ACTH, α-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio

Abstract

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.

Experimental hyperthyroidism and central mediators of stress axis and thyroid axis activity in common carp (Cyprinus carpio L.)

“…30G0 . 11 nM (nZ19), which values are, again, within the range measured in unstressed carp (van den Burg et al 2003, 2005a, Metz et al 2005. In hyperthyroid carp, basal a-MSH levels had increased by 30% (P!0 .…”

“…Tissues were superfused with a Hepes-Tris-buffered medium (pH 7 . 4) saturated with carbogen (95% O 2 -5% CO 2 ), at a rate of 30 ml/min as described in detail by Metz et al (2005). When a stable baseline of cortisol secretion was obtained, T 4 (at 1 mM), 3,5,3 0 -triiodothyronine (T 3 ; 1 mM), or human ACTH (1-39) (hACTH; 0 .…”

“…Baker, Phillipsburg, NJ, USA). Cortisol was measured by RIA, as described by Metz et al (2005), using a commercially available antiserum (Campro Scientific, Veenendaal, The Netherlands) and 125 I-cortisol (Amersham). Plasma-free T 4 (fT 4 ) was measured using a commercially available enzyme-linked immunoassay (Research Diagnostics, Inc., Flanders, NJ, USA) according to manufacturer's instructions.…”

Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution

“…The initial screen revealed two leptin-like sequences, one of which was already predicted in an earlier screen of the zebrafish genome (third (Table 1), based on these partial leptin sequences, two cDNA sequences were obtained from the liver and gonads respectively. RNA isolation, cDNA synthesis, cloning, and sequencing was carried out as previously described (Metz et al 2005). Briefly, PCR products were ligated and cloned in TOP10 chemically competent Escherichia coli in the pCR4-TOPO vector (Invitrogen).…”

Physiological responses and non-specific immune parameters in Epinephelus moara exposed to repeated physical stress

“…The blood was placed in a sterile centrifuge tube at room temperature for 2 h, placed at 4 °C overnight, centrifuged at 2717 g for 20 min, and the upper serum was collected. Serum and liver tissues were stored at Analytical procedures: Plasma cortisol was measured by radioimmunoassay as described by Metz et al (2005). Plasma glucose and lactic acid were assessed using commercially available kits (Jiancheng, Nanjing -China).…”