in duplicate using NIAMDD-RP-I (ii IU/mg) as standard. The concentrations of prolactin were expressed as either ng/ml or ~g/mg wet tissue weight. In addition, the prolactin released into the incubation medium during the second hour was expressed as % of prolactin available for release. Prolactin available for release was calculated by subtracting the amount of prolactin released into incubation medium during the first hour from the average prolactin content of the non-incubated pituitary fragments from the same rat. Statistical Analysis One way ANOVA and Tukey's hsd multiple comparison test were used to compare means across times of estradiol treatment. The comparison of the effects of dopamine vs. no dopamine and the differences between strains at each period of estradiol treatment was done using an F-test. Significant differences were assessed at P<0.05. Results Total pituitary weight, plasma levels of prolactin and pituitary prolactin content in both F-344 and Holtzman rats from 2 to 8 weeks of estradiol treatment are shown in Figure IA, B and C respectively. Significant increases in pituitary weight and plasma levels of prolactin were observed in the F-344 rats over the duration of estradiol treatment, but pituitary prolactin content significantly decreased over the same period. Holtzman rats showed a significantly smaller increase in pituitary weight over the 8 week period of estradiol treatment compared to F-344 rats. Plasma levels of prolactin increased significantly over this period in Holtzman rats but the level at 8 weeks was significantly lower than plasma levels in F-344 rats. Pituitary content of prolactin in the Holtzman strain was not significantly different from the F-344 rats at 2 or 8 weeks of estradiol treatment and a decrease across the duration of steroid treatment was also observed. The release of prolactin from pituitary fragments into medium in the presence or absence of dopamine (expressed as ~g/mg AP) is shown in Figure 2. At 2 weeks of estradiol treatment pituitaries of Holtzman rats released significantly more prolactin in the absence than in the presence of dopamine, but a similar difference was not present at 8 weeks of treatment (Figure 2A). Prolactin release in the presence of dopamine was not different between weeks 2 and 8 in Holtzman rats. Prolactin release from pituitary fragments of F-344 rats was not significantly increased by the removal of dopamine at 2 or 4 weeks but at 6 and 8 weeks of estradiol treatment dopamine withdrawal significantly increased prolactin release (Figure 2B). The data in Figure 2B also show that dopamine was more effective in inhibiting prolactin release at 6 and 8 weeks of estradiol treatment than at 2 or 4 weeks in F-344 rats. When in vitro prolactin release was expressed as a % of that available for release and hence corrected for the decrease in pituitary content seen with increased duration of estrogen treatment, the same qualitative differences in responsiveness to dopamine were observed in both strains at
238
Lactotroph
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50, No.
A. TOTAL AP WEIGHT 50g v
E 3 F-344 Ho
40-
P
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m
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1
P I T U I T A R Y WEIGHT, P L A S M A A N D P I T U I T A R Y P R O L A C T I N IN O V A R I E C T O M I Z E D F I S C H E R 344 (F-344) A N D H O L T Z M A N S P R A G U E - D A W L E Y (HO) R A T S F O L L O W I N G E S T R A D I O L TREATMENT. A. T o t a l p i t u i t a r y weight, B. P l a s m a levels of p r o l a c t i n , C. Pituitary prolactin concentration. +Bars r e p r e s e n t m e a n ± S.E.M. M e a n s w i t h d i f f e r e n t letters (a, b, a, S) w i t h i n strains are s t a t i s t i c a l l y d i f f e r e n t P<0.05 (n=6-12).
3, 1992
Vol. 50, NO. 3, 1992
Lactotroph Responsiveness After Estrogen
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FIG. 2 EFFECT OF DOPAMINE ON PROLACTIN RELEASE IN VITRO FROM PITUITARY EXPLANTS OF OVARIECTOMIZED HOLTZMAN (A) AND FISCHER 344 (B) RATS AT VARIOUS TIMES OF ESTRADIOL TREATMENT. Bars represent mean ± S.E.M. (n=6). Means within strains with different letters (a,b) are significantly different. *,*** +DA vs. -DA at each estrogen treatment period (P<0.05 or P<0.001).
239
240
Lactotroph Responsiveness After Estrogen
Vol. 50, No. 3, 1992
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Vol. 50, No. 3, 1992
Lactotroph Responsiveness After Estrogen
241
each period of estrogen treatment examined (Figure 3A and B). However, the differences across weeks of estrogen treatment were less dramatic than seen in Figure 2. In fact, there were no significant differences in prolactin release in the presence of dopamine across the 8 weeks of estrogen treatment in the F-344 rats (Figure 3B). Discussion The results of the present study indicate that F-344 and Holtzman rats are different with respect to the temporal changes in pituitary weight, plasma concentrations of prolactin and in vitro responsiveness to dopamine during an 8 week period of estradiol treatment. On the other hand, both strains showed similar changes in pituitary concentration of prolactin over the period. It is clear from the current study that F-344 rats show a more marked pituitary hyperplasia to estradiol than do Holtzman rats. This confirms the observations of Wiklund et al. ( I ) . Rats of the F-344 strain also released significantly more prolactin into the plasma than did Holtzman females. The large difference in plasma prolactin between strains extends the previous report of Stone et al. (16). Since the pituitary concentrations of prolactin were not significantly different between the strains, the large difference in plasma levels of prolactin could have been due in part to the larger mass of the F-344 pituitaries. However, the large difference in plasma levels could have also been due to a greater decrease in hypophysial portal blood dopamine or a greater reduction in the inhibitory effects of dopamine in F-344 than in Holtzman rats. Dopamine did not inhibit in vitro prolactin release in F-344 rats at 2 and 4 weeks of estradiol treatment whereas the inhibitory effects were restored by 6 and 8 weeks of estrogen treatment. These observations suggest that the long-lasting hyperprolactinemia in this strain cannot be completely explained by a sustained reduction in the effects of dopamine on the lactotroph. One must be cautious about extrapolating results obtained in vitro to the in vivo condition. However, we do have unpublished data which show that bromocryptine does not significantly inhibit prolactin release in vivo in F344 rats treated with DES for 1 week but is effective at 4 weeks of DES treatment. Perhaps the long-lasting hyp~rprolactinemia seen in vivo is due to a reduced concentration of dopamine arriving at the lactotroph or to an increase in prolactin releasing hormones from the hypothalamus or stimulatory paracrine or autocrine factors from the pituitary itself. The local production of stimulatory factors within the pituitary could explain the results of Wiklund et al. (I) that pituitary transplants from F-344 rats, but not those from Holtzman rats, become hyperplastic in common recipients treated with estrogen. However, there is also strong evidence that dopamine reaching the lactotroph is reduced by long term estrogen treatment either by decreased release from tuberoinfundibular neurons (i0) or by the dilution of portal blood by systemic blood carried to the pituitary by non-portal vessels formed by arteriogenesis (17). The pituitaries of the F-344 rats in the current study began to show gross vascular changes at 6 weeks of estradiol treatment suggesting that arteriogenesis may play a role in the later stages of the hyperplasia and hyperprolactinemia noted in this strain. That pituitaries of Holtzman rats ultimately showed a decreased responsiveness to the inhibitory effects of dopamine in vitro indicates that Holtzman and F-344 rats do respond similarly to estradiol treatment albeit over a different time course. Early reports indicated that Sprague-Dawley rats do develop pituitary hyperplasia if estrogen treatment is continued for a sufficiently long period (18). Clearly, the strains showed marked temporal differences in responsiveness to dopamine but it is not clear what mechanisms
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Lactotroph Responsiveness After Estrogen
account for the differences. to address these mechanisms.
Vol. 50, No. 3, 1992
Ongoing studies in this laboratory will attempt
One possible factor that may influence the observed temporal changes in the responsiveness to dopamine is the level of prolactin in the intracellular pool that can be inhibited by dopamine. Mena and his coworkers (19) have shown that newly synthesized prolactin is strongly inhibited by dopamine whereas the prolactin that has been stored in the lactotroph for more than one hour is not as susceptible to dopamine inhibition. In the F-344 rat, it is possible that late in estrogen treatment much of the intracellular prolactin was newly synthesized and strongly inhibited by dopamine whereas in the early periods of estradiol treatment a greater proportion of the intracellular prolactin was in the stored component and not so easily inhibited by dopamine. On the other hand, in the Holtzman rats more pituitary prolactin may have been in the newly synthesized, dopamine responsive pool, early in estradiol treatment, but as the treatment was continued a greater proportion of stored prolactin may have been present. In summary, the data obtained in the current study show that there is an association between the loss of pituitary responsiveness to dopamine and the susceptibility of rat strains to show pituitary hyperplasia in response to DES treatment. Acknowledgements The authors wish to thank Drs. Raiti and Parlow of the National Pituitary Agency and the NIDDK for rat prolactin used for standards and for iodination. We also wish to thank Dr. Richard Gala for the use of equipment to do the radioiranunoassay. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17.
J. WIKLUND, N. WERTZ and J. GORSKI, Endocrinology 109 1700-1707 (1981). V. RAYMOND, M. BEAULIEU, F. LABRIE and J. BOISSIER, Science 200 11731175 (1978). S. JAQUES and R.R. GALA, Acta Endocrinol. 92 437-447 (1979). L. FERLAND, F. LABRIE, C. EUVRARD and J-P. RAYNAUD, Mol. Cell. Endocrinol. 14 199-204 (1979). B. WEST and P.S. DANNIES, Endocrinology 106 1108-1113 (1980). G.A. GUDELSKY, D.D. NANSEL and J.C. PORTER, Endocrinology 108 440-444 (1981). D.D. NANSEL, G.A. GUDELSKY, M.J. REYMOND and J.C. PORTER, Endocrinology 108 903-907 (1981). V. GIGUERE, H. MEUNIER, R. VEILLEUX and F. LABRIE, Endocrinology 111 857-862 (1982). D. BRESSION, A.M. BRANDI, P. PAGESY, M. LE DAFNIET, M. MARTINET, S. BRAILLY, M. MICHARD and F. PEILLON, Endocrinology 116 1905-1911 (1985). K.T. DEMAREST, G.D. RIEGLE and K.E. MOORE, Neuroendo. 39 193-200 (1984). N.S. PILOTTE, D.R. BURT and C.E. BARRACLOUGH, Endocrinology 114 23062311 (1984). M.L. HEIMAN and N. BEN-JONATHAN, Endocrinology Iii 1057-1060 (1982). T. DIPAOLO, R. CARMICHAEL, F. LABRIE and J-P. RAYNAUD, Mol. Cell. Endocrinol. 16 99-112 (1979). A. ENJALBERT, Horm. Res. 31 6-12 (1989). E.Y.H. KUO and R.R. GALA, Biochim. Biophys. Acta 264 462-471 (1972). J.P. STONE, S. HOLTZMAN and C.J. SHELLABARGER, Cancer Res. 39 773-778 (1979). J. SCHECTER, N. AHMAD and R. WEINER, Amer. J. Anat. 179 315-323 (1987).
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18. 19.
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C.W. WELSCH, T. JENKINS, Y. AMENOMORI and J. MEITES, Experientia 27 1350-1352 (1971). F. MENA, C. CLAPP, D. AGUAYO, M.T. MORALES, C.E. GROSVENOR and G. MARTINEZ DE LA ESCALERA, Endocrinology 125 1814-1820 (1989).
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