D

* *v

  1. 2. Effect of acute restraint stress for 1 h on VP hnRNA levels in the PVN of naive rats (B) or 24 h after the last restraint in rats subjected to 1-h repeated restraint for 14 days (D). Basal VP hnRNA levels were undetectable in the parvocellular region of naive rats (A), while they were markedly elevated 24 h after the last stress in repeatedly restrained rats (C). Darkfield photographs of emulsion-dipped slides were taken through the medial parvocellular subdivision of the PVN (MP). 3rd V, third ventricle; PM, posterior magnocellular; MP, medial parvocellular; RR x 14, repeated restraint, 1 h daily for 14 days.
  2. 2. Effect of acute restraint stress for 1 h on VP hnRNA levels in the PVN of naive rats (B) or 24 h after the last restraint in rats subjected to 1-h repeated restraint for 14 days (D). Basal VP hnRNA levels were undetectable in the parvocellular region of naive rats (A), while they were markedly elevated 24 h after the last stress in repeatedly restrained rats (C). Darkfield photographs of emulsion-dipped slides were taken through the medial parvocellular subdivision of the PVN (MP). 3rd V, third ventricle; PM, posterior magnocellular; MP, medial parvocellular; RR x 14, repeated restraint, 1 h daily for 14 days.

with pituitary VP receptor down-regulation, whereas somatosensory stressors leading to ACTH hyperresponsiveness to a novel stress (repeated immobilization or repeated i.p. hypertonic saline injections, a painful stress with an osmotic component) are associated with VP receptor up-regulation (Aguilera, 1994). In general, changes in VP receptors reflect changes in the number of binding sites with no significant alteration in binding affinity (Aguilera et al., 1994).

In contrast to VP receptors, there is a poor correlation between pituitary responsiveness and the number of CRH receptors in the anterior pituitary (Aguilera, 1998). In this regard, following adrenalectomy and chronic stress, there is marked down-regulation and desensitization of pituitary CRH receptors, with decreases in both receptor number and CRH-stimulated adenylate cyclase (Hauger et al., 1988; Aguilera, 1998). It is interesting to note that VP plays an important role on the loss of CRH receptors. In the regard, CRH receptor down-regulation following adrena-lectomy is markedly attenuated in the VP-deficient Brattleboro rat, and minipump infusion of VP and

CRH accentuate the down-regulation induced by infusion of CRH alone (Holmes et al., 1987; Hauger and Aguilera, 1993). It is unlikely that decreases in pituitary CRH receptors during chronic stress account for the desensitization of the ACTH responses to the heterotypic stimulus, since responses to a novel stress are enhanced in these conditions (Aguilera, 1994) and CRH receptor down-regulation also occurs in paradigms associated with sustained ACTH responses to the homotypic stressor. The molecular mechanisms involved in the regulation of both CRH and V1b receptors include transcriptional, translational and post-translational events controlled by the interactive effect of glucocorticoids and CRH and VP themselves. This has been extensively reviewed elsewhere (Aguilera, 1994; Volpi et al., 2004).

The positive correlation between the content of pituitary VP receptors but not CRH receptors and pituitary ACTH responsiveness to a novel stimulus, suggests that VP receptor regulation is part of the mechanism controlling corticotroph responses and supports the concept that during chronic stress regulation of HPA axis activity switches from CRH to VP (Ma et al., 1997; Aguilera and Rabadan-Diehl, 2000).

Does VP mediate ACTH responsiveness during chronic stress?

As discussed above, the parallel changes in VP expression in parvocellular neurons and pituitary V1bR and ACTH responsiveness during chronic stress have suggested that the increase in vaso-pressinergic activity is a major determinant of ACTH responsiveness to a novel stress. However, the hypothesis that VP becomes the primary regulator of ACTH responses during chronic stress has been difficult to demonstrate in studies using genetic models of VP or V1bR deficiency or pharmacological blockade of VP receptors. For example, the VP-deficient Brattleboro rat shows normal responses to most acute stressors and only a transient reduction in ACTH responses during repeated restraint (Baertschi et al., 1984; Zelena et al., 2004). On the other hand, studies in V1bR knockout mice show clear compromise of HPA axis responses to some stressors. Studies using a mouse carrying a deletion of the 3' end of the coding region of the V1bR show reduced ACTH responses to acute hypoglycemia, lipopolysaccha-ride and ethanol administration but normal basal and acute restraint-stimulated ACTH (Wersinger et al., 2002; Tanoue et al., 2004; Lolait et al., 2007a, b). It is noteworthy that in contrast to rats (Aguilera, 1998; Ma et al., 1999), wild-type mice showed no habituation of ACTH responses to the repeated homotypic stress of restraint. However, V1bR knockout mice showed no response to restraint stress on day 14, suggesting that V1bR are required for sustained responses to repeated stress (Lolait et al., 2007a). Although ACTH responses are reduced, these mice are able to display sustained corticosterone responses to the repeated stimuli. Other investigators find severely deficient HPA axis responses to forced swim stress in a mouse model with a full deletion of the V1bR coding region (Tanoue et al., 2004). However, it is important to consider that the development of compensatory mechanisms due to the functional disruption of the gene since embryonic life could obscure the interpretation of the findings in models of non-inducible gene ablation.

An alternative approach is the use of VP receptor antagonists, but to date only a selective V1bR antagonist, SSR149415, is available (Serradeil-Le Gal et al., 2005). This non-peptide, orally active antagonist binds to the V1bR with nM affinity, totally blocks radiolabelled VP binding to the V1bR in transfected cells, and inhibits VP-stimulated ACTH secretion in cultured pituitary cells (Serradeil-Le Gal et al., 2002). In vivo studies have shown that SSR149415 administered intravenously, intraperitoneally or orally effectively blocked VP-induced ACTH secretion and also restraint stress-stimulated ACTH secretion (Serradeil-Le Gal et al., 2002, 2005). However, recent studies in repeatedly restrained rats show only minor effects of SSR149415 on ACTH responses to a novel stress, when given either as a single intravenous (i.v.) injection preceding a novel stress, or by repeated oral daily administration during the 14-day restraint stress (Chen et al., 2008). The same study suggests that lack of a significant effect of the selective V1b antagonist is due to a rather short biological half life of the antagonist in the experimental conditions used in the study, as shown by partial reduction of ACTH responses to exogenously injected VP (Chen et al., 2008). An additional issue to consider when interpreting the latter experiments is the fact that SSR149415 crosses the blood brain barrier and that blockade of central V1bR by the compound may influence the HPA axis responses.

In contrast, chronic osmotic minipump administration of the non-selective peptide V1 receptor antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP, has been shown to effectively block ACTH and corticosterone responses to exogenous VP (Subburaju and Aguilera, 2007; Chen et al., 2008), indicating reasonable blockade of V1bR in the corticotroph. In contrast to the lack of effect of the orally active V1bR antagonist, chronic administration of the non-selective V1 antagonist caused a significant reduction of ACTH responses to i.p. hypertonic saline injection, suggesting that VP contributes to the response in this acute stress paradigm (Fig. 3A). The remarkable finding in the latter study was the total inability of the

  1. 3. The effect of chronic osmotic minipump administration of the non-selective V1 receptor antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP (V1R Ant), or vehicle for 14 days on plasma ACTH responses to the novel stress of i.p. hypertonic saline injection (ipHS) in conscious naïve control rats (A), or rats subjected to restraint stress for 1 h daily for 14 days (B). Data are the mean and SE of values obtained in 8 to 10 rats per group. Both groups showed a significant effect of ipHS on plasma ACTH levels (*^< 0.001). ACTH responses to ipHS in naïve rats were significantly reduced by the V1R Ant compared with the vehicle-infused controls ($p<0.04). ACTH responses to ipHS in repeatedly restrained rats were significantly higher than those in naïve rats (#p<0.02), and these enhanced responses were unaffected by the V1R Ant.
  2. 3. The effect of chronic osmotic minipump administration of the non-selective V1 receptor antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP (V1R Ant), or vehicle for 14 days on plasma ACTH responses to the novel stress of i.p. hypertonic saline injection (ipHS) in conscious naïve control rats (A), or rats subjected to restraint stress for 1 h daily for 14 days (B). Data are the mean and SE of values obtained in 8 to 10 rats per group. Both groups showed a significant effect of ipHS on plasma ACTH levels (*^< 0.001). ACTH responses to ipHS in naïve rats were significantly reduced by the V1R Ant compared with the vehicle-infused controls ($p<0.04). ACTH responses to ipHS in repeatedly restrained rats were significantly higher than those in naïve rats (#p<0.02), and these enhanced responses were unaffected by the V1R Ant.

antagonist to inhibit ACTH responses to i.p. hypertonic saline injection in repeatedly stressed rats (Fig. 3B). The fact that partial (with SSR149415) or complete pituitary V1bR blockade (peptide non-selective V1 antagonist) failed to inhibit acute stress responses in repeatedly restrained rats suggest that the VP receptor up-regulation observed following repeated restraint is not required for the sensitization of ACTH responses to a novel stress.

Overall, the available evidence supports the view that VP contributes to the full ACTH response during some types of acute stress. The loss of a sustained ACTH response to repeated restraint in V1bR knockout mice suggest that VP is required for long-term HPA axis responses. However, the normal corticosterone responses indicate that ACTH secretion in these mice is sufficient to elicit full adrenocortical activation. In addition, the ineffectiveness of the peptide antagonist to modify ACTH responses during chronic stress indicates that VP does not mediate the hyperresponsiveness of ACTH responses to a novel stress and suggests alternative roles for the peptide during stress adaptation.

Alternative actions of VP in the pituitary on mitogenesis

The disproportionality between the minor effects of genetic and pharmacologic VP blockade and the prominent increase in vasopressinergic activity (manifested as an increase in parvocellular VP and pituitary V1bR expression) raises the possibility that VP not only modulates ACTH secretion but has additional functions in the pituitary. It has been shown that VP stimulates mitogenesis in a number of systems, including mouse Swiss 3T3 cells (Rozengurt et al., 1979), rat bone marrow cells following haemorrhage (Hunt et al., 1977), rat liver cells (Russell and Bucher, 1983), mesangial cell (Ghosh et al., 2001), human osteoblast-like cells (Lagumdzija et al., 2004) and the murine corticotroph tumour cell line, AtT20 (van Wijk et al., 1995). VP has also been shown to increase the number of cells incorporating deoxybromour-idine (BrdU) in primary cultures of rat anterior pituitary cells (McNicol et al., 1990). Since chronic stress and adrenalectomy induce an increase in the number of corticotrophs, it is likely that VP could mediate mitogenic responses in the pituitary. This question was examined in a recent study using long-term infusion of a V1 antagonist in long-term adrenalectomized rats (Subburaju and Aguilera, 2007). As previously shown (Crane and Loomes, 1967; Rappay and Makara, 1981; Childs et al., 1989; Nolan et al., 1998), the latter study showed significant increases in the number of BrdU- and ACTH-labelled cells at 3 and 6 days, and a much larger increase at 28 days. Minipump infusion of the peptide V1 antagonist, dGly[Phaa1, D-tyr(et), Lys, Arg]VP at doses blocking the increases in ACTH and corticosterone induced by exogenous VP, for the duration of the experiment starting at the time of adrenalectomy, prevented the increases in BrdU incorporation (Fig. 4A), but not irACTH cells induced by adrenalectomy (Fig. 4B; Subburaju and Aguilera, 2007). This suggests that VP mediates mitogenic responses to adrenalectomy but that differentiation

  1. 4. The effect of chronic administration of the non-selective V1 receptor antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP (V1R Ant), or vehicle on adrenalectomy (ADX)-induced changes in the number of cells immunostained for ACTH or deoxybromouridine (BrdU). Rats were subjected to adrenalec-tomy and implantation of osmotic minipumps (Alza model 2004) containing the V1 antagonist (230ng/h/28 days) or vehicle, and BrdU injections for 7 days before sacrifice at day 28. Rats were killed by decapitation and pituitaries fixed for BrdU and ACTH immunohistochemistry. Bars represent the mean and SE of immunopositive cells counted in pituitary sections of four rats per experimental group. *^<0.05 vs. sham;
  2. 002 vs. sham; 0.001 vs. sham; $^<0.05 vs. ADX.
  3. 4. The effect of chronic administration of the non-selective V1 receptor antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP (V1R Ant), or vehicle on adrenalectomy (ADX)-induced changes in the number of cells immunostained for ACTH or deoxybromouridine (BrdU). Rats were subjected to adrenalec-tomy and implantation of osmotic minipumps (Alza model 2004) containing the V1 antagonist (230ng/h/28 days) or vehicle, and BrdU injections for 7 days before sacrifice at day 28. Rats were killed by decapitation and pituitaries fixed for BrdU and ACTH immunohistochemistry. Bars represent the mean and SE of immunopositive cells counted in pituitary sections of four rats per experimental group. *^<0.05 vs. sham;
  4. 002 vs. sham; 0.001 vs. sham; $^<0.05 vs. ADX.

can occur in the absence of the peptide. However, in contrast to the findings in rats, in V1bR knockout mice, adrenalectomy for 6 or 14 days failed to increase either the number of cells incorporating BrdU or the number of irACTH cells, while inducing the expected increase in wildtype mice. This suggests that lifetime deficient pituitary vasopressinergic activity has a more profound impact on the corticotroph population. Other studies showing a reduced number of corticotrophs in the VP-deficient Brattleboro rats compared with control Long Evans rats (Tankosic et al., 1982; Schmale and Richter, 1984) also support this view.

The effects of the V1 antagonist and V1bR ablation on the number of cells undergoing proliferation during long-term adrenalectomy discussed above support the hypothesis that VP mediates the mitogenic activity in the pituitary following glucocorticoid withdrawal. In contrast, in another study, PVN lesions were unable to prevent the increase in pituitary cell proliferation induced by adrenalectomy (Nolan et al., 2004), suggesting that pituitary mitogenesis could be a direct consequence of glucocorticoid withdrawal in the pituitary. However, it is possible that VP of supraoptic origin with access to the pituitary portal circulation promotes mitogenesis (Antoni, 1993). Thus, it is conceivable that VP becomes a critical pituitary mitogenic agent during longer-term adrenalectomy.

A question still outstanding is that of the origin of the cells undergoing mitogenesis during adre-nalectomy. Since adrenalectomy increases the number of corticotrophs, it would be expected that BrdU-stained nuclei co-localize with ACTH-immunoreactive cells. However, Subburaju and Aguilera (2007) found that only a minor proportion of BrdU-labelled nuclei corresponded to cells stained with ACTH or the corticotroph precursor marker, T-pit (Pulichino et al., 2004). Reports of co-localization of ACTH in pituitary cell types other than corticotrophs have suggested that mature pituitary cells can cross-differentiate (Denef et al., 2005). However, the lack of co-localization of BrdU-stained nuclei in lactotrophs, thyrotrophs, somatotrophs or gonadotrophs shown in this study is against this possibility. These observations render unlikely that the increase in corticotrophs originates from the division of existing corticotrophs or already differentiated corticotroph precursors, but suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells. Other studies have also shown lack of co-localization of BrdU in corticotrophs following adrena-lectomy (Gulyas et al., 1991; Taniguchi et al., 1995; Nolan and Levy, 2001). Nolan and Levy (2006) also reported a minor incidence of mitogenesis in corticotrophs or gonadotrophs following 3- and 6-days adrenalectomy or gona-dectomy in rats. In the latter study, there was no additivity of mitogenic responses to adrenalectomy and gonadectomy, supporting the view that mitogenesis in response to both stimuli occurs in an undifferentiated progenitor population (Nolan and Levy, 2006).

Of the two cell types examined as potential corticotroph progenitor cells, neither S100P-stained folliculo-stellate nor nestin-labelled stem cells have been found to co-localize BrdU. This suggest that folliculo-stellate cells do not act as precursors for the newly-formed corticotroph following adrenalectomy, and that non-nestin expressing stem cells are probably responsible for the mitogenic responses to long-term adrena-lectomy.

The fact that the increase in cells undergoing mitogenesis is VP-dependent raises the question of whether pituitary cell types other than cortico-trophs express VP receptors. While the main VP receptor subtype found in the pituitary is the V1bR, the major supporting evidence for VP-mediation of mitogenic responses was obtained using an antagonist equally effective for V1aR and V1bR (Rabadan-Diehl et al., 1995). Thus, it is possible that blockade of V1aR located in pituitary cells other than corticotrophs or in the periphery could contribute to the effects of the antagonist. In addition, the published in situ hybridization image showing co-localization of V1bR mRNA and POMC mRNA (Lolait et al., 1995b), shows clusters of V1bR mRNA grains not overlaying POMC stained cells, suggesting that not only corticotrophs may express V1bR. Whether these cells correspond to pituitary progenitor cells remains to be elucidated.

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