Cortisol regulates nitric oxide synthase in freshwater and seawater acclimated rainbow trout, Oncorhynchus mykiss

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Abstract

Cortisol and nitric oxide (NO) are regulators of ion transport and metabolic functions in fish. In the gill, they show opposite effects on Na+/K+-ATPase (NKA) activity: cortisol stimulates NKA activity while NO inhibits NKA activity. We hypothesized that cortisol may impact NO production in osmoregulatory tissues by regulating NO synthase (NOS) expression. We evaluated the influence of cortisol treatment on mRNA expression of Nos1 and Nos2 in gill, kidney and middle intestine of both freshwater (FW) and seawater (SW) acclimated rainbow trout and found both tissue- and salinity-dependent effects. Nos2 expression was down-regulated in the gill by cortisol injection in both FW and SW trout. This was substantiated by incubating gill tissue with cortisol ex vivo. Similarly, cortisol injection significantly down-regulated Nos2 expression in kidney of SW fish but not in FW fish. In the middle intestine, Nos2 expression was up-regulated by cortisol injection in FW but unchanged in SW fish. Nos1 expression was up-regulated by cortisol injection in FW kidney and down-regulated in SW kidney, whereas it was unaffected in gill and middle intestine of FW and SW fish. Our data provide the first evidence that cortisol may influence NO production in fish by regulating Nos expression. Indeed, the down-regulation of Nos2 expression by cortisol in the gill may prevent the inhibitory effect of NO on NKA activity thereby furthering the stimulatory effect of cortisol on ion-transport.

Introduction

Cortisol is the major corticosteroid in fish and has a wide range of actions including ionoregulatory and metabolic functions (Mommsen et al., 1999). Interestingly, the gasotransmitter nitric oxide (NO) is also known to affect such processes (Cooper and Giulivi, 2007, Fago and Jensen, 2015, Perry et al., 2016) and it seems appropriate to expect that the two regulatory pathways may interact locally in various tissues. Whereas cortisol is a relatively slow-acting hormone, typically involved in transcriptional changes on a relatively long time scale (hours-days), NO is a gasotransmitter synthetized in various tissues that operates in seconds-minutes, characteristic of a paracrine/autocrine regulator. Even though they work on different time scales, the potential interaction between the two mediators remains to be elucidated. In salmonids, both in vivo and ex vivo studies have documented that cortisol mediates long-term osmoregulatory adjustments by stimulating branchial, renal and intestinal Na+/K+-ATPase (NKA) activity (Kiilerich et al., 2007, Kiilerich et al., 2011b, Madsen, 1990, McCormick, 2001, McCormick et al., 1991, McCormick et al., 2008, McCormick and Bern, 1989, Shrimpton and McCormick, 1999, Veillette and Young, 2005). Yet the recent discovery of NO as a rapid inhibitory modulator of ion transport and of NKA activity in fish challenges our understanding of how these regulatory processes are integrated. Evidence of NO involvement in ion transport in fish was first given by Tipsmark and Madsen (2003), who observed an inhibition of NKA activity by NO donors in gill and kidney of freshwater (FW)-acclimated brown trout. A similar inhibitory effect was subsequently reported in the gill of seawater (SW)-acclimated Atlantic salmon (Ebbesson et al., 2005). Furthermore, Evans et al. (2004) and Trischitta et al. (2007) observed a significant down-regulation of ion transport by NO in the opercular epithelium of SW-acclimated killifish and the middle intestine of SW eel, respectively. In addition, we recently showed a strong inhibition of Cl secretion by both endogenous NO production and NO donors in the opercular membrane of SW killifish, which was primarily mediated by activation of guanylyl cyclase and cGMP signalling (Gerber et al., 2016), but suggesting S-nitrosation of ion transporting proteins as an additional mechanism. Based on these former studies in fish, it seems that cortisol and NO may have antagonistic effects on epithelial ion transport. In mammals there is evidence of such interaction. Glucocorticoids inhibit NO production in various cell and tissue types, including lung, liver, kidney, cardiac and vascular endothelial cells and macrophages (Balligand et al., 1994, Di Rosa et al., 1990, Knowles et al., 1990, Lou et al., 2001, Radomski et al., 1990, Simmons et al., 1996) by mechanisms that include inhibition of the expression and activity of nitric oxide synthases and regulation of its cofactors and substrate (Whitworth et al., 2002). Thus, some of the physiological and pharmacological effects of cortisol may be due to interactions with NO synthesis. Nitric oxide synthase (NOS) is the enzyme that catalyses the production of NO. In fish, two distinct isoforms of the enzyme have been identified: NOS1 (also called neuronal NOS, nNOS) and NOS2 (also called inducible NOS, iNOS) (Andreakis et al., 2011). The two isoforms share common domains but also present distinctive features (in structures, expression/localisation, enzymatic functions and activities) that are well-described in mammals (Alderton et al., 2001, Andrew and Mayer, 1999, Michel and Feron, 1997). In fish, the characteristics of NOS1 and NOS2 are not clearly defined. Yet, the cofactor binding domains of mammalian and teleost NOSs are well conserved (Øyan et al., 2000, Hyndman et al., 2006, Andreakis et al., 2011) suggesting some common features. For instance, the NOS1 isoform requires Ca2+ for its enzymatic activity, whereas NOS2 is Ca2+-independent and can be induced in various physiological conditions to produce NO (Nathan, 1997). Hence, the two isoforms can be activated and regulated differentially and may have distinct biological roles when expressed in different tissues.

In the present study, we hypothesized an interaction between cortisol and Nos expression in fish. First, we examined a comprehensive organ distribution of Nos1 and NOS2 in FW rainbow trout. Then, we examined the influence of cortisol, the major teleost corticosteroid (Takahashi and Sakamoto, 2013), on mRNA expression of Nos1 and Nos2 in gill, kidney and middle intestine of cortisol-treated rainbow trout acclimated to FW and SW. This study is the first to report an interaction of cortisol with NOS expression in osmoregulatory tissues of a euryhaline teleost.

Section snippets

Animals

Juvenile female rainbow trout of body mass ~ 40 g were obtained from a local fish farm (Lihme, Randbøl, Denmark). The fish were acclimated for two months to 15 °C and a 12 h:12 h light:dark cycle in aerated bio-filtered, recirculated freshwater or artificial seawater at 25 ppt (Red Sea Salts, Verneuil s/Avre, France). Fish were fed with commercial trout pellets every second day. Feeding was withheld three days prior the experiments. The experimental work followed the guidelines of the Danish Law on

Tissue distribution of Nos isoforms mRNA in FW rainbow trout

Both Nos2 and Nos1 isoforms were constitutively expressed in all organs tested. However, the mRNA level of Nos2 (Fig. 1A) and Nos1 (Fig. 1B) varied greatly among organs. Ovary and gill had relatively high levels of Nos2 mRNA, approximately 10 to 100-fold higher than in the other organs tested (Fig. 1A). The highest relative expression of Nos1 was, by far, detected in ovary followed by middle intestine and brain which was 25 to 1000-fold higher than in other organs tested (Fig. 1B).

In vivo influence of cortisol on branchial, renal and intestinal Nos mRNA expression

In FW fish,

Discussion

Cortisol, the major corticosteroid in teleosts, has a multitude of gluco- and mineralocorticoid actions (Mommsen et al., 1999, Takahashi and Sakamoto, 2013). Some of these effects relate to salinity stress, where cortisol is known to stimulate cellular proliferation and expression of specific ion-transporter genes in the gill and intestine. Less is known about such mechanisms in the kidney. NO is also a modulator of many physiological processes; its roles in vasodilation and immune function are

Acknowledgements

We thank Lene Jakobsen for technical assistance. This work was supported with grants to S.S.M. (DFF-4181-00020) and F.B.J. (10-084565) from the Danish Council for Independent Research.

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