Neurokinin A and neurokinin B in the human retina

peptides 27 (2006) 3370–3376 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/peptides Neurokinin A and neurokinin B in the human retina Eduard Schmid a, Johannes Leierer b, Gerhard Kieselbach a, Barbara Teuchner a, Martina Kralinger a, Reiner Fischer-Colbrie b, James E. Krause c, Quynh Anh Nguyen a, Gertrud Haas a, Katrin Stemberger a, Josef Troger a,* a Department of Ophthalmology, Innsbruck Medical University, Innsbruck, Austria Department of Pharmacology, Innsbruck Medical University, Innsbruck, Austria c Neurogen Corporation, 135 Northeast Industrial Road, Brandford, USA b article info abstract Article history: Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the Received 24 April 2006 human retina which were more than five times higher than those of substance P (SP). The Received in revised form present study aimed to find out how many of these immunoreactivities can be attributed to 28 July 2006 NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in Accepted 28 July 2006 the human retina and compared with that of SP. For this purpose, NKA-like immunor- Published on line 7 September 2006 eactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Further- Keywords: more, the retinae from both a 22- and 70-year-old donor were processed for double- Neurokinin A immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to Neurokinin B approximately two thirds and NKB to approximately one third of the immunoreactivities Retina measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in Immunofluorescence the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique. # 2006 Elsevier Inc. All rights reserved. 1. Introduction Neurokinin (NK) A and NKB are neuropeptides which belong to the family of tachykinins and which share the common Cterminal amino acid sequence Phe-X-Gly-Leu-Met-NH2 [18]. The tachykinin family includes substance P (SP), NKA, NKB and two NKA-related peptides (neuropeptide K and neuropeptide g). SP, NKA and NKA-related peptides are encoded by the preprotachykinin (PPT) I gene, whereas NKB is encoded by the PPT II gene [29,36]. The PPT I gene generates three distinct PPTA mRNAs by alternative RNA processing [22,26,33,34]. band g-PPTA mRNAs encode SP/NKA-containing precursors which account for the majority of the PPTA mRNAs derived from the SP/NKA gene, and a mRNA, a minor species encodes a SP-containing precursor only. SP, NKA and NKB act preferentially on distinct neurokinin (NK) receptors, termed * Corresponding author at: Universitätsklinik für Augenheilkunde, Anichstraße 35, 6020 Innsbruck, Austria. Tel.: +43 512 504 23758; fax: +43 512 504 23768. E-mail address: [email protected] (J. Troger). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.07.021 peptides 27 (2006) 3370–3376 NK1, NK2 and NK3, respectively, which belong to the superfamily of G-protein-coupled receptors [36] and are distributed differentially throughout the nervous system and peripheral tissues [30]. In the retina, SP has been localized to amacrine cells in the proximal inner nuclear layer (INL) and to displaced amacrine cells in the ganglion cell layer (GCL) in various species which represent interneurons with dendrites ramifying in the inner plexiform layer (IPL) [2,35,38,47] and this is also valid for primates [1,14,32]. The peptide is also present in ganglion cells in the rabbit [3], rat [6,43] and chicken [16]. Furthermore, previous studies also demonstrated the presence of SP in the human retina [13,28,44] where SP-immunoreactivity has been localized to large-field amacrine and displaced amacrine cells with processes ramifying not only in stratum 3 but also 1 and 5 of the IPL and to sparse large-field ganglion cells [13]. Functionally, SP is well known to modulate the excitability of innerretinal neurons [15,19,48], to participate in retinocentral projections at least in certain species [3,6,16,43] and possibly to play an important role in the development of correct innerretinal circuits and of correct retinocentral projections [8,10]. While SP has been extensively studied in the retina, there is little knowledge on the other main tachykinins NKA and NKB available at present. However, these tachykinins are present in the bovine retina [41], levels of NKA have been reported to be higher than those of SP in the porcine retina [21], the expression of SP/NKA mRNA and NKB mRNA has been demonstrated in the rat retina by in situ hybridization [4] and the localization of NKA is distinct from that of SP in the anuran retina [39]. A detailed study of the presence and distribution of NKA and NKB in the retina of higher primates has not been published so far. Very recently, the authors found more than 1000 fmol/mg protein of NKA-like immunoreactivities in the human retina measured by radioimmunoassay and this concentration is more than five times higher than that of SP [40]. In the present study, the authors aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB in the human retina and then the distribution pattern of both NKA and NKB was evaluated by immunofluorescence in the human retina and compared with that of SP. 2. Materials and methods 2.1. Reversed phase HPLC and radioimmunoassay Since the antibody K12 used in the recent study (donated from Theodorsson E, Department of Clinical Chemistry, University Hospital, Linkoping, Sweden) recognizes both NKA and NKB and other tachykinins including neuropeptide K, NKA (3–10) and NKA (4–10), a reversed phase HPLC of the human retina was performed followed by radioimmunoassay as described previously [45] to separate the tachykinins. In brief, an extract of two human retinae was loaded into a reversed phase HPLC column (LiChrospher WP 300 RP-18 5 mm, Merck, Darmstadt) and eluted with a gradient ranging from 20% to 60% acetonitrile in 0.1% trifluoroacetic acid/water over 50 min at 3371 a flow rate of 1 ml/min. Fractions (1.0 ml) were collected, lyophilized, reconstituted in assay buffer and analyzed by radioimmunoassay using the K12 antibody [40]. The elution position of NKA and NKB was determined in a separate run with synthetic NKA and NKB as standard (Peninsula Laboratories, 601 Taylor Way San Carlos, CA 94002, US). The radioimmunoassay was performed as described by our previous study [45]. 2.2. Immunofluorescence The eyes of a 22- and 70-year-old donor dedicated for corneal transplantation were removed immediately after death because of an accident and a bronchial carcinoma, respectively. The eyes had no signs of pathologies and were not pseudophacic. The cornea was trephaned in the local cornea bank. Then the iris/ciliary body complex, lens and vitreous were carefully removed, the whole retina was detached from the retinal pigment epithelium and cut from the periphery. The retinae were immersed with 4% paraformaldehyde in phosphate buffered saline (PBS) overnight, then placed in a solution containing 15% sucrose in PBS for one hour and in 30% sucrose overnight, frozen in cold ( 60 8C) isopentane and stored at 70 8C. Ten to 30-mm-thick sections were cut from the specimens on a Reichert Jung cryostat (LeicaReichert, Vienna, Austria) at 20 8C and mounted on poly-Llysine-coated slides. The immunofluorescence was performed as described by the authors recently [40]. The sections were incubated for 72 h at 4 8C with either the antibody SK2 for NKA (donated from Theodorsson E, Department of Clinical Chemistry, University Hospital, Linkoping, Sweden) or the antibody Peptide 2 for NKB Fig. 1 – Analysis of NKA-like immunoreactivities by reversed phase HPLC. A total of 100 ml from an extract of two human retinae was loaded into a reversed phase HPLC column and eluted with 0.1% trifluoroacetic acid/ water over 50 min at a flow rate of 1.0 ml/min. The dotted line indicates the gradient profile (percent acetonitrile, right ordinate). One milliliter per minute fractions were collected, lyophilized, reconstituted in an assay buffer and immunoreactivities were determined by radioimmunoassay using the K12 antibody. The elution positions of synthetic NKA and NKB are indicated above the peaks. 3372 peptides 27 (2006) 3370–3376 Fig. 2 – Demonstration of the distribution pattern of NKA- (a, c, e, and g) in comparison with SP-immunoreactivities (b, d, f, and h) in the human retina. (a) NKA-immunoreactivity is shown in one ovally-shaped amacrine cell in the proximal INL (arrow) and in one displaced amacrine cell in the GCL (arrowhead) and a continuous immunoreactive band is shown in peptides 27 (2006) 3370–3376 (generated by Krause JE, Neurogen Corporation, 135 Northeast Industrial Road, Brandford, CT06405, USA) at a dilution of 1:1000 each in tris buffered saline (TBS) containing 2% normal goat serum and 0.3% Triton X-100. The SK2 antibody was raised in New Zealand White rabbits with NKA coupled to bovine serum albumine using carbodiimide hydrochloride as coupling agent [5]. The antibody features no crossreactivity with SP but 63% with NKB [5]. The Peptide 2 antibody was raised in rabbits against a synthetic peptide corresponding to the amino acids 50–79 of rat preprotachykinin B. It was coupled to poly-L-lysine and glutarylaldehyde was used to couple via amino groups [31]. The antibody does not cross-react with SP and NKA [31]. Furthermore, double immunofluorescence of NKA with SP and NKB with SP was performed using a SP-antibody raised in guinea pigs against the immunogen CRPKPQQFFGLM (dilution of 1:1000) (Research Diagnostics Inc., Pleasant Hill Road, Flanders NJ 07836, USA). After three washes with TBS, the sections were incubated with the secondary antibody for NKA and NKB (Cy3-conjugated AffiniPure goat anti-rabbit IgG; code 111165-006; Jackson Immuno Research Labs, West Grove, PA, USA) and diluted 1:1000 for 24 h at 4 8C. For SP, a goat antiguinea pig antibody at a dilution of 1:1000 was taken (Alexa Fluor1 488 goat anti-guinea pig IgG, Medac GmbH, 22880 Wedel, Germany). In control experiments, no immunoreactivity could be detected with the SP-antibody adsorbed with an excess of SP (1 mM) or when the primary antibody was omitted. When the SP-antibody was adsorbed with an excess of NKA or NKB the distribution pattern was identical to that without preadsorption. 3. Results 3.1. Reversed phase HPLC followed by radioimmunoassay Reversed phase HPLC of an extract of the human retina followed by radioimmunoassay using the K12 antibody revealed two peaks (Fig. 1): one higher peak which appeared in the position of synthetic NKA and a smaller one which appeared in the position of synthetic NKB. These results indicate that in the human retina the NKA-like immunoreactivities measured by radioimmunoassay in the recent study attribute exclusively to the tachykinins NKA and NKB. NKA makes up 65.5% and NKB 34.5% of immunoreactivities. The absolute levels deduced from the recent study are therefore 660 fmol/mg protein and 350 fmol/mg protein, respectively, and the ratio of the levels of SP, NKA and NKB is approximately 1:3,5:2. 3373 3.2. Distribution pattern of NKA and NKB in the human retina The distribution pattern of NKA and NKB was evaluated in many sections of the human retina and the most important findings are summarized in Figs. 2 and 3. For NKA, immunoreactivities were present in all retinal regions in varicose processes, which were most prominently seen in sublamina 3 of the IPL where they formed a continuous band (Fig. 2a, c and e) but also sparsely in sublamina 1 and 5 (Fig. 2e). The ramification pattern in the IPL showed full colocalization with SP (Fig. 2b, d and f) but the bands in sublamina 3 and also in sublamina 1 and 5 were more prominent for NKA than for SP (Fig. 2e and f). NKA was also present in cells of the human retina. The cells were observed sparsely in the INL at the boarder to the IPL (Fig. 2a) and also in the GCL at the boarder to the IPL (Fig. 2a and c). The cells were approximately round or oval in shape with a diameter of 13–17 mm in the INL and also 13–17 mm in the GCL and are therefore amacrine cells and displaced amacrine cells, respectively. As seen for the processes in the IPL, the cells which were immunoreactive for NKA both in the INL and GCL were also immunoreactive for SP (Fig. 2a–d). Very rarely, NKA-like immunoreactive cells in the GCL with a larger diameter (approximately 20 mm) resembling most likely ganglion cells were also immunoreactive and as observed for the amacrine cells, these cells were also immunoreactive for SP (Fig. 2g and h). For NKB, cells in the proximal INL resembling amacrine and cells in the GCL resembling displaced amacrine cells were not immunoreactive and a stained continuous or discontinuous band in the IPL was not observed. In contrast, immunoreactive cells were only found sparsely in cells of the GCL with a diameter of more than 17 mm which therefore are most obviously ganglion cells (Fig. 3a) since a clear immunoreactive band was also observed in the nerve fiber layer (Fig. 3c). Colocalization studies with SP revealed that the ganglion cells immunoreactive for NKB are also immunoreactive for SP (Fig. 3a and b) whereas the stained band in the nerve fiber layer was only seen for NKB but not for SP (Fig. 3c and d). 4. Discussion Our results revealed two main novel findings: firstly, the presence of both NKA and NKB has been unequivocally verified in the human retina by reversed phase HPLC in this study providing evidence that at least quantitatively NKA predominates NKB. Considering the absolute levels of NKAlike immunoreactivities of 1010 fmol/mg protein [40] and the sublamina 3 of the IPL (asterisk). (b) The same section incubated with the SP-antibody shows SP-immunoreactivity in the same cells and IPL-band as in (a). (c) NKA-immunoreactivity is shown in two displaced amacrine cells (arrows) and in sublamina 3 and 5 of the IPL (asterisk). (d) The same section as in (c) but incubated with the SP-antibody demonstrates immunoreactivity for SP in the same cells (arrows) and in sublamina 3 of the IPL (asterisk). (e) NKA-immunoreactivity is prominently present in sublamina 3 (asterisk) but also in sublamina 1 (arrowhead) and sublamina 5 (arrow). (f) Colocalization with SP revealed a band in sublamina 3 (asterisk) but scarcely in sublamina 1 (arrowhead) and 5 (arrow). (g) A larger NKA-like immunoreactive cell with a diameter of more than 15 mm is stained in the GCL which obviously represents a ganglion cell (arrow). (h) The same cell as in (g) is also positive for SP (arrow). GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer. Scale bar = 50 mm. 3374 peptides 27 (2006) 3370–3376 Fig. 3 – Demonstration of the distribution pattern of NKB (a and c) with that of SP (b and d) in the human retina. (a) An immunoreactive cell stained for NKB in the GCL resembling a ganglion cell is indicated by the arrow. (b) The same cell as in (a) is also immunoreactive for SP (arrow) but the immunoreactive band in the IPL is only seen for SP (asterisk). (c) A stained band in the nerve fiber layer is immunoreactive for NKB (arrows). (d) The same section as in (c) but stained for SP. Note that the nerve fiber layer is devoid of immunoreactivity (arrows). GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer. Scale bar = 50 mm. results of the reversed phase HPLC of the present study, there are more than three times higher concentrations of NKA and two times higher concentrations of NKB when compared with SP. Although the tachykinins are colocalized, their concentrations are different which can simply be explained methodologically since the immunofluorescence provides a qualitative approach allowing no conclusions about the absolute levels. The second main finding of this study concerns the histochemical results. There is no doubt that most neuropeptides are expressed by amacrine and displaced amacrine cells in the vertebrate retina due to their classic localization and ramification of their processes in the IPL and this is also true for NKA in the human retina whereas NKB-immunoreactivities are only found in ganglion cells. The latter is based on the observation that a clear immunoreactive band was found in the nerve fiber layer although processes of displaced amacrine cells can also run into this layer as previously described for displaced GABAergic amacrine cells [24]. However, since a clear immunoreactive band in the IPL was never observed for NKB, they are most likely true ganglion cells. This is in agreement with the study of Brecha et al. who found a signal for SP/NKA mRNA in the INL and for NKB mRNA in the GCL by in situ hybridization in the rat retina [4]. The most important feature of our study is that both NKA and NKB are colocalized with SP in cells of the INL and GCL. This is in contrast to the anuran retina where NKA and SP were found in distinct subsets of amacrine cells [39]. This can only be explained by species differences as is the case for the tachykinin receptors (see below). In contrast to NKB, there is no immunoreactivity for SP in the nerve fiber layer which is most obviously the result of the lower levels and consequently of too low anterograde transport of the peptide to become detectable by immunofluorescence. Whether the SK2 antibody indeed features 63% cross-reactivity with NKB remains open since an immunoreactive band in the nerve fiber layer was never observed with this antibody which is so typical for NKB. By examining wholemounts additionally to cryostat sections, SP-IR was most prominently found in one type of large-field amacrine cell which showed many similarities with thorny, type 2 amacrine cells described from Golgi studies and one type of ganglion cell, a wide-field ganglion cell, resembling G21 from a Golgi study [13,23]. Because of the colocalization of the tachykinins, the cells positive for NKA and NKB are simply of the same type. peptides 27 (2006) 3370–3376 The functional significance of NKA and NKB in the retina has not been evaluated in this study but is worth discussing. Both the NK1 and NK3 receptor have been localized in the rat retina by immunohistochemistry [7,8,12,37] and in situ hybridization [25,42] whereas staining for the NK2 receptor was negative both in the adult and postnatal rat retina indicating an absence of this receptor in this tissue [37]. The NK1 receptor was found to be localized mainly on neurons of the inner retina including GABA- and tyrosine hydroxylase (TH)-positive amacrine cells and displaced amacrine cells and furthermore, an intense staining of each lamina in the IPL was evident [7,8]. At least in rats, there is a clear mismatch between the prominent expression of the NK1 receptor in the inner retina and a much weaker staining of its preferred ligand, the tachykinin SP. Since the preferred receptor of NKA, the NK2 receptor, is obviously absent in the retina and the NK1 receptor is so prominently present in the rat, NKA may also act on this receptor. This would not be unique for the retina since NKA contracts the sphincter muscle in the rabbit via the NK1 receptor rather than the NK2 receptor [27]. This hypothesis has functional consequences. SP is well known to exert excitatory influences on innerretinal neurons in carp and mudpuppy [15,19] and in rabbits without affecting ganglion cell receptive field properties [48] and is proposed to act as a developmental factor in the immature retina [8,10]. Hence, if NKA also acts on the NK1 receptor then this peptide would exert similar functional roles in the retina to SP and due to the much higher levels, would functionally represent the much more important peptide when compared with SP. However, the NK1 receptor must be further characterized in the human retina since there are species differences. In rabbits for instance, the NK1 receptor is expressed much less in the inner retina but by a subset of On-type cone bipolar cells [9,10] and in the mouse also by On-type cone bipolar cells and by certain amacrine cells [11]. A common feature in all species is the presence of the NK1 receptor on dopaminergic amacrine cells which is consistent with the reported enhancement of dopamine release by SP in the rat [46] and rabbit retina [10]. On the other hand, the cellular expression of the NK3 receptor on Off-cone bipolar cells and TH-positive amacrine cells in the rat [8,12] may indicate that NKB has a direct action on Off-type cone pathways and indirect actions mediated by the dopamine-containing amacrine cells given that the expression pattern of the NK3 receptor is similar in humans. But it must be emphasized that there is a discrepancy between the localization of the NK3 receptor in the mid retina in rats [8,12] and the localization of its preferred ligand NKB in the inner retina in humans (this study) and rats [4]. Thus, NK3mediated effects of NKB may play a minor role since there is a distance for NKB to act in a paracrine manner. The staining of ganglion cells and the nerve fiber layer indicates that NKB mainly participates in the transfer of visual information into the central nervous system rather than in visual processing within the retina. Nevertheless, the localization of NKB exclusively in ganglion cells and the nerve fiber layer is atypical and unique for neuropeptides although other peptides have previously been shown to be mainly expressed by ganglion cells, in particular PACAPs (review, see [20]) or LANT-6 [17,28]. 3375 references [1] Brecha NC, Hendrickson A, Floren I, Karten HJ. 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