Neurokinin A Is a Main Constituent of Sensory Neurons Innervating the Anterior Segment of the Eye Eduard Schmid,1 Johannes Leierer,2 Alfred Doblinger,2 Andrea Laslop,2 Reiner Fischer-Colbrie,2 Christian Humpel,3 Elvar Theodorsson,4 Barbara Teuchner,1 Djavad Lalehabbasi,1 Ernst Dragosits,1 Christian Kunze,1 Wolfgang Philipp,1 Wolfgang Go¨ttinger,1 and Josef Troger1 PURPOSE. To study the innervation pattern of the anterior seg- Ophthalmol Vis Sci. 2005;46:268–274) DOI:10.1167/iovs.04- ment of the eye by neurokinin (NK)-A–immunoreactive nerves 0608 and to determine their sensory origin. METHODS. The presence and distribution of NKA was examined eurokinin (NK)-A, a 10-amino-acid neuropeptide, belongs in human eyes by radioimmunoassay and immunofluores- Nto the group of tachykinins that share the common C- cence. The source of nerves was determined by measuring the 1 terminal amino acid sequence Phe-X-Gly-Leu-Met-NH2 and that concentration of NKA in the trigeminal ganglion (TG) in com- have a variety of similar biological effects. The best known parison with that of the classic sensory peptides substance P tachykinin is the undecapeptide substance P (SP), which is a (SP) and calcitonin gene-related peptide (CGRP) and in eye well-known constituent of sensory nerves (for review, see Ref. tissues in capsaicin-pretreated rats versus control subjects. The 2). For a long time, SP was thought to be the only tachykinin NKA-like immunoreactivities were further characterized by re- but so far, more than 40 tachykinins have been isolated from versed phase HPLC in the rat TG and the human iris–ciliary invertebrate (insects, worms, and molluscs), protochordate, ␥ body complex. The presence of -PPT-A mRNA was studied in and vertebrate (skin, gastrointestinal tract, peripheral, and cen- the rat TG by in situ hybridization. tral nervous system) tissues.3 Three main tachykinins have RESULTS. The levels of NKA in human eye tissues were approx- been identified in mammals, apart from SP: NKA; neuropeptide imately 10 times higher than those of SP but lower than those K (NPK), an N-terminally extended form of NKA; and NKB.4–7 of CGRP. Nerve fibers were visualized in the cornea, the tra- SP and NKA are expressed by the same gene, which gives becular meshwork, the iridial stroma, and, prominently, in the rise to ␣-, ␤-or␥-preprotachykinin (PPT)-A as a result of sphincter muscle, the ciliary body stroma and muscle and alternative splicing of a common transcription product. SP processes, and the choroidal stroma and surrounding blood alone is present in the ␣-PPT-A sequence, whereas ␤- and vessels. In the rat TG, the concentration of NKA was approx- ␥-PPT-A contain both SP and NKA. In contrast, NKB is ex- imately five times higher than that of SP. Capsaicin led to a pressed by a second gene that gives rise to PPT-B. PPT-A and -B Ͼ60% decrease of the concentration of the peptide in the rat are structurally very similar, suggesting evolution from a com- TG and rat eye tissues except for the retina. NKA-like immu- mon ancestor gene.8–11 In accordance with the existence of a noreactivities were present in a single peak corresponding to common precursor for SP and NKA, NKA is known to coexist synthetic NKA, both in the rat TG and in the human iris–ciliary with SP in sensory neurons in a large number of tissues.12,13 body complex, and numerous ganglion cells of small size were The tachykinins exert their effects by interacting with specific labeled by a ␥-PPT-A probe in the rat TG. receptors termed the NK receptors. There have been three CONCLUSIONS. The present results clearly demonstrate that NKA NK-receptors identified: the NK-1 receptor having the highest is a main constituent of sensory neurons innervating the ante- affinity for SP, the NK-2 receptor having the highest affinity for rior segment of the eye. The presence of the peptide in C fibers NKA, and the NK-3 receptor with the highest affinity for NKB in ocular tissues indicates a participation in sensory transmis- (for review, see Ref. 14). sion and an involvement in the irritative response in the eye, a With respect to the eye, SP has been extensively studied. In model for neurogenic inflammation in lower mammals. (Invest the anterior segment, it represents a classic sensory neuropep- tide that features a distinct innervation pattern (for review, see Ref. 15). There is little knowledge about the two other mam- malian tachykinins NKA and -B. High levels of NKA were From the Departments of 1Ophthalmology and 2Pharmacology, reported in the porcine retina indicating participation in visual the 3Laboratory of Psychiatry, University Clinic of Psychiatry, Inns- 16 17,18 4 processing. Furthermore, NKA innervates the rabbit iris, bruck Medical University, Innsbruck, Austria; and the Department of and it has been detected by immunohistochemistry in the rat Clinical Chemistry, University Hospital, Linkoping, Sweden. trigeminal ganglion (TG) supporting the idea that it also may be Supported by Grant P14022-Med from the Austrian Science Foun- 19 dation (JT). a sensory peptide for cranial tissues. Detailed studies on the Submitted for publication May 27, 2004; revised July 14, 2004; innervation pattern of the anterior segment of the eye have not accepted July 26, 2004. been performed, and experimental evidence is lacking about Disclosure: E. Schmid, None; J. Leierer, None; A. Doblinger, whether this peptide indeed contributes to the sensory inner- None; A. Laslop, None; R. Fischer-Colbrie, None; C. Humpel, vation of the eye. None; E. Theodorsson, None; B. Teuchner, None; D. Lalehabbasi, In the present study, we investigated the innervation pat- None; E. Dragosits, None; C. Kunze, None; W. Philipp, None; W. tern of the anterior segment of the eye by NKA. We evaluated Go¨ttinger, None; J. Troger, None the presence and distribution of the peptide in human eyes in The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- detail and investigated the source of nerves, by using animal experiments. The common precursor for SP and NKA and the ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 19 Corresponding author: Josef Troger, Universita¨tsklinik fu¨r Augen- detection of NKA in the rat TG indicate a sensory origin of heilkunde, Anichstraße 35, 6020 Innsbruck, Austria; NKA-positive ocular nerves, which should be confirmed by this [email protected]. study. Capsaicin is an ideal tool to characterize sensory neu- Investigative Ophthalmology & Visual Science, January 2005, Vol. 46, No. 1 268 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/26/2021 IOVS, January 2005, Vol. 46, No. 1 Neurokinin A in Sensory Neurons in the Anterior Segment 269 rons, because it is known to provoke selectively the loss of 50% was 0.1 to 0.2 femtomoles. Differences between control and capsaicin to 90% of these neurons when injected into newborn rats, in data were calculated with the Mann-Whitney U test. particular small neurons giving rise to unmyelinated C-fi- bers.20–22 The levels of NKA, SP, and calcitonin gene-related Reversed Phase HPLC peptide (CGRP) were measured in the rat TG and in human eye ␥ The antibody K12 used for the measurements by RIA recognizes both tissues in a comparative manner. The expression of -PPT-A NKA and certain other tachykinins, including NKB, neuropeptide K, mRNA was studied in the rat TG by in situ hybridization. NKA (3-10), and NKA (4-10). To differentiate between NKA and other immunoreactive peptides, we separated the tissue extracts of the rat MATERIALS AND METHODS TG and the human iris-ciliary body complex by reversed phase HPLC, as described recently.23 For this purpose, the peptides were extracted Capsaicin Treatment by sonication in 2 M acetic acid, the supernatant was lyophilized and reconstituted in 500 ␮L distilled water. A total of 100 ␮L was loaded Newborn Sprague-Dawley rats were injected at the first day after birth into a reversed phase HPLC column (LiChrospher WP 300 RP-18, 5 ␮m; with a single dosage of 50 mg/kg capsaicin (Sigma-Aldrich, Vienna, Merck, Darmstadt, Germany) and eluted with a gradient ranging from Austria), which was dissolved in saline containing 10% ethanol and 20% to 60% acetonitrile in 0.1% trifluoroacetic acid-water over 50 10% Tween-80. Untreated rats served as control subjects. The injection minutes at a flow rate of 1 mL/min. Fractions (1.0 mL) were collected, was performed subcutaneously, under the neck fold. The animals were lyophilized, reconstituted in assay buffer, and analyzed for NKA by RIA, housed in cages with a dark–light cycle of 12 hours each (lights on at as just described. The elution position of NKA was determined in a Ϯ 7AMandoffat7PMin23 1°C) and fed a commercial chow and separate run with synthetic NKA as standard (Peninsula Laboratories). water ad libitum. All experimental and animal care procedures were performed in compliance with the Guide for the Care and Use of In Situ Hybridization Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the animal experiments were For in situ hybridization, the ganglia of three untreated rats were approved by the Ministry of Science in Austria. The animals were dissected, mounted on optimal cutting temperature (OCT) medium allowed to grow, and they were killed by an overdose of CO after 3 (Tissue-Tec; Sakura, Loeterwoude, The Netherlands), and frozen in 2 Ϫ ␮ months. The eyes then were removed, followed by dissection of the 2-methylbutane ( 30°C).