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Endocrinology Vol. 138, No. 12 5189-5194
Copyright © 1997 by The Endocrine Society

ARTICLES

Regulation of Pituitary V1b Vasopressin Receptor Messenger Ribonucleic Acid by Adrenalectomy and Glucocorticoid Administration

Cristina Rabadan-Diehl, Gabor Makara, Alexander Kiss1, Stephen Lolait, Dora Zelena, Tomasz Ochedalski and Greti Aguilera

Section on Endocrine Physiology (C.R-D., A.K., T.O., G.A), Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; Institute of Experimental Medicine (G.M., D.Z.), Hungarian Academy of Science, Budapest, H-1450 Hungary; Laboratory of Molecular and Cellular Regulation (S.L.), National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Greti Aguilera, M.D., Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892. E-mail: AguilerG{at}cc1.nichd.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of the number of pituitary vasopressin (VP) receptors plays an important role in controlling pituitary responsiveness during alterations of the hypothalamic pituitary adrenal axis. The mechanisms regulating these VP receptors were studied by analysis of the effects of adrenalectomy and glucocorticoid administration on V1b receptor (V1b-R) messenger RNA (mRNA) by Northern blot and by in situ hybridization in the rat. Adrenalectomy transiently decreased V1b-R mRNA levels by 18 h (77% and 62% for the 3.7-kb and 3.2-kb bands in the Northern blots, and 50% by in situ hybridization), returning to basal levels after 6 days. The decrease in V1b-R mRNA after 18 h adrenalectomy was fully prevented by dexamethasone (100 µg sc) but not by elimination of hypothalamic CRH and VP by paraventricular nucleus lesions or median eminence deafferentation. In sham-operated rats, dexamethasone increased receptor mRNA by 50% after 6 days. In contrast to Sprague-Dawley rats, in Brattleboro rats (di/di), which lack hypothalamic VP, adrenalectomy caused a sustained decrease in V1b-R mRNA levels (<50% of controls by 6 days). The data show that pituitary V1b-R mRNA is positively regulated by glucocorticoids and that the recovery of V1b-R mRNA levels after prolonged adrenalectomy is probably mediated by VP. In addition, the data suggest that the down-regulation of VP binding after long-term adrenalectomy is due to posttranscriptional events rather than to changes in V1b-R mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASOPRESSIN (VP) and CRH are major regulators of pituitary ACTH secretion during stress (1). While VP is a weak ACTH secretagogue on its own, it has an important physiological role modulating the effects of CRH on the corticotrope (1, 2, 3, 4). VP is secreted into the pituitary portal circulation from axon terminals projecting from the parvicellular division of the paraventricular nucleus (PVN) to the external zone of the median eminence (1). Studies in the rat have shown that in basal conditions 50% of CRH containing perikarya in the parvicellular area of the PVN coexpress VP (5). The proportion of VP-expressing cells increases substantially during chronic stress paradigms associated with increased pituitary ACTH responsiveness to a novel stress (6, 7). On the other hand, vasopressinergic magnocellular neurons of the PVN and supraoptic nuclei project axons to the neural pituitary from which VP is released to the peripheral circulation (1). Although magnocellular VP can access the pituitary portal circulation and stimulate the corticotrope in some experimental conditions (8, 9, 10, 11), it is not thought to play a major role in corticotrope regulation (12).

As is the case for other peptide hormones, VP exerts its regulatory effects through interaction with specific plasma membrane receptors of which two major subtypes, V1 and V2, have been identified (13). In contrast to the renal V2 receptor, which is coupled to adenylate cyclase, the smooth muscle and liver VP (V1a) and the pituitary (V1b) receptors are coupled to calcium/phospholipid-dependent signaling systems (14). Previous studies have shown a correlation between the number of binding sites for VP in the pituitary and corticotrope responsiveness (12, 13, 14, 15). In a number of chronic stress paradigms associated with enhanced pituitary ACTH responses to a novel stress, increases in VP binding are accompanied by increases in V1b-R messenger RNA (mRNA) (16). While this suggests that VP receptor regulation is a determining factor controlling pituitary corticotrope responses, the mechanisms of V1b-R mRNA regulation are not understood.

Glucocorticoids, key regulators of hypothalamus-pituitary-adrenal axis activity (17), are known to influence the number of pituitary VP receptors (18, 19, 20). Therefore it is likely that elevations in circulating glucocorticoid levels play a role in the regulation of pituitary V1b-R during stress. The objective of the present studies is to further investigate glucocorticoid actions on pituitary VP receptor regulation by analysis of the effects of adrenalectomy and glucocorticoid administration on V1b-R mRNA levels in the pituitary. Since hypothalamic expression of CRH and VP increases after adrenalectomy, the role of these peptides in mediating the changes induced by adrenalectomy was also studied after ablating their source through surgical PVN lesions or median eminence deafferentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and in vivo procedures
Adult male Sprague-Dawley rats weighing 280–320 g were purchased from Taconic Farms (Germantown, NY) or Charles River (Budapest, Hungary) [anterolateral cuts (ALC) studies]. Rats were maintained under standard conditions of lighting (lights on from 0700–1900 h) and temperature (22–24 C) with free access to food and water for 4–7 days before the experiments. Brattleboro rats (di/di) were purchased from Harlan Sprague Dawley (Indianapolis, IN). All animal protocols were approved by the Animal Users Care Committee, NICHD.

Bilateral adrenalectomy or sham operations were performed through a dorsal incision, under ketamine/xylazine anesthesia, 18 h, 4 days, or 6 days before tissue collection. Adrenalectomized rats had access to both drinking water and 0.9% saline. When indicated, glucocorticoid replacement was accomplished by daily sc injection of dexamethasone (Sigma Chemical Co., St. Louis, MO), 100 µg/day.

To determine the role of endogenous CRH and VP on the decrease of V1b-R mRNA after adrenalectomy, the endogenous source of these peptides was eliminated by PVN lesions or by anterolateral hypothalamic cuts disconnecting the median eminence from the PVN. For the PVN lesions, rats were anesthetized with ketamine/xylazine and placed in a stereotaxic apparatus, and the skull was exposed through a dorsal skin incision. After a slit had been drilled along the medial suture, 4 mm caudal, starting at the bregma, a specially designed wire knife (0.5 mm thick with a triangular shaped point, 1.5 mm) was inserted 1 mm caudal to the bregma and lowered 9.5 mm deep from the surface of the skull. In this position the knife was rotated two times, producing an inverted-cone lesion in the PVN area (19). After removal of the knife, the skin incision was closed with metal clips. Sham-operated rats were subjected to identical procedures but without rotation of the knife. Rats were maintained in their cages for 12 days to achieve complete depletion of CRH stores in the median eminence before adrenalectomy. The effectiveness of the lesion was evaluated by three criteria: 1) absence of PVN cells by light microscopic examination of thionine-stained hypothalamic sections, 2) absence of hybridization for CRH mRNA throughout the PVN, and 3) absence of histochemical evidence of immunoreactive CRH in the median eminence. Only rats with complete lesions, as judged by the above criteria, were included in the study.

To prevent the delivery of hypothalamic peptides to the median eminence, anterolateral cuts around the medial basal hypothalamus were performed with a bayonet-shaped Halasz-type knife with a blade radius and height of 1.8 mm, as described previously (20). Briefly, the knife was lowered in the midline 1.8 mm behind the bregma to the base of the skull, and a semicircular cut was made, with a further 2-mm posterior extension of the cut on each side of the medial hypothalamus. The cut started immediately behind the optic chiasma and extended backward to a coronal level between the middle of the median eminence and the attachment of the pituitary stalk. The cut deafferented the median eminence and the basal hypothalamus from all anterior and lateral connections, eliminating CRH, VP, and oxytocin inputs to the median eminence. Control rats were subjected to sham operations, and all animals were left to recover for 4 or 7 days before being subjected to adrenalectomy or sham adrenalectomy and killed by decapitation 18 h later. Trunk blood was collected for plasma hormone measurements, and the hypothalamus was fixed for histological analysis. Only rats with cuts transecting all anterior input and lateral input extending at least to the level of the middle of the median eminence were included in the study. Rats with successful ALC showed increased diuresis from 30.9 ± 3.1 to 133 ml/24 h.

After decapitation, pituitaries were rapidly removed and collected in ribonuclease (RNase)-free 1.5-ml plastic tubes on dry ice for poly(A) preparation, or mounted in cryoembedding medium (Tissue-Tek, O.T.C. compound, Miles, IN) for in situ hybridization.

Northern blot analysis of V1b-R mRNA
Poly(A) RNA from pools of four to five pituitaries was isolated using FastTrack (Invitrogen, San Diego, CA) kit reagents and separated in a denaturing formaldehyde agarose gel for 16 h at 20 V. After being transferred to Nytran plus membranes (Schleicher & Schuller, Keene, NH) using a pressure blotter (Stratagene, La Jolla, CA) at 70 mm Hg for 1 h and UV cross-linking, mRNA was hybridized overnight at 42 C with 20 million cpm of a 32P random-primed V1b-R cDNA probe as previously described (16). To correct the results for the amount of RNA loaded, membranes were further hybridized with 2 million cpm of a random primed 700-bp cyclophilin cDNA probe for an additional 3 h. Preliminary in situ hybridization experiments using a 48-mer oligonucleotide (bp 30 to 78 of rat cyclophilin) revealed that treatment of rats with dexamethasone, 100 µg/day, sc, had no effect on cyclophilin mRNA levels in the anterior pituitary. Therefore, cyclophilin was considered a suitable marker by which to normalize the Northern blot results. After the posthybridization washes (16), membranes were air dried and exposed to a Fuji imaging plate type BAS-III (Fuji Medical Systems USA, Inc., Stamford, CT) overnight, and the hybridized radioactivity was measured using a Fuji Bio imaging analyzer. Data are expressed in arbitrary Fuji Units normalized per 100 cyclophilin Fuji Units (16). In some experiments, membranes were exposed to X-OmatAR Kodak film (Eastman Kodak, Rochester, NY) for 6–10 days to obtain hard copy images.

In situ hybridization
High specific activity antisense probes directed to 464 bases of the 5'-untranslated region, immediately upstream from the putative initiating methionine, were synthesized as previously described (16). Slide-mounted pituitary sections were thawed for 10 min at room temperature. Prehybridization, hybridization, and posthybridization treatment of the slide-mounted sections was performed as described by Luo et al. (21) with the exception that posthybridization washes were performed at 70 C. Slides were exposed to Hyperfilm-beta Max (Amersham, Arlington Heights, IL) for 14–21 days. Sections from controls and experimental groups were processed in the same hybridization reaction, and light transmittance of the autoradiographs were quantitated using a computerized image analysis system (Imaging Research, Inc., St Catherine, Ontario, Canada). Measurements in at least three sections for each rat were averaged and used in the statistical calculations.

Statistical analysis
Data are presented as the mean ± SE of the values in the number of observations indicated in Results or in the figure legends. Statistical significance of the differences between experimental groups was determined by ANOVA followed by the Fisher test for multiple group comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of adrenalectomy on pituitary V1b-R mRNA
Northern blot analysis of pituitary poly(A) RNA hybridized with a complementary DNA (cDNA) probe corresponding to 363 bp of the coding region of the rat V1b-R revealed two mRNA populations with molecular sizes of 3.7 and 3.2 kb (Fig. 1BGo). As shown in Fig 1Go., A and B, hybridization of both bands decreased markedly 18 h after adrenalectomy (19% and 34% of the values in sham-adrenalectomized rats, for the 3.7, P < 0.01, and 3.2, P < 0.05, bands, respectively). This decrease in V1b-R mRNA levels was fully prevented by glucocorticoid replacement with dexamethasone, 100 µg, at the time of adrenalectomy.



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  		Figure 1. Autoradiogram of a representative Northern blot of V1b-R mRNA in pituitary poly(A) RNA from adrenalectomized (ADX) for 18 h and sham-operated rats with or without a single injection of dexamethasone (100 µg) or vehicle at the time of surgery. The arrows indicate the 3.7- and 3.2-kb bands corresponding to two populations of V1b-R mRNA, and a 1.0-kb band corresponding to cyclophilin mRNA. Lane 1, Sham adrenalectomy; lane 2, sham adrenalectomy-dexamethasone injection; lane 3, adrenalectomy; lane 4, adrenalectomy-dexamethasone injection.

 
V1b-R mRNA levels returned to values not significantly different from those in sham-operated rats after long-term adrenalectomy for 6 days (Fig. 2Go). Dexamethasone administration for 6 days increased the intensity of the 3.7-kb band to 181% and 168% of the values in vehicle-injected rats, in sham-adrenalectomized, and adrenalectomized rats, respectively, P < 0.01 (Fig. 2Go). The changes in the 3.2-kb band after dexamethasone administration were not statistically significant.



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  		Figure 2. A, Northern blot analysis of V1b-R mRNA in adrenalectomized (ADX) for 18 h and sham-operated rats with or without a single injection of 100 µg dexamethasone (Dex) or vehicle at the time of surgery. Bars represent the mean ± SE of the data obtained in the number of groups indicated in the bars, using pools of four or five pituitaries per group in each experiment. *, P < 0.01 vs. sham-ADX; #, P < 0.05 vs. sham-ADX. Values are arbitrary units (U) corrected per 100 U of cyclophilin. B, Northern blot analysis of V1b-R mRNA in 6-day adrenalectomized or sham-operated rats with or without daily injections of dexamethasone (100 µg). Bars represent the mean ± SE of the data obtained in the number of experiments indicated in the bars, using poly(A) mRNA from pools of five pituitaries per group in each experiment. Values are arbitrary units (U) normalized per 100 cyclophilin units. *, P < 0.01 vs. vehicle-injected controls.

 
A similar transient inhibitory effect of adrenalectomy on V1b-R mRNA levels was seen by in situ hybridization. Pituitary V1b-R mRNA levels decreased to 50% of the values in sham-operated rats 18 h after adrenalectomy (P < 0.05), but they were no different from those in sham-operated rats by 4 or 6 days (Fig. 3Go).



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  		Figure 3. Changes in pituitary V1b-R mRNA after adrenalectomy measured by in situ hybridization. Bars are the mean ± SE of the values for five rats per experimental group. *, P < 0.01 lower than sham-operated rats; **, P < 0.01 vs. vehicle-injected controls. Images show representative pituitary sections from each experimental group.

 
Effect of PVN lesion or median eminence deafferentation on V1b-R mRNA
To investigate the role of CRH and VP, major corticotrope regulators, in the decrease in V1b-R mRNA after early adrenalectomy, experiments were performed in rats subjected to PVN lesions to eliminate the source of these peptides. Additional groups of rats were subjected to median eminence deafferentation by hypothalamic ALC to prevent peptides from the PVN or supraoptic nucleus from reaching the pituitary portal circulation. PVN lesions for 12 days or ALC for 7 days had no effect on plasma ACTH levels but prevented the increases after 18 h adrenalectomy (Table 1Go). Plasma corticosterone was elevated in sham-lesioned rats, and these levels were not affected by PVN lesions or ALC. As expected, plasma corticosterone levels were undetectable after adrenalectomy (Table 1Go).


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  		Table 1. Effect of median eminence deafferentation by hypothalamic ALC or PVN lesion on plasma ACTH and corticosterone levels 18 h after adrenalectomy

 
The effect of 18 h adrenalectomy on V1b-R mRNA measured by in situ hybridization in PVN-lesioned rats is shown in Fig. 4Go. Consistent with the results above, in sham PVN-lesioned rats, V1b-R mRNA decreased to 34% of the values in sham-adrenalectomized rats 18 h after adrenalectomy. PVN lesions alone had no effect of V1b-R mRNA levels with transmittance values of 28 ± 2.3 in sham-lesioned rats and 27 ± 2.2 in PVN-lesioned rats (arbitrary units). In PVN-lesioned rats, adrenalectomy decreased V1b-R mRNA to 64% of the values in sham-adrenalectomized rats, a reduction that was not statistically significant from that in sham-lesioned rats. Similar to sham-lesioned rats, dexamethasone administration prevented the V1b-R mRNA loss 18 h after adrenalectomy in PVN- lesioned rats (Fig. 4Go).



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  		Figure 4. Effect of adrenalectomy (ADX) for 18 h on pituitary V1b-R mRNA in rats subjected to sham PVN lesions (control) or PVN lesions (PVN-L) 12 days before adrenalectomy. Bars represent the mean ± SE of the values obtained by in situ hybridization in the number of rats indicated in the bars. *, P < 0.01 vs. sham-adrenalectomized controls or adrenalectomized/sham lesioned rats.

 
As with PVN lesions, no changes in basal levels of pituitary V1b-R mRNA were observed after 7 days ALC alone. Similarly, median eminence deafferentation by ALC did not prevent the decrease in V1b-R mRNA observed 18 h after adrenalectomy (Fig. 5Go).



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  		Figure 5. Effect of median eminence deafferentation by ALC on the changes in pituitary V1b-R mRNA after adrenalectomy (ADX). Rats were adrenalectomized 7 days after sham cut or hypothalamic ALC and killed 18 h after adrenalectomy. Bars represent the mean ± SE of the values obtained by in situ hybridization in the number of rats indicated in the bars. *, P < 0.01 lower than sham-adrenalectomy or sham-cut controls.

 
Effect of adrenalectomy in di/di Brattleboro rats
In contrast to Sprague-Dawley rats, in di/di Brattleboro rats, which lack hypothalamic VP, adrenalectomy caused sustained decreases in V1b-R mRNA. As shown by Northern blot analysis, hybridization to both V1b-R mRNA bands was markedly reduced 7 days after adrenalectomy, with values of 31% and 41% of those observed in sham-adrenalectomized rats for the 3.7- and 3.2-kb bands, respectively (Fig. 6Go). Similarly, in in situ hybridization studies, V1b-R mRNA decreased to 51% of the values in sham-operated rats 6 days after adrenalectomy (66 ± 7.2 and 34 ± 2.1 arbitrary transmittance units for sham-operated and 6 days adrenalectomized rats, respectively, P < 0.001).



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  		Figure 6. Northern blot analysis of V1b-R mRNA in 7-day adrenalectomized or sham-operated Brattleboro (di/di) rats with or without daily injections of dexamethasone (100 µg). Bars represent the mean ± SE of the data obtained in four experiments using poly(A) mRNA from pools of five pituitaries per group in each experiment. Values are arbitrary units (U) normalized per 100 cyclophilin units. *, P < 0.001 vs. 6-day sham ADX.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In contrast with the sustained decreases in VP binding reported in adrenalectomized rats (22, 23, 24), this study, using Northern blot and in situ hybridization procedures, shows that adrenalectomy causes marked but only transient decreases in V1b-R mRNA levels in the pituitary. As previously reported during stress (16), no consistent differences in the regulation pattern of the 3.2- and 3.7-kb bands by glucocorticoids were observed in the present experiments. The significance of the two species of V1b receptor mRNA is not yet understood. Clearly, complete isolation and characterization of the V1b receptor gene will be required to determine whether different transcriptional start sites, alternate splicing, and/or differential polyadenylation of the mRNA account for the two transcript populations.

The lack of correlation between V1b-R mRNA and the number of binding sites indicates that changes in VP binding in the pituitary during adrenalectomy are not determined by the steady state levels of receptor mRNA. Since the expression of VP in the parvicellular hypothalamus is markedly increased after long-term adrenalectomy (25, 26, 27, 28), it is possible that the decrease in VP binding is due to an increase in receptor occupancy and receptor internalization rather than a decrease in receptor synthesis. However, an inhibition of receptor synthesis at the posttranscriptional level cannot be excluded. A similar VP receptor down-regulation with normal V1b-R mRNA levels was observed after pituitary stalk compression (29), an experimental model with high pituitary exposure to VP obtained through shunting the peptide from magnocellular fibers to the pituitary portal circulation (30, 31). In contrast, during chronic stress, a condition associated with increased expression of parvicellular VP (6, 7, 12), pituitary VP binding is increased (16). VP secretion into the pituitary portal circulation during stress is probably episodic and differs from the higher and more sustained levels likely to occur during adrenalectomy and pituitary stalk compression. Such dissimilar VP levels and mode of secretion of the peptide may explain the different regulation of pituitary VP receptors in these conditions.

The mechanism of the decrease in pituitary V1b-R mRNA observed after short-term adrenalectomy may include the loss of glucocorticoid action in the pituitary and/or increased secretion of hypothalamic regulators, secondary to glucocorticoid withdrawal. It is unlikely that an increase in VP plays a role in the transient V1b-R mRNA down-regulation. Recent work in this laboratory has not shown any down-regulatory effect of acute or chronic VP administration on pituitary V1b-R mRNA levels (Aguilera G., T. Ochedalski, and C. Rabadan-Diehl, manuscript in preparation). Similarly, pituitary V1b-R mRNA levels are not reduced by exposure to high VP levels in pituitary portal circulation after pituitary stalk compression (29). On the other hand, in some experimental conditions it appears that CRH can induce V1b-R mRNA down-regulation. For example, recent studies show decreases in V1b-R mRNA after injection of interleukin-1 in the rat, an effect that was prevented by pretreatment of the rats with a CRH antibody (F. Tilders and G. Aguilera, manuscript in preparation). In addition, studies in progress show rapid but transient decreases in V1b-R mRNA after an injection of CRH. Since the median eminence is a site of glucocorticoid feedback (32), and adrenalectomy results in rapid release of median eminence CRH stores to the pituitary portal circulation (33), CRH could induce V1b-R mRNA down-regulation after early adrenalectomy. However, the present demonstration that the decrease in V1b-R mRNA after 18 h adrenalectomy was fully prevented by glucocorticoid replacement, but not by PVN lesions and median eminence deafferentation, indicates that CRH is not responsible for the early effect of adrenalectomy on pituitary levels of V1b-R mRNA. On the other hand, the fact that glucocorticoids prevented the effect of adrenalectomy in rats with PVN lesions, in conjunction with the ability of long-term dexamethasone administration to increase V1b-R mRNA, strongly suggests that removal of a positive regulatory influence by glucocorticoids alone can account for the decrease in V1b receptor mRNA. Similar to the present findings with the V1b-R, V1a receptor mRNA levels have been shown to increase after in vitro incubation with glucocorticoids in vascular smooth muscle cells (34) or rat mammary tumor cell line, WRK-1 (35), or in the septum after in vivo administration of dexamethasone in the rat (36).

In contrast to the results presented here for V1b-R mRNA, it has been reported that the decrease in VP binding after adrenalectomy is prevented by PVN lesions or hypothalamic cuts (22, 24). This indicates that VP receptor content in the pituitary is regulated at multiple sites by different mechanisms. Additional studies are required to determine whether the alterations in pituitary V1b-R mRNA are due to direct effects on gene transcription or alterations in receptor mRNA turnover. The stimulatory effects of glucocorticoids on V1a receptor mRNA and VP responsiveness in vascular smooth muscle cells appear to be due to an increase in V1a-R mRNA stability rather than gene transcription (34).

If pituitary V1b-R mRNA levels are stimulated by glucocorticoids as suggested by the data, the fact that mRNA levels return to normal after long-term adrenalectomy, in spite of the continuing glucocorticoid deficiency, is intriguing. Since long-term adrenalectomy results in marked increases in VP expression in parvicellular neurons (25, 26, 27, 28), exposure of the corticotrope to increased levels of VP or VP/CRH ratios may compensate for the lack of glucocorticoids and be responsible for the recovery of the early loss of V1b-R mRNA after adrenalectomy. This possibility is supported by the marked and sustained loss of V1b-R mRNA observed after adrenalectomy in Brattleboro rats that lack hypothalamic VP. The possibility that the lack of VP in Brattleboro rats delays the recovery of V1b receptor mRNA only after long-term adrenalectomy is unlikely since the studies in Sprague-Dawley rats show that V1b mRNA level are fully restored after 4 days’ adrenalectomy. In any case, further studies will be needed to confirm the role of VP during long-term adrenalectomy because preliminary attempts to restore V1b-R mRNA with minipump infusions of VP in Brattleboro rats, or to induce sustained decreases with V1 receptor antagonists in Sprague-Dawley rats, have not been successful (data not shown). One of the difficulties in interpreting results obtained from manipulation of pituitary exposure to VP is the marked hemodynamic effects of systemic administration of VP or V1 agonists at doses required to reach levels in the range of those observed in the pituitary portal circulation (37, 38). In addition, the biological effects of sustained elevated circulating levels of VP obtained with minipump infusion are likely to differ from those caused by the episodic endogenous increases observed in physiological conditions (39, 40).

Overall, this study shows that while glucocorticoids induce pituitary VP receptor down-regulation, they positively control V1b-R mRNA levels. Increased secretion of hypothalamic regulators immediately after adrenalectomy do not appear to play a role in the transient decreases in V1b-R mRNA, whereas the progressive increases in parvicellular VP expression and secretion during long-term glucocorticoid withdrawal are likely to mediate the recovery of pituitary V1b-R mRNA levels. In addition, the lack of correlation between V1b-R mRNA and VP binding indicates that steady state levels of V1b-R mRNA are not a major determinant of VP receptor content in the pituitary.


   Footnotes
 
1 Present address: Institute of Experimental Endocrinology, Slovak Academy of Science, 833-06 Bratistava, Slovak Republic. Back

Received May 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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