Proopiomelanocortin and melanocortin receptors in the adult rat retino-tectal system and their regulation after optic nerve transection
Introduction
The melanocortins are a family of bioactive peptides derived from proopiomelanocortin (POMC) (Eipper and Mains, 1980). The melanocortins are formed by post-translational modifications of POMC in a tissue-specific manner. Adreno-corticotropic hormone, α-melanocyte-stimulating hormone (α-MSH), β-MSH, or γ-MSH are formed by proteolytic cleavages of POMC by two main proteolytic enzymes, proconvertases 1 and 2 (Benjannet et al., 1991). Other biologically active peptides derived from POMC are β-lipotropin, γ-lipotropin and β-endorphin (Eberle, 1988). The melanocortins are synthesised in various regions in the central nervous system and in peripheral tissues and participate in multiple physiological functions (Adan and Gispen, 2000). The biological effects of melanocortins are mediated by the activation of melanocortin receptors. This activation is antagonised and modulated by two endogenous physiological antagonists, agouti-signaling protein and agouti-related protein, that bind the melanocortin receptors (Dinulescu and Cone, 2000). In all, five melanocortin receptors have been cloned and characterised (melanocortin MC1–5 receptors) Abdel-Malek, 2001, Chhajlani and Wikberg, 1992, Gantz et al., 1993, Mountjoy et al., 1992 that mediate the physiological functions, including stimulatory effects on pigmentation, adrenal function, energy homeostasis, feeding, sebaceous gland lipid production, steroidogenesis in immune and sexual function Butler and Cone, 2002, Wikberg et al., 2000. Melanocortin receptors represent a distinct family of G-protein-coupled receptors and all five receptors are functionally coupled to adenylate cyclase and mediate their effects by activating cAMP-dependent signaling pathways. Melanocortin receptors differ from each other in their tissue distribution and in their binding affinity for the melanocortins and their antagonists (Abdel-Malek, 2001). The main sites of melanocortin receptor expression include: melanocytes, immuno- and inflammatory cells for melanocortin MC1 receptor; adrenal cortex and adipocytes for melanocortin MC2 receptor; central nervous system, stomach, duodenum and pancreas for melanocortin MC3 receptor; central nervous system for melanocortin MC4 receptor and exocrine gland; sebaceous glands and adipocytes for melanocortin MC5 receptor Fathi et al., 1995, Wikberg et al., 2000, Wong et al., 1997, Xia et al., 1995. Notably for this study is that POMC and melanocortin receptors have been found in the developing chicken eye indicating presence of the melanocortin system in the eye and retinal cells (Teshigawara et al., 2001).
Previous work has shown that melanocortins may have activities on developing and injured neurons that resemble those of neurotrophic factors Gispen et al., 1994, Hol et al., 1995, Muller et al., 1994, Plantinga et al., 1995, Van de Meent et al., 1997, Van der Neut et al., 1992. The classical neurotrophic factors with the neurotrophin gene family members as prototypes are secreted proteins that exert their trophic functions via receptor tyrosine kinases Barde, 1988, Lewin and Barde, 1996. Recent results show that retinal ganglion cells respond with increased neurite growth and survival to the stimulation of classical neurotrophic factors if they are co-stimulated with agents that increase intracellular cAMP Meyer-Franke et al., 1995, Meyer-Franke et al., 1998. The described neurotrophic activity of melanocortins and the fact that their main signalling pathway involves modulation of intracellular cAMP concentration prompted us to study the expression of the melanocortin system in the retina. The expression has not previously been characterised in the retina and this work shows that melanocortin receptor and POMC mRNA are expressed in the rat retina and in particular in retinal ganglion cells. In addition to the normal expression, we have studied retina and superior colliculus, which is one of the main central retinal ganglion cell targets, after optic nerve transection. This axotomy leads to subsequent retinal ganglion cell death (Villegas-Perez et al., 1993). The induced death occurs via apoptosis (Berkelaar et al., 1994) and can be postponed but not halted by classical neurotrophic factors Aguayo et al., 1996, Di Polo et al., 1998, Peinado-Ramón et al., 1996. Responsiveness of retinal ganglion cells to neurotrophic factors is decreased after axotomy but is restored by cAMP elevation (Meyer-Franke et al., 1998). The survival of axotomised retinal ganglion cells can be improved if they are simultaneously stimulated by several trophic factors together with cAMP elevation. Thus, this suggest that the death of retinal ganglion cells after axotomy is not caused solely by the loss of retrograde trophic stimuli but also by a loss of complementing intracellular signaling and trophic responsiveness (Shen et al., 1999). In addition to the characterization of the retinal localization of melanocortin receptors, the aim of this work was to study whether the melanocortin receptors contribute to an injury-induced receptor system. We have also used retinal explant cultures to study if retina responds to α-MSH stimulation in a similar way as has been shown for other peripheral and central neurons.
Section snippets
Animals and surgical procedures
We used adult female DA rats (225–250 g; B&K Universal, Sollentuna, Sweden). For all experimental manipulations, animals were anesthetised with an intraperitoneal injection mixture of xylazine (Rompun; Bayer) and ketamine (Ketalar; Parke-Davis), 10–15 and 30–100 mg/kg body weight, respectively. Rats were housed in standard cages, fed ad libitum and maintained in temperature-controlled rooms with a 12:12-h light/dark cycle with light intensity ranging from 8 to 24 lx. Experiments were carried
Melanocortin MC3, MC4, MC5 receptor and POMC mRNA expression in retina and superior collicuclus
Among the melanocortin receptors, the melanocortin MC3 and MC4 receptors are mainly expressed in the nervous system whereas melanocortin MC1 and MC2 receptor are expressed in melanocytes and in the adrenal cortex. In order to study the melanocortin receptor mRNA expression in the rat retina and superior colliculus, we used quantitative real-time PCR with primers for melanocortin MC2–5 receptors (Table 1). The melanocortin MC1 receptor sequence is not available from rat and was not included in
Discussion
In this work we show that the melanocortin system is present in the adult rat retina. Melanocortin MC3, MC4 and MC5 receptors as well as the POMC mRNA are expressed in retina although at different levels. Melanocortin MC4 receptor mRNA is clearly the most abundant melanocortin receptor mRNA in the retina. Melanocortin MC4 receptor mRNA was also abundant in the superior colliculus, which is one of the main regions in the rat brain that receive visual information via the optic nerve. Injury to
Acknowledgments
The work was supported by the Swedish Research Council (12187), Mattsons and KMA research foundations. NL was supported by the National Network for Neuroscience, UN was supported by the Academy of Finland, Glaukooma Tukisäätiö Lux and De Blindas Vänner r.y. We thank A. Kylberg and P.H. Edqvist for technical assistance.
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