0013-7227/92/1304-1796$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society
Vol. 130, No. 4 Printed
in U.S.A.
Tissue-Specific Regulation of Vasoactive Intestinal Peptide Messenger Ribonucleic Acid Levels by Estrogen in the Rat* S. KASPER, R. A. POPESCU, AND H. G. FRIESEN
A. TORSELLO?,
M. E. VRONTAKIS,
C. IKEJIANI,
University of Manitoba, Department of Physiology, Faculty of Medicine, Winnipeg, Manitoba, Canada R3E 0 W3; and Dipartimento Di Farmuco~gia R4 (A. T.), 20129 Miluno, Italy
ABSTRACT. The early and chronic effects of 17@estradiol on vasoactive intestinal peptide (VIP) gene expression in rats were examined. Total RNA of four VIP-producing tissues were subjected to Northern blot analysis 15 and 30 min, and 1, 3, 6, and 24 h after a single injection of 17fl-estradiol (100 pg/kg ip). Pituitary, hypothalamus, brain, and ileum VIP messenger RNA (mRNA) levels rose in a time-dependent manner after estrogen treatment. In the pituitary, the increase was maximal at 30-60 min, whereas in the hypothalamus, the increase reached significance only at 3 h but then persisted until at least 24 h. In the brain, a transient increase in VIP mRNA was observed at 30
T
HE anatomical distribution of vasoactive intestinal peptide (VIP) throughout the central nervous system suggests that VIP functions as a neuroendocrine regulatory peptide (1, 2). High concentrations of immunoreactive VIP are found in the brain, especially in the cortex, amygdala, hippocampus, and the hypothalamus (3). Immunoreactive VIP is present in cells of the paraventricular nucleus with fibers projecting down into the median eminence (4). Detectable VIP serum levels in the hypophyseal portal system (5,6) and specific VIP receptors on lactotrophs (7, 8) suggest that VIP may act as a hypothalamic regulatory factor influencing normal pituitary function. One of the more important functions of VIP in the anterior pituitary appears to be as a physiological mediator of PRL release (9, 10). Direct measurements of VIP effects on PRL secretion have been obtained from isolated rat anterior pituitary tissue in culture (11) as well Received October 3,199l. Address all correspondence and requests for reprints to: Dr. S. Kasper, University of Manitoba, Department of Physiology, Faculty of Medicine, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E ow3. * This work was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada. t Recinient of a Manitoba Health Research Council Postdoctoral Fellowship.
min, whereas VIP mRNA levels in the ileum responded in a biphasic pattern; the initial early increase was followed by a second elevation occurring at 6 h. A smaller 1-kilobase VIPrelated transcript particularly abundant in the pituitary was regulated in parallel with the 1.7-kilobase mature VIP mRNA species. Continuous estrogen stimulation for 7 weeks dramatically increased both mRNA species in the pituitary but did not affect VIP mRNA levels in the other tissues. These data suggest that the regulation of VIP gene expression by transient increases in estrogen levels is rapid and that the pattern of induction is tissue specific. (Endocrinology 130: 1796-1801,1992)
as from pituitary cells such as the GHs-derived cell lines (12,13). Moreover, recent reports suggest that the pituitary also synthesizes VIP. Lam and coworkers (14) have shown that primary rat pituitary cell cultures secrete basal levels of VIP and that pituitary VIP messenger RNA (mRNA) content was markedly increased in hypothyroidism (15, 16). The identity of the cell type producing VIP was investigated using immunohistochemical techniques. Steel et al. (17) found that lactotrophs contained immunoreactive VIP and that VIP was also present in a subset of galanin-containing lactotrophs after estrogen treatment. Other immunohistochemical studies have reported that VIP is localized in large stellate cells which are noticeably different than lactotrophs (16, 18). These findings suggest that the relationship between pituitary VIP and PRL secretion may be more paracrine than autocrine (16,18) and that the VIP-producing cells display plasticity of expression which may be under the influence of estrogen (17). Pituitary VIP gene expression appears to be regulated by several hormones that also regulate PRL synthesis and secretion. Lam and Reichlin (14) have reported that TRH stimulates the secretion of both VIP and PRL in hypothyroid pituitary cell cultures. Estrogen, another regulator of PRL synthesis and secretion, also raises pituitary VIP mRNA levels (9, 10, 17). Changes in VIP
1796
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ESTROGEN ACTION content may therefore be essential in mediating the action of estrogen on PRL secretion. In this study, we have attempted to determine how rapidly estrogen treatment increases VIP mRNA levels in the pituitary and to compare this rate of increase with the effects of estrogen on other VIP-producing tissues.
Materials
and Methods
Animals All animal studies were conducted in accordance with the principles and procedures outlined by the Canadian Council on Animal Care, Male Sprague Dawley rats (Central Animal Care, University of Manitoba, Manitoba, Canada) weighing 250-300 g were housed in a temperature-controlled environment (21-23 C) with lights on from 0600-1800 h. Food and water were available ad libitum. Intact animals (four animals per group) were given a single ip injection of 100 pg/kg 17j3-estradiol (Sigma Chemical Co., St. Louis, MO) and were killed after 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h. The anterior pituitary as well as the medial basal hypothalamus, whole brain (minus the medial basal hypothalamus), and ileum were removed and frozen in liquid nitrogen. The corresponding tissues from the four animals in each group were pooled for the RNA extraction procedure. This experiment was repeated on three individual occasions. Animals (eight per group) in the control groups were killed at the same time points after receiving a single injection of the vehicle alone. The basal or zero time point was determined by killing one group of animals which did not receive an injection. Fischer 344 female rats were obtained from Charles River, Canada Inc. (St. Constant, Quebec, Canada). Intact animals weighing 180-200 g were implanted SCwith a single 50-mg diethylstilbestrol (DES, Sigma Chemical Co.) pellet (silastic medical adhesive, Dow Corning Co., Midland, MI) as reported by Vrontakis et al. (19). The controls were sham-operated but did not receive an implant. After 7 weeks the animals were killed, the pituitary, hypothalamus, brain, and ileum were removed and frozen in liquid nitrogen. RNA extraction and Northern blot analysis Total RNA was extracted using the guanidinium isothiocyanate method (20), and 20 rg RNA from each sample were subjected to electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde, transferred to Nitroplus hybridization transfer membrane (Micron Separations Inc., Westboro, MA) and baked at 80 C for 2 h. Hybridization was performed at 65 C in 50% formamide using lo6 dpm/ml of a 32P-labeled 380-base rat prepro VIP complementary RNA probe containing both exon 4 and exon 5 which encode for peptide histidine isoleucine and VIP, respectively (21). Final washing conditions were 0.2~ SSC (30 mM sodium chloride, 3 mM sodium citrate final concentration), 0.1% sodium dodecyl sulfate at 65 C for 30 min. The blots were placed on Kodak XAR film (Eastman Kodak Co., Rochester, NY) at -70 C in the presence of an intensifying screen, and the autoradiograms were analyzed by a scanning densitometer (BioRad, Richmond, CA; model 620 Video Densitometer).
ON VIP mRNA LEVELS
1797
Statistical analysis Basal tissue VIP mRNA levels were determined from control animals, and all values were normalized to the 28s ribosomal subunit mRNA. Since the effect of the vehicle on VIP mRNA levels was negligible over a 24-h period, the l-h time point was chosen as the basal level for each individual experiment and assigned a value of one. The 17/3-estradiol-treated groups were compared to the control groups using one-way analysis of variance and the significance of differences determined by Duncan’s multiple range test. The results were expressed as the mean & SEM, and probability values less than 0.05 were considered significant. RNase digestion of poly(A) tracts RNase H will selectively degrade poly(A) tracts of mRNA when they are present as a double-stranded form after hybribization with oligo(dT). Twenty micrograms of rat pituitary, brain, and ileum total RNA were hybridized to oligo(dT) 1218 (Pharmacia, Piscataway, NJ) and digested with RNase H (Pharmacia) as described by Sippel et al. (22). The digestion reaction was subjected to formaldehyde gel electrophoresis and Northern blot analysis as described above.
Results Estrogen action on VIP mRNA levels in the anterior pituitary, medial basal hypothalamus, brain, and ileum The treatment of normal male rats with a single injection of 17&estradiol resulted in a time-dependent increase of VIP mRNA levels in the anterior pituitary, medial basal hypothalamus, brain, and ileum. A representative autoradiogram and analysis of the fold induction in mRNA levels of each tissue is shown in Figs. l4. A representative autoradiogram for the vehicle-treated animals is shown for the 1.7-kilobase (kb) and 1-kb VIP transcripts in the pituitary (Fig. 1B). Pituitary VIP mRNA (1.7 kb) levels began to rise after 15 min in response to estrogen (Fig. l), peaked at 1 h (up to a &fold increase compared to control) and returned to basal levels within 3 h. A smaller 1-kb VIPlike transcript increased 3-fold in 1 h, returning to basal levels within 3 h. In the hypothalamus, VIP mRNA levels increased 4- to &fold after 30 min and remained elevated even at 24 h (Fig. 2). The smaller 1-kb transcript was not detected in the hypothalamus. Estrogen treatment resulted in a marked increase in brain VIP gene expression (Fig. 3), mRNA levels increasing 12-fold within 30 min, and falling to basal levels within 1 h. Again, the smaller transcript did not appear to be present, or was present only at minimal levels, in the brain. Ileum VIP mRNA levels appeared to respond to estrogen treatment in a biphasic pattern (Fig. 4). The first peak occurred within 15-30 min of the injection and a second elevation at 6 h. The increases were 6-fold and 12-fold, respectively.
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ESTROGEN
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ON VIP
mRNA
Endo. 1992
LEVELS
Voll30.
No 4
n 15’
C
30’
lh
3h
24h
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FIG. 2. Effect of estrogen on hypothalamic VIP mRNA levels. A, Northern blot analysis was performed as described in Fig. 1. B, Graphic representation of the fold increase in VIP mRNA levels in response to estrogen (0). Significant differences from controls (0): *, P < 0.05.
VIP mRNA levels after long term exposure to estrogen
01
,I
0
1
2
3
4
5
6
, 24
TIME (h)
FIG. 1. Effect of estrogen treatment on pituitary mRNA levels. A, Northern blot analysis of pituitary VIP mRNA. Total RNA (20 rg) was run in each lane. The control group (C) received the vehicle alone and were killed after 1 h. RNA from estrogen-treated animals was prepared at 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h after a single injection of 17@-estradiol (100 rg/kg ip) and were run in subsequent lanes. Each sample represents the pooled RNA of tissues from four rats. Total RNA was prepared from DES-induced pituitary tumors (PT). The autoradiogram of the 28s ribosomal subunit shows the homogeneous loading and transfer. B, VIP mRNA levels in response to vehicle alone over a 24-h time period. C, Fold increase in both the 1.7-kb (0) and 1-kb (W) VIP mRNA after estrogen treatment. Each point represents the mean and SEM from three independent experiments including four animals each. Statistically significant differences from the control (0) are indicated by asterisks, *, P < 0.05.
Under continuous estrogen stimulation of female rats (Fig. 5), the levels of both the 1.7-kb and 1-kb transcripts increased in the pituitary, whereas no change was noted in the other tissues. Furthermore, the 1-kb transcript was only easily detected in the pituitary. Analysis of the 1.7-kb and 1-kb transcripts digestion
by RNase H
As noted previously, the 1-kb transcript was detected primarily in the pituitary and hardly at all in the other VIP-producing tissues. Upon RNase H digestion (Fig. 6), removal of the poly(A) tract decreases the size of both the 1.7-kb and the 1-kb transcript to the same degree. Removal of the poly(A) tract from the 1.7-kb transcript clearly does not reduce the transcript size to 1 kb.
In this study, we have demonstrated that the increase in VIP mRNA in response to estrogen administration is
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ESTROGEN ACTION A c
ON VIP mRKA LEVELS
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A 13’
30’
th
fh
6h
24h
c
18s _
15’
30’
th
3h
6h
24h
mm
-1-7kb
28 s-
TIME (h)
FIG. 3. Effect of estrogen treatment on bratn VIP mRNA levels. A, Northern blot analysis was performed as described in Fig. 1 B, Fold increase in VIP mRNA levels in estrogen-treated animals (e) IX controls (0). *, P < o.I15.
rapid and that the pattern of induction is tissue-specific. The presence of immunoreactive VIP and VIP mRNA in the rat anterior pituitary has been reported by several investigators (9, 10, 23). Moreover, VIP levels increased when estrogen was administered either by daily injections (24) or by silastic implant (23). We were therefore interested in determining the time interval needed to elicit an increase in VIP mRNA levels in response to a single injection of 17P-e&radio). Koves et ~1. (23) reported that immunoreactive VIP was present in both normal male and estrogen-treated female anterior pituitaries. Furthermore, total RNA extracted from male anterior pituitaries consistently showed detectable levels of VIP message in our hands. This animal model was therefore chosen to reduce any variabilty due to changing serum estrogen levels during the estrous cycle. Pituitary VIP mRNA levels increased rapidly in a time-dependent manner, increasing after 30 min to give a S-fold induction of VIP mR?JA levels at 1. h and returning to basal levels after 3 h in response to estrogen treatment. These results suggest that the control of VIP
FIG. 4. Effect of estrogen treatment on ileum VIP mRKA levels. A, Nofihern biot analysis wa8 performed as described in Fig. 1. H, Graphic representation of the VIP mRMA induction by estmgen (0) compared to controls (0). *, P c 0.05, except for the 30-min and 6-h time points, where P < 0.01.
0 1.7
. -”
-1 7 kD
kb-
-f.O
28 s
-e
bcl,
kb
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FIG. 5. Northern blot analysts of VIP RNA levels in pituitary (API, me&o-basal hypothalamus IMBH), brain (BR), and ileum (Gut) after long term exposure of female rats to estrogen. A, The control group was sham-operated animals which did not receive s IX-S implant. B, The long term estrogen-treated rats received a 50.mg DES srlastic implant for 7 weeks.
expression may be at the level of transcription. Whether the respunse is mediated through an estrogen
gene
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ESTROGEN ACTION
1800 PT RNaseH
_
SR +
_
Gut +
_
+
*-
1.7kb -
l.Okb
FIG. 6. Analysis of the 1.7-kb and l-kb transcripts in the RNaseH digestion assay. The RNase H assay was performed according to the method of Sippel et al. (22). A total of 20 pg RNA from pituitary, brain, or ileum was used for each assay point. l&S marks the position of the 18s ribosomal subunit.
response element remains to be established. The human VIP gene has been cloned and sequenced (25, 26) and using a DNA analysis program, we have identified several estrogen response element-like consensus sequences. Whether the increase in VIP mRNA levels occurred as a result of increased gene transcription, increased mRNA stability, or through a secondary mechanism under the regulation of estrogen could not be determined from this study. Prolonged exposure to DES resulted in a dramatic increase in pituitary VIP mRNA content over the controls. Under chronic estrogen stimulation, it is possible that other regulatory mechanisms in the hypothalamic/ pituitary axis have contributed to the increase in VIP mRNA levels. Of particular interest was a smaller 1-kb transcript present under basal conditions almost exclusively in the pituitary. This message gave a more modest response (3fold increase) to estrogen treatment, implying that the 1-kb transcript was regulated in parallel with the larger 1.7-kb mRNA, the mature VIP mRNA species in the rat (24). Although the transcript appeared predominantly in the pituitary, a faint band was occasionally seen in the brain or small intestine. Moreover, the 1-kb transcript was upregulated by long term exposure to estrogen in the pituitary alone and not in the other VIP-producing tissues. Recent studies by Lam and collaborators (14, 15) have shown that pituitary content of a 1-kb transcript was increased in hypothyroidism, implying that the regulation of VIP gene expression is also dependent on thyroid status. The generation of the two VIP mRNA species is not due to differences in the length of the poly(A) tail since relative transcript size of both the 1.7kb and the 1-kb mRNA was approximately the same after RNase H digestion. Thus it may be speculated that the difference arises from differential use of 3’-poly(A)adenylation signals, differential mRNA processing, or from the possibility that the 1-kb transcript encodes for a different, but VIP-related, peptide. VIP mRNA levels also rose within the 15-min to l-h period in response to estrogen treatment in the hypo-
ON VIP mRNA LEVELS
Endo. 1992
Voll30. No4
thalamus, brain, and ileum, suggesting that the transient increase in VIP mRNA content was a result of increased gene transcription. VIP mRNA levels in the hypothalamus only reached a level of significance at 3 h but then remained elevated for 24 h. A second elevation in VIP mRNA content in the small intestine was seen at 6 h, suggesting that there is tissue-specific regulation of the VIP gene. In contrast, prolonged estrogen exposure did not increase VIP gene expression in these tissues. These data confirm previous reports that chronic estrogen treatment does not change VIP mRNA content in the medial basal hypothalamus, brain, and jejunum (27, 28). In fact, our data suggest that hypothalamic and intestinal VIP mRNA levels might even decrease in the presence of high estrogen levels. Maletti et al. (29) reported that hypothalamic VIP mRNA content also decreased in rats after a l-week DES implant. In conclusion, estrogen regulated VIP gene expression in a time-dependent manner in the pituitary, hypothalamus, brain, and ileum. A second, smaller VIP-related transcript was also regulated in parallel with the mature 1.7-kb VIP species which was present primarily in the pituitary. In the pituitary, both transcripts were upregulated after sustained estrogen treatment, whereas VIP mRNA levels remained unchanged or slightly decreased in the other VIP-producing tissues. Whether the 1-kb message encodes another form of VIP or is translated into a unique peptide remains to be established. Acknowledgments We thank Ingo Schroedter for his excellent technical help and Drs. Newman Stephens and Jiang He for their advice and help with the statistical analysis of the data. The authors are indebted to Debbie Tsuyuki for her critical reading of this manuscript.
References 1. Prysor-Jones RA, Jenkins JS 1988 Vasoactive intestinal peptide and anterior pituitary function. Clin Endocrinol29:677-688 2. Gazes I, Brenneman DE 1989 VIP: molecular biologv and neurobiological function. Mol Neurobiol3:201-236 -3. Morrison JH. Maaistretti PJ. Benoit R. Bloom FE 1984 The distribution and m&phological characteristics of the intracortical VIP-positive cells: an immunohistochemical analysis. Brain Res 292:269-282 4. Mezey E, Kiss JZ 1985 Vasoactive intestinal peptide-containing neurons in the paraventricular nucleus may participate in the secretion of prolactin. Proc Nat1 Acad Sci USA 82:245-247 5. Said SI, Porter JC 1979 Vasoactive intestinal DeDtide: release into hypophyseal portal blood. Life Sci 24:227-230‘ 6. Shimatsu A. Kato Y. Matsushita N. Katakami H. Yanaihara N. Imura H 1982 Stimulation by serotbnin of vasoactive intestinal polypeptide release into rat hypophyseal portal blood. Endocrinology 111:338-340 7. Robberecht P, Deschodt-Lanckman M, Camus J-C, de Neef P, Lambert M, Christoph J 1979 VIP activation of rat anterior pituitary adenylate cyclase. FEBS Lett 103:229-233 8. Wanke IE, Rorstad OP 1990 Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: localization of binding to lactotrophs. Endocrinology 126:1981-1988
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ESTROGEN ACTION 9. Reichlin S 1988 Neuroendocrine significance of vasoactive intestinal polypeptide. Ann NY Acad Sci 527:431-449 10. Lam KSL 1991 Vasoactive intestinal peptide in the hypothalamus and pituitary. Neuroendocrinology 53(suppl1):45-51 11. Maas DL, Arnaout MA, Martinson DR, Erdmann MD, Hagen TC 1991 Vasoactive intestinal polypeptide and thyrotropin-releasing hormone stimulate newly synthesized, not stored, prolactin. Endocrinology 128:1015-1020 12. Gourdji D, Bataille D, Vauclin N, Grouselle D, Rosselin G, TixierVidal A 1979 Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and CAMP production in a rat pituitary cell line (GH./B.). Additive effects of VIP and TRH on PRL release. FEBS Lett 104:165-168 13. Nagy G, Mulchahey JJ, Neil1 JD 1988 Autocrine control of prolactin secretion by vasoactive intestinal peptide. Endocrinololgy 1223364-366 14. Lam KSL, Reichlin S 1989 Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat. Neuroendocrinology 50:524-528 15. Lam KSL, Lechan RM, Minamitani N, Segerson TL, Reichlin S 1989 Vasoactive intestinal peptide in the anterior pituitary is increased in hypothyroidism. Endocrinology 124:1077-1084 16. Segerson TP, Lam KSL, Cacicedo L, Minamitani N, Fink JS, Lechan RM, Reichlin S 1989 Thyroid hormone regulates vasoactive intestinal peptide (VIP) mRNA levels in the rat anterior pituitary gland. Endocrinology 125:2221-2223 17. Steel JH, Gon G, O’Halloran DJ, Jones PM, Yanaihara N, Ishikawa H, Bloom SR, Polak JM 1989 Galanin and vasoactive intestinal peptide are colocalized with classical pituitary hormones and show plasticity of expression. Histochemistry 93:183-189 18. Carrillo AJ, Phelps CJ, Short-term e&radio1 benzoate treatment increases pituitary intracellular content of vasoactive intestinal peptide. Program of the 73rd Annual Meeting of The Endocrine Society, Washington, DC, 1991, p 390 (Abstract) 19. Vrontakis ME, Thliveris JA, Friesen HG 1987 Influence of bromocriptine and oestrogen on prolactin synthesis, secretion and tumour growth in uiuo in rats. J Endocrinol 113:383-388 20. Chirgwin JM, Prsybyla AE, MacDonald RJ, Rutter WJ 1979
ON VIP mRNA LEVELS
21.
22.
23.
24. 25.
26. 27.
28. 29.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry l&5294-5299 Fink JS, Montminy MR, Tsukada T, Hoefler H, Specht LA, Lechan RM. Wolfe H. Mandel G. Goodman RH 1986 In situ hybridization of somatbstatin and’ vasoactive intestinal nentide mRNA in the rat nervous system: contrasting patterns of ontogeny. In: Uhl GR (ed) In Situ Hvbridixation in Brain. Plenum Publishine Corp., New York, p lSl-i91 Sippel AE, Stavrianopoulos JG, Schutz G, Feigelson P 1974 Translational properties of rabbit globii mRNA after specific removal of poly(A) with ribonuclease H. Proc Nat1 Acad Sci USA 71:46354639 Koves K, Gottshall PE, Gores T, Scammell JG, Arimura A 1990 Presence of immunoreactive vasoactive intestinal polypeptide in anterior pituitary of normal male and long term estrogen-treated female rats: a light microscopic immunohistochemical study. Endocrinology 12617561763 Lam KSL, Srivastava G, Lechan RM, Lee T, Reichlin S 1990 Estrogen regulates the gene expression of vasoactive intestinal peptide in the anterior pituitary. Neuroendocrinology 52417-421 Yamagami T, Ohsawa K, Nishixawa M, Inoue C, Gotoh E, Yanaihara N, Yamamoto H, Okamoto H 1988 Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter. Ann NY Acad Sci 527:87-102 Tsukada T, Horovitch SJ, Montminy MR, Mandel G, Goodman RH 1985 Structure of the human vasoactive intestinal polypeptide gene. DNA 4293-300 Carrillo AJ, Doherty PC, Guan X, Sturtevant JR, Waho DG 1991 Preferential increase in pituitary prolactin uersus vasoactive intestinal peptide as a function of estradiol benzoate dose in the ovariectomised rat. Endocrinology 128131-138 Prysor-Jones PA, Silverlight JJ, Kennedy SJ, Jenkins JS 1988 Vasoactive intestinal peptide and the stimulation of lactotroph growth by oestradiol in rats. J Endocrinol116:259-265 Maletti M, Rostene WH, Carr L, Scherrer H, Rotten D, Kordon C, Rosselin G 1982 Interaction between estradiol and prolactin on vasoactive intestinal peptide concentrations in the hypothalamus and in the anterior pituitary of the female rat. Neurosci Lett 32307-313
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Vol. 130, No. 4 Printed
in U.S.A.
Tissue-Specific Regulation of Vasoactive Intestinal Peptide Messenger Ribonucleic Acid Levels by Estrogen in the Rat* S. KASPER, R. A. POPESCU, AND H. G. FRIESEN
A. TORSELLO?,
M. E. VRONTAKIS,
C. IKEJIANI,
University of Manitoba, Department of Physiology, Faculty of Medicine, Winnipeg, Manitoba, Canada R3E 0 W3; and Dipartimento Di Farmuco~gia R4 (A. T.), 20129 Miluno, Italy
ABSTRACT. The early and chronic effects of 17@estradiol on vasoactive intestinal peptide (VIP) gene expression in rats were examined. Total RNA of four VIP-producing tissues were subjected to Northern blot analysis 15 and 30 min, and 1, 3, 6, and 24 h after a single injection of 17fl-estradiol (100 pg/kg ip). Pituitary, hypothalamus, brain, and ileum VIP messenger RNA (mRNA) levels rose in a time-dependent manner after estrogen treatment. In the pituitary, the increase was maximal at 30-60 min, whereas in the hypothalamus, the increase reached significance only at 3 h but then persisted until at least 24 h. In the brain, a transient increase in VIP mRNA was observed at 30
T
HE anatomical distribution of vasoactive intestinal peptide (VIP) throughout the central nervous system suggests that VIP functions as a neuroendocrine regulatory peptide (1, 2). High concentrations of immunoreactive VIP are found in the brain, especially in the cortex, amygdala, hippocampus, and the hypothalamus (3). Immunoreactive VIP is present in cells of the paraventricular nucleus with fibers projecting down into the median eminence (4). Detectable VIP serum levels in the hypophyseal portal system (5,6) and specific VIP receptors on lactotrophs (7, 8) suggest that VIP may act as a hypothalamic regulatory factor influencing normal pituitary function. One of the more important functions of VIP in the anterior pituitary appears to be as a physiological mediator of PRL release (9, 10). Direct measurements of VIP effects on PRL secretion have been obtained from isolated rat anterior pituitary tissue in culture (11) as well Received October 3,199l. Address all correspondence and requests for reprints to: Dr. S. Kasper, University of Manitoba, Department of Physiology, Faculty of Medicine, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E ow3. * This work was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada. t Recinient of a Manitoba Health Research Council Postdoctoral Fellowship.
min, whereas VIP mRNA levels in the ileum responded in a biphasic pattern; the initial early increase was followed by a second elevation occurring at 6 h. A smaller 1-kilobase VIPrelated transcript particularly abundant in the pituitary was regulated in parallel with the 1.7-kilobase mature VIP mRNA species. Continuous estrogen stimulation for 7 weeks dramatically increased both mRNA species in the pituitary but did not affect VIP mRNA levels in the other tissues. These data suggest that the regulation of VIP gene expression by transient increases in estrogen levels is rapid and that the pattern of induction is tissue specific. (Endocrinology 130: 1796-1801,1992)
as from pituitary cells such as the GHs-derived cell lines (12,13). Moreover, recent reports suggest that the pituitary also synthesizes VIP. Lam and coworkers (14) have shown that primary rat pituitary cell cultures secrete basal levels of VIP and that pituitary VIP messenger RNA (mRNA) content was markedly increased in hypothyroidism (15, 16). The identity of the cell type producing VIP was investigated using immunohistochemical techniques. Steel et al. (17) found that lactotrophs contained immunoreactive VIP and that VIP was also present in a subset of galanin-containing lactotrophs after estrogen treatment. Other immunohistochemical studies have reported that VIP is localized in large stellate cells which are noticeably different than lactotrophs (16, 18). These findings suggest that the relationship between pituitary VIP and PRL secretion may be more paracrine than autocrine (16,18) and that the VIP-producing cells display plasticity of expression which may be under the influence of estrogen (17). Pituitary VIP gene expression appears to be regulated by several hormones that also regulate PRL synthesis and secretion. Lam and Reichlin (14) have reported that TRH stimulates the secretion of both VIP and PRL in hypothyroid pituitary cell cultures. Estrogen, another regulator of PRL synthesis and secretion, also raises pituitary VIP mRNA levels (9, 10, 17). Changes in VIP
1796
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ESTROGEN ACTION content may therefore be essential in mediating the action of estrogen on PRL secretion. In this study, we have attempted to determine how rapidly estrogen treatment increases VIP mRNA levels in the pituitary and to compare this rate of increase with the effects of estrogen on other VIP-producing tissues.
Materials
and Methods
Animals All animal studies were conducted in accordance with the principles and procedures outlined by the Canadian Council on Animal Care, Male Sprague Dawley rats (Central Animal Care, University of Manitoba, Manitoba, Canada) weighing 250-300 g were housed in a temperature-controlled environment (21-23 C) with lights on from 0600-1800 h. Food and water were available ad libitum. Intact animals (four animals per group) were given a single ip injection of 100 pg/kg 17j3-estradiol (Sigma Chemical Co., St. Louis, MO) and were killed after 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h. The anterior pituitary as well as the medial basal hypothalamus, whole brain (minus the medial basal hypothalamus), and ileum were removed and frozen in liquid nitrogen. The corresponding tissues from the four animals in each group were pooled for the RNA extraction procedure. This experiment was repeated on three individual occasions. Animals (eight per group) in the control groups were killed at the same time points after receiving a single injection of the vehicle alone. The basal or zero time point was determined by killing one group of animals which did not receive an injection. Fischer 344 female rats were obtained from Charles River, Canada Inc. (St. Constant, Quebec, Canada). Intact animals weighing 180-200 g were implanted SCwith a single 50-mg diethylstilbestrol (DES, Sigma Chemical Co.) pellet (silastic medical adhesive, Dow Corning Co., Midland, MI) as reported by Vrontakis et al. (19). The controls were sham-operated but did not receive an implant. After 7 weeks the animals were killed, the pituitary, hypothalamus, brain, and ileum were removed and frozen in liquid nitrogen. RNA extraction and Northern blot analysis Total RNA was extracted using the guanidinium isothiocyanate method (20), and 20 rg RNA from each sample were subjected to electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde, transferred to Nitroplus hybridization transfer membrane (Micron Separations Inc., Westboro, MA) and baked at 80 C for 2 h. Hybridization was performed at 65 C in 50% formamide using lo6 dpm/ml of a 32P-labeled 380-base rat prepro VIP complementary RNA probe containing both exon 4 and exon 5 which encode for peptide histidine isoleucine and VIP, respectively (21). Final washing conditions were 0.2~ SSC (30 mM sodium chloride, 3 mM sodium citrate final concentration), 0.1% sodium dodecyl sulfate at 65 C for 30 min. The blots were placed on Kodak XAR film (Eastman Kodak Co., Rochester, NY) at -70 C in the presence of an intensifying screen, and the autoradiograms were analyzed by a scanning densitometer (BioRad, Richmond, CA; model 620 Video Densitometer).
ON VIP mRNA LEVELS
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Statistical analysis Basal tissue VIP mRNA levels were determined from control animals, and all values were normalized to the 28s ribosomal subunit mRNA. Since the effect of the vehicle on VIP mRNA levels was negligible over a 24-h period, the l-h time point was chosen as the basal level for each individual experiment and assigned a value of one. The 17/3-estradiol-treated groups were compared to the control groups using one-way analysis of variance and the significance of differences determined by Duncan’s multiple range test. The results were expressed as the mean & SEM, and probability values less than 0.05 were considered significant. RNase digestion of poly(A) tracts RNase H will selectively degrade poly(A) tracts of mRNA when they are present as a double-stranded form after hybribization with oligo(dT). Twenty micrograms of rat pituitary, brain, and ileum total RNA were hybridized to oligo(dT) 1218 (Pharmacia, Piscataway, NJ) and digested with RNase H (Pharmacia) as described by Sippel et al. (22). The digestion reaction was subjected to formaldehyde gel electrophoresis and Northern blot analysis as described above.
Results Estrogen action on VIP mRNA levels in the anterior pituitary, medial basal hypothalamus, brain, and ileum The treatment of normal male rats with a single injection of 17&estradiol resulted in a time-dependent increase of VIP mRNA levels in the anterior pituitary, medial basal hypothalamus, brain, and ileum. A representative autoradiogram and analysis of the fold induction in mRNA levels of each tissue is shown in Figs. l4. A representative autoradiogram for the vehicle-treated animals is shown for the 1.7-kilobase (kb) and 1-kb VIP transcripts in the pituitary (Fig. 1B). Pituitary VIP mRNA (1.7 kb) levels began to rise after 15 min in response to estrogen (Fig. l), peaked at 1 h (up to a &fold increase compared to control) and returned to basal levels within 3 h. A smaller 1-kb VIPlike transcript increased 3-fold in 1 h, returning to basal levels within 3 h. In the hypothalamus, VIP mRNA levels increased 4- to &fold after 30 min and remained elevated even at 24 h (Fig. 2). The smaller 1-kb transcript was not detected in the hypothalamus. Estrogen treatment resulted in a marked increase in brain VIP gene expression (Fig. 3), mRNA levels increasing 12-fold within 30 min, and falling to basal levels within 1 h. Again, the smaller transcript did not appear to be present, or was present only at minimal levels, in the brain. Ileum VIP mRNA levels appeared to respond to estrogen treatment in a biphasic pattern (Fig. 4). The first peak occurred within 15-30 min of the injection and a second elevation at 6 h. The increases were 6-fold and 12-fold, respectively.
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ESTROGEN
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n 15’
C
30’
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2
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4
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TIME (h)
FIG. 2. Effect of estrogen on hypothalamic VIP mRNA levels. A, Northern blot analysis was performed as described in Fig. 1. B, Graphic representation of the fold increase in VIP mRNA levels in response to estrogen (0). Significant differences from controls (0): *, P < 0.05.
VIP mRNA levels after long term exposure to estrogen
01
,I
0
1
2
3
4
5
6
, 24
TIME (h)
FIG. 1. Effect of estrogen treatment on pituitary mRNA levels. A, Northern blot analysis of pituitary VIP mRNA. Total RNA (20 rg) was run in each lane. The control group (C) received the vehicle alone and were killed after 1 h. RNA from estrogen-treated animals was prepared at 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h after a single injection of 17@-estradiol (100 rg/kg ip) and were run in subsequent lanes. Each sample represents the pooled RNA of tissues from four rats. Total RNA was prepared from DES-induced pituitary tumors (PT). The autoradiogram of the 28s ribosomal subunit shows the homogeneous loading and transfer. B, VIP mRNA levels in response to vehicle alone over a 24-h time period. C, Fold increase in both the 1.7-kb (0) and 1-kb (W) VIP mRNA after estrogen treatment. Each point represents the mean and SEM from three independent experiments including four animals each. Statistically significant differences from the control (0) are indicated by asterisks, *, P < 0.05.
Under continuous estrogen stimulation of female rats (Fig. 5), the levels of both the 1.7-kb and 1-kb transcripts increased in the pituitary, whereas no change was noted in the other tissues. Furthermore, the 1-kb transcript was only easily detected in the pituitary. Analysis of the 1.7-kb and 1-kb transcripts digestion
by RNase H
As noted previously, the 1-kb transcript was detected primarily in the pituitary and hardly at all in the other VIP-producing tissues. Upon RNase H digestion (Fig. 6), removal of the poly(A) tract decreases the size of both the 1.7-kb and the 1-kb transcript to the same degree. Removal of the poly(A) tract from the 1.7-kb transcript clearly does not reduce the transcript size to 1 kb.
In this study, we have demonstrated that the increase in VIP mRNA in response to estrogen administration is
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ESTROGEN ACTION A c
ON VIP mRKA LEVELS
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A 13’
30’
th
fh
6h
24h
c
18s _
15’
30’
th
3h
6h
24h
mm
-1-7kb
28 s-
TIME (h)
FIG. 3. Effect of estrogen treatment on bratn VIP mRNA levels. A, Northern blot analysis was performed as described in Fig. 1 B, Fold increase in VIP mRNA levels in estrogen-treated animals (e) IX controls (0). *, P < o.I15.
rapid and that the pattern of induction is tissue-specific. The presence of immunoreactive VIP and VIP mRNA in the rat anterior pituitary has been reported by several investigators (9, 10, 23). Moreover, VIP levels increased when estrogen was administered either by daily injections (24) or by silastic implant (23). We were therefore interested in determining the time interval needed to elicit an increase in VIP mRNA levels in response to a single injection of 17P-e&radio). Koves et ~1. (23) reported that immunoreactive VIP was present in both normal male and estrogen-treated female anterior pituitaries. Furthermore, total RNA extracted from male anterior pituitaries consistently showed detectable levels of VIP message in our hands. This animal model was therefore chosen to reduce any variabilty due to changing serum estrogen levels during the estrous cycle. Pituitary VIP mRNA levels increased rapidly in a time-dependent manner, increasing after 30 min to give a S-fold induction of VIP mR?JA levels at 1. h and returning to basal levels after 3 h in response to estrogen treatment. These results suggest that the control of VIP
FIG. 4. Effect of estrogen treatment on ileum VIP mRKA levels. A, Nofihern biot analysis wa8 performed as described in Fig. 1. H, Graphic representation of the VIP mRMA induction by estmgen (0) compared to controls (0). *, P c 0.05, except for the 30-min and 6-h time points, where P < 0.01.
0 1.7
. -”
-1 7 kD
kb-
-f.O
28 s
-e
bcl,
kb
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FIG. 5. Northern blot analysts of VIP RNA levels in pituitary (API, me&o-basal hypothalamus IMBH), brain (BR), and ileum (Gut) after long term exposure of female rats to estrogen. A, The control group was sham-operated animals which did not receive s IX-S implant. B, The long term estrogen-treated rats received a 50.mg DES srlastic implant for 7 weeks.
expression may be at the level of transcription. Whether the respunse is mediated through an estrogen
gene
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ESTROGEN ACTION
1800 PT RNaseH
_
SR +
_
Gut +
_
+
*-
1.7kb -
l.Okb
FIG. 6. Analysis of the 1.7-kb and l-kb transcripts in the RNaseH digestion assay. The RNase H assay was performed according to the method of Sippel et al. (22). A total of 20 pg RNA from pituitary, brain, or ileum was used for each assay point. l&S marks the position of the 18s ribosomal subunit.
response element remains to be established. The human VIP gene has been cloned and sequenced (25, 26) and using a DNA analysis program, we have identified several estrogen response element-like consensus sequences. Whether the increase in VIP mRNA levels occurred as a result of increased gene transcription, increased mRNA stability, or through a secondary mechanism under the regulation of estrogen could not be determined from this study. Prolonged exposure to DES resulted in a dramatic increase in pituitary VIP mRNA content over the controls. Under chronic estrogen stimulation, it is possible that other regulatory mechanisms in the hypothalamic/ pituitary axis have contributed to the increase in VIP mRNA levels. Of particular interest was a smaller 1-kb transcript present under basal conditions almost exclusively in the pituitary. This message gave a more modest response (3fold increase) to estrogen treatment, implying that the 1-kb transcript was regulated in parallel with the larger 1.7-kb mRNA, the mature VIP mRNA species in the rat (24). Although the transcript appeared predominantly in the pituitary, a faint band was occasionally seen in the brain or small intestine. Moreover, the 1-kb transcript was upregulated by long term exposure to estrogen in the pituitary alone and not in the other VIP-producing tissues. Recent studies by Lam and collaborators (14, 15) have shown that pituitary content of a 1-kb transcript was increased in hypothyroidism, implying that the regulation of VIP gene expression is also dependent on thyroid status. The generation of the two VIP mRNA species is not due to differences in the length of the poly(A) tail since relative transcript size of both the 1.7kb and the 1-kb mRNA was approximately the same after RNase H digestion. Thus it may be speculated that the difference arises from differential use of 3’-poly(A)adenylation signals, differential mRNA processing, or from the possibility that the 1-kb transcript encodes for a different, but VIP-related, peptide. VIP mRNA levels also rose within the 15-min to l-h period in response to estrogen treatment in the hypo-
ON VIP mRNA LEVELS
Endo. 1992
Voll30. No4
thalamus, brain, and ileum, suggesting that the transient increase in VIP mRNA content was a result of increased gene transcription. VIP mRNA levels in the hypothalamus only reached a level of significance at 3 h but then remained elevated for 24 h. A second elevation in VIP mRNA content in the small intestine was seen at 6 h, suggesting that there is tissue-specific regulation of the VIP gene. In contrast, prolonged estrogen exposure did not increase VIP gene expression in these tissues. These data confirm previous reports that chronic estrogen treatment does not change VIP mRNA content in the medial basal hypothalamus, brain, and jejunum (27, 28). In fact, our data suggest that hypothalamic and intestinal VIP mRNA levels might even decrease in the presence of high estrogen levels. Maletti et al. (29) reported that hypothalamic VIP mRNA content also decreased in rats after a l-week DES implant. In conclusion, estrogen regulated VIP gene expression in a time-dependent manner in the pituitary, hypothalamus, brain, and ileum. A second, smaller VIP-related transcript was also regulated in parallel with the mature 1.7-kb VIP species which was present primarily in the pituitary. In the pituitary, both transcripts were upregulated after sustained estrogen treatment, whereas VIP mRNA levels remained unchanged or slightly decreased in the other VIP-producing tissues. Whether the 1-kb message encodes another form of VIP or is translated into a unique peptide remains to be established. Acknowledgments We thank Ingo Schroedter for his excellent technical help and Drs. Newman Stephens and Jiang He for their advice and help with the statistical analysis of the data. The authors are indebted to Debbie Tsuyuki for her critical reading of this manuscript.
References 1. Prysor-Jones RA, Jenkins JS 1988 Vasoactive intestinal peptide and anterior pituitary function. Clin Endocrinol29:677-688 2. Gazes I, Brenneman DE 1989 VIP: molecular biologv and neurobiological function. Mol Neurobiol3:201-236 -3. Morrison JH. Maaistretti PJ. Benoit R. Bloom FE 1984 The distribution and m&phological characteristics of the intracortical VIP-positive cells: an immunohistochemical analysis. Brain Res 292:269-282 4. Mezey E, Kiss JZ 1985 Vasoactive intestinal peptide-containing neurons in the paraventricular nucleus may participate in the secretion of prolactin. Proc Nat1 Acad Sci USA 82:245-247 5. Said SI, Porter JC 1979 Vasoactive intestinal DeDtide: release into hypophyseal portal blood. Life Sci 24:227-230‘ 6. Shimatsu A. Kato Y. Matsushita N. Katakami H. Yanaihara N. Imura H 1982 Stimulation by serotbnin of vasoactive intestinal polypeptide release into rat hypophyseal portal blood. Endocrinology 111:338-340 7. Robberecht P, Deschodt-Lanckman M, Camus J-C, de Neef P, Lambert M, Christoph J 1979 VIP activation of rat anterior pituitary adenylate cyclase. FEBS Lett 103:229-233 8. Wanke IE, Rorstad OP 1990 Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: localization of binding to lactotrophs. Endocrinology 126:1981-1988
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ESTROGEN ACTION 9. Reichlin S 1988 Neuroendocrine significance of vasoactive intestinal polypeptide. Ann NY Acad Sci 527:431-449 10. Lam KSL 1991 Vasoactive intestinal peptide in the hypothalamus and pituitary. Neuroendocrinology 53(suppl1):45-51 11. Maas DL, Arnaout MA, Martinson DR, Erdmann MD, Hagen TC 1991 Vasoactive intestinal polypeptide and thyrotropin-releasing hormone stimulate newly synthesized, not stored, prolactin. Endocrinology 128:1015-1020 12. Gourdji D, Bataille D, Vauclin N, Grouselle D, Rosselin G, TixierVidal A 1979 Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and CAMP production in a rat pituitary cell line (GH./B.). Additive effects of VIP and TRH on PRL release. FEBS Lett 104:165-168 13. Nagy G, Mulchahey JJ, Neil1 JD 1988 Autocrine control of prolactin secretion by vasoactive intestinal peptide. Endocrinololgy 1223364-366 14. Lam KSL, Reichlin S 1989 Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat. Neuroendocrinology 50:524-528 15. Lam KSL, Lechan RM, Minamitani N, Segerson TL, Reichlin S 1989 Vasoactive intestinal peptide in the anterior pituitary is increased in hypothyroidism. Endocrinology 124:1077-1084 16. Segerson TP, Lam KSL, Cacicedo L, Minamitani N, Fink JS, Lechan RM, Reichlin S 1989 Thyroid hormone regulates vasoactive intestinal peptide (VIP) mRNA levels in the rat anterior pituitary gland. Endocrinology 125:2221-2223 17. Steel JH, Gon G, O’Halloran DJ, Jones PM, Yanaihara N, Ishikawa H, Bloom SR, Polak JM 1989 Galanin and vasoactive intestinal peptide are colocalized with classical pituitary hormones and show plasticity of expression. Histochemistry 93:183-189 18. Carrillo AJ, Phelps CJ, Short-term e&radio1 benzoate treatment increases pituitary intracellular content of vasoactive intestinal peptide. Program of the 73rd Annual Meeting of The Endocrine Society, Washington, DC, 1991, p 390 (Abstract) 19. Vrontakis ME, Thliveris JA, Friesen HG 1987 Influence of bromocriptine and oestrogen on prolactin synthesis, secretion and tumour growth in uiuo in rats. J Endocrinol 113:383-388 20. Chirgwin JM, Prsybyla AE, MacDonald RJ, Rutter WJ 1979
ON VIP mRNA LEVELS
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22.
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24. 25.
26. 27.
28. 29.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry l&5294-5299 Fink JS, Montminy MR, Tsukada T, Hoefler H, Specht LA, Lechan RM. Wolfe H. Mandel G. Goodman RH 1986 In situ hybridization of somatbstatin and’ vasoactive intestinal nentide mRNA in the rat nervous system: contrasting patterns of ontogeny. In: Uhl GR (ed) In Situ Hvbridixation in Brain. Plenum Publishine Corp., New York, p lSl-i91 Sippel AE, Stavrianopoulos JG, Schutz G, Feigelson P 1974 Translational properties of rabbit globii mRNA after specific removal of poly(A) with ribonuclease H. Proc Nat1 Acad Sci USA 71:46354639 Koves K, Gottshall PE, Gores T, Scammell JG, Arimura A 1990 Presence of immunoreactive vasoactive intestinal polypeptide in anterior pituitary of normal male and long term estrogen-treated female rats: a light microscopic immunohistochemical study. Endocrinology 12617561763 Lam KSL, Srivastava G, Lechan RM, Lee T, Reichlin S 1990 Estrogen regulates the gene expression of vasoactive intestinal peptide in the anterior pituitary. Neuroendocrinology 52417-421 Yamagami T, Ohsawa K, Nishixawa M, Inoue C, Gotoh E, Yanaihara N, Yamamoto H, Okamoto H 1988 Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter. Ann NY Acad Sci 527:87-102 Tsukada T, Horovitch SJ, Montminy MR, Mandel G, Goodman RH 1985 Structure of the human vasoactive intestinal polypeptide gene. DNA 4293-300 Carrillo AJ, Doherty PC, Guan X, Sturtevant JR, Waho DG 1991 Preferential increase in pituitary prolactin uersus vasoactive intestinal peptide as a function of estradiol benzoate dose in the ovariectomised rat. Endocrinology 128131-138 Prysor-Jones PA, Silverlight JJ, Kennedy SJ, Jenkins JS 1988 Vasoactive intestinal peptide and the stimulation of lactotroph growth by oestradiol in rats. J Endocrinol116:259-265 Maletti M, Rostene WH, Carr L, Scherrer H, Rotten D, Kordon C, Rosselin G 1982 Interaction between estradiol and prolactin on vasoactive intestinal peptide concentrations in the hypothalamus and in the anterior pituitary of the female rat. Neurosci Lett 32307-313
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