Distribution of the regulatory peptide alarin in the eye of various species

Abstract

Alarin is a recently discovered regulatory peptide with vasoconstrictive properties in murine skin. Control of vasoconstriction/-relaxation is essential for ocular blood flow and hence the eye's homeostasis, and regulatory peptides are involved in regulation of ocular blood flow. Here we describe the existence and distribution of alarin in the eye of human and potential experimental animals (rat, mouse). Eyes of rat, mouse, and human were prepared for immunohistochemistry against murine and human alarin, respectively. Additionally, double staining experiments for alarin and CD31 were performed in human choroidal flat-mount preparations. For documentation, confocal laser scanning microscopy was used while quantitative real-time-PCR was applied to confirm immunohistochemical data and to detect alarin mRNA expression in human retina and choroid. Alarin-like immunoreactivity (alarin-LI) was detected in corneal epi- and endothelium of human, mouse, and rat, as well as in the conjunctiva of mouse and rat. Alarin-LI was found in the iris of all the species investigated and, in humans, was concentrated around blood vessels. All three species showed distinctive alarin-LI in the non-pigmented epithelium of the ciliary body. In the retina of mouse and rat, maximum signals were detected in the outer nuclear and ganglion cell layer, whereas in humans a strong alarin-LI was found around retinal blood vessels and in intrinsic choroidal neurons (ICN). Quantitative RT-PCR in human confirmed alarin mRNA expression retina and choroid. The existence of alarin in cornea and conjunctiva might indicate a role in immune defense, while its presence in the non-pigmented ciliary epithelium favors an involvement in aqueous humor production. Alarin around blood vessels/in ICN might indicate an involvement in ocular blood flow regulation. Since alarin is found widely distributed in the eyes of species investigated, we were able to establish the basis for further functional experiments.

Introduction

To achieve proper homeostasis for optimal visual function, the eye is controlled by the autonomic nervous system (Neuhuber and Schrödl, 2011). This autonomic control is achieved via nerve fibers of sympathetic, parasympathetic as well as primary afferent origin, each using specific “modulators” to adequately set ocular blood flow or aqueous humor dynamics. These modulators include classical transmitters like catecholamines or acetylcholine for the sympathetic or parasympathetic pathways, respectively, however, during the last decades many other neuroactive substances have been introduced, such as the gas nitric oxide (Boeckxstaens et al., 1991), purinergic compounds (Burnstock, 2010), or regulatory peptides. Regulatory peptides represent a class of small protein-like molecules involved not only in neurosignalling or co-transmission of the classical neurotransmitters (Troger et al., 2007), but also in inflammatory processes and pain sensation (Raddant and Russo, 2011), immune defense (Souza-Moreira et al., 2011), osmoregulation (Kozniewska and Romaniuk, 2008), and behavior (Nixon et al., 2012) or memory function (Crawley, 2010).

In the eye, however, the interaction of the various neurotransmitters and regulatory peptides in ocular regulation is poorly understood.

Alarin is a recently discovered small peptide of 25 amino acids (Santic et al., 2006) and belongs to the galanin family of peptides (Lang et al., 2007). It was described in human neuroblastic tumor as a splice variant lacking exon3 of the galanin-like peptide (GALP) mRNA (Santic et al., 2006), thereby also missing the galanin-receptor binding domain and thus possibly using different receptors for its action. Splice variants exist for other regulatory peptides, e.g. CGRP (Rosenfeld et al., 1983), or neuropeptide Y (Melas et al., 2012), which leads to a diversification of proteins resulting from a single gene and hence to a reduction of the underlying genetic code (Matlin et al., 2005).

Alarin is detectable in neurons of various CNS regions (Eberhard et al., 2012) as well as in epithelial cells and around blood vessels (Eberhard et al., 2012; Santic et al., 2007), but is also found in neuroblastic tumors (Santic et al., 2006). In animal experiments in mice and rats it shows endocrine functions stimulating appetite and reproductive hormone secretion (Boughton et al., 2010; Fraley et al., 2012; Van Der Kolk et al., 2010). Furthermore, alarin inhibits neurogenic inflammation by vasoconstriction in the murine skin (Santic et al., 2007).

Since the regulation of the vessel diameter is a key player in ocular blood flow, and therefore ocular homeostasis, alarin is of potential interest for experimental ophthalmology. Here, we investigate the existence of alarin in eyes of rat, mouse and human by morphological and molecular biological methods.