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PE-11, a peptide derived from chromogranin B, in the rat eye
Peptides 32 (2011) 1201–1206
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PE-11, a peptide derived from chromogranin B, in the rat eye Katrin Lorenz a , Josef Troger b,∗ , Oliver Gramlich a , Franz Grus a , Rosa Hattmannstorfer c , Reiner Fischer-Colbrie d , Stephanie Joachim e , Eduard Schmid b , Barbara Teuchner b , Gertrud Haas b , Nikolaos Bechrakis b a
Experimental Ophthalmology, Department of Ophthalmology, University Medical Center, Johannes Gutenberg-University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany Department of Ophthalmology, Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria c Clinical Department of Restorative and Prosthetic Dentistry, Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria d Department of Pharmacology, Medical University of Innsbruck, Peter Mayrstraße 1a, 6020 Innsbruck, Austria e Center for Vision Science, Ruhr University Eye Hospital, In der Schornau 23–25, 44892 Bochum, Germany b
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Article history: Received 16 December 2010 Received in revised form 14 March 2011 Accepted 14 March 2011 Available online 23 March 2011 Keywords: PE-11 Chromogranin B Rat Eye Capsaicin
a b s t r a c t The aim of the study was to investigate the presence and distribution of PE-11, a peptide derived from chromogranin B, in the rat eye. For this purpose, newborn rats were injected with a single dosage of 50 mg/kg capsaicin subcutaneously under the neck fold and after three months, particular eye tissues were dissected and the concentration of PE-11-like immunoreactivity was determined by radioimmunoassay. Furthermore, PE-11-like immunoreactivities were characterized in an extract of the rat eye by reversed phase HPLC. Then, the distribution pattern of PE-11 was investigated in the rat eye and rat trigeminal ganglion by immunofluorescence. As a result, PE-11 was present in each tissue of the rat eye and capsaicin pretreatment led to a 88.05% (±7.07) and a 64.26% (±14.17) decrease of the levels of PE-11 in the cornea and choroid/sclera, respectively, and to a complete loss in the iris/ciliary body complex. Approximately 70% of immunoreactivities detected by the PE-11 antiserum have been found to represent authentic PE-11. Sparse nerve fibers were visualized in the corneal and uveal stroma, surrounding blood vessels at the limbus, ciliary body and choroid and in association with the dilator and sphincter muscle. Furthermore, immunoreactivity was present in the corneal endothelium. In the retina and optic nerve, glia was labeled. In the rat trigeminal ganglion, PE-11-immunoreactivity was visualized in small and medium sized ganglion cells with a diameter of up to 30 m. In conclusion, there is unequivocal evidence that PE-11 is a constituent of capsaicin-sensitive sensory neurons innervating the rat eye and the distribution pattern is typically peptidergic in the peripheral innervation but in the retina completely atypical for neuropeptides and unique. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The chromogranins are the acidic proteins of secretory granules and comprise at least three subtypes: chromogranins A and B (for review see [36]) and secretogranin II (for review see [9]). Whereas secretogranin II has been discovered in the anterior pituitary [29], the other granins have been first found in the chromaffin granules of the adrenal medulla where they are coreleased with catecholamines [3,8]. Subsequent studies revealed that the granins are widely distributed throughout the neuroendocrine system where they are stored in large dense core vesicles [9,36]. The function is still not completely clear but it has been proposed that they may play a fundamental role in secretory granule formation and neurotransmitter/hormone release [5,27]. Furthermore, the granins
∗ Corresponding author. Tel.: +43 512 504 23758; fax: +43 512 504 23768. E-mail address: [email protected] (J. Troger). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.03.011
are characterized by numerous pairs of basic amino acids in the primary sequence as potential sites for intra- and extragranular processing indicating that they are precursors of functional active neuropeptides [10]. And indeed, chromogranin-derived peptides are generated in vivo and are functionally active. In particular, the chromogranin-A-derived peptide pancreastatin can inhibit glucose-stimulated insulin release from perfused rat pancreas or isolated acini [7]. Vasostatin, a peptide also derived from chromogranin A, exerts effects on vascular smooth muscle [1], and another chromogranin A-derived peptide, catestatin, was shown to be an effective inhibitor of catecholamine secretion from chromaffin cells [24]. A peptide derived from chromogranin B, secretolytin (CgB 614–626), was found to display antibacterial properties [31] and the secretogranin II-derived peptide secretoneurin (SN) exerts a variety of functional effects (review see [35]). PE-11 is another granin-derived peptide, in particular it is generated in vivo by proteolytical processing of chromogranin B [19]. Although no biological effect has been described for this peptide
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so far, it is widely distributed throughout the rat [19] and human brain [25], human brainstem [15], human hippocampus [26] and its presence in the fetal human vagal/nucleus solitary complex has been well documented [2]. Furthermore, PE-11 is present in the peripheral nervous system, in particular it has been found in large sensory axons in the dorsal roots of the spinal cord indicating that it is a sensory transmitter [22]. And it is also present in rat pelvic ganglia and vas deferens [21]. There is evidence that the processing of chromogranin B is quite extensive in the nervous system, in the rat brain and fetal human vagal/nucleus solitary complex it is completely processed [2,19], in the sciatic nerve and vas deferens to a high degree [21,22] and to varying degrees in particular tissues of the human brain [25]. With respect to the eye, the secretogranin II-derived peptide SN has been extensively studied. It is present in the human retina in amacrine and displaced amacrine cells [28] and it is a typical sensory transmitter with nerve fibers deriving from the trigeminal ganglion [32]. Furthermore, it is present in high concentrations in the human [30] and rabbit aqueous humor [18] and also in significant levels in the human vitreous of patients with various vitreoretinal diseases [23]. On the other hand, the presence and distribution of WE-14, a peptide derived from chromogranin A, has been investigated in porcine ocular tissues [6], but chromogranin B has not been studied in the eye so far. The present study concentrates on examinations on chromogranin B using an antibody raised against PE-11. It aims to find out whether it is present in capsaicin-sensitive sensory nerves of the rat eye and since this was found to be the case, to detect the peptide in the rat trigeminal ganglion. Capsaicin is the pungent ingredient of red pepper and when it is injected subcutaneously into newborn rats it destroys more than a half of the sensory neurons which makes it an ideal tool to investigate these neurons [11–13]. And then, the presence and distribution of PE-11 has been investigated in the rat eye in detail. 2. Materials and methods 2.1. Animal experiments In one set of experiments, capsaicin pretreatment in rats has been performed. For this purpose, newborn Sprague–Dawley rats were injected subcutaneously under the neck fold with a single dosage of 50 mg/kg capsaicin (obtained from Sigma–Aldrich, Vienna, Austria) on the first day after birth. Capsaicin was dissolved in saline containing 10% ethanol and 10% Tween 80. Animals without treatment served as controls. The animals were housed in cages with a dark–light cycle of 12 h each (lights on at 7 AM and off at 7 PM, in 23 ± 1 ◦ C) and fed a commercial chow and water ad libitum. The animals were allowed to grow for 3 months and were then sacrificed by an overdose of CO2 . All experimental and animal care procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the animal experiments were approved by the Ministry of Science in Austria. 2.2. Radioimmunoassay The radioimmunoassay (RIA) was performed with a specific antiserum and the generation of the antiserum was described previously [19]. In brief, it was generated against a synthetic peptide, PE-11, corresponding to rat chromogranin B 552–562. The antiserum only reacts with the free C-terminal part of PE-11 since an elongated peptide (chromogranin B 552–574) reacts exclusively within the RIA when it is first subjected to trypsin digestion. No cross-reactivity was found with peptides derived from chromo-
granin A and secretogranin II nor with the following neuropeptides: galanin, substance P, neuropeptide Y, neurotensin and calcitonin gene-related peptide. For determination of the concentration by RIA, rat eye tissues were prepared. For this purpose, the eyes were immediately removed after the rats were killed. A small incision was made at the limbus and the cornea inclusive limbus was circumferentially excised. Then, the iris/ciliary body-complex was removed and finally, the retina was detached from the underlying choroid/scleracomplex. The tissues were weighed and immediately frozen and kept frozen at −70 ◦ C. The tissues were then processed by RIA as described previously in detail [19]. In brief, tissues were sonicated in 300 L distilled water for 10 s and then immediately boiled for 10 min. After centrifugation (20 min at 12,000 g) the supernatant was analyzed for PE-11-immunoreactivity. For the RIA, samples, RIA buffer and antiserum (final dilution: 1:4500) were incubated for 24 h at 4 ◦ C. Then, the tracer (specific activity 77,000 d.p.m./ng) obtained by iodination of PE-11 with the chloramine T method was added and samples were incubated for a further 24 h at 4 ◦ C. Bound/free separation was performed with dextran-coated charcoal. 2.3. Characterization of PE-11-like immunoreactivities (LI) by reversed phase-HPLC followed by RIA The eyes of two Sprague–Dawley rats were enucleated, the lens was removed and the extraction from the residual tissues was performed by sonication as described above. For the reversed phase-HPLC, an aliquot of the extract was loaded into a reversed phase-HPLC column (LiChrospherWP300RP-18.5 m; Merck, Darmstadt, Germany) and eluted with a gradient ranging from 0% to 80% acetonitrile in 0.1% trifluoracetic acid–water over 60 min at a flow rate of 1 ml/min. Fractions (1.0 ml) were collected, lyophilized, reconstituted in assay buffer and analyzed for PE-11 by RIA as just described. The elution position of PE-11 was determined in a separate run with synthetic PE-11 as standard. 2.4. Immunofluorescence for PE-11 in the rat eye Nerve fibers in the rat eye were visualized by immunofluorescence. For this purpose, the eyes were enucleated and under a dissecting microscope, an incision was made in the cornea. Then, the eye ball was immersed in 4% paraformaldehyde for 1 h at room temperature followed by preparation of particular tissues inclusive removal of the lens and vitreous. Subsequently, the eyes were washed with PBS for a few times and then cryoprotection was performed. In particular, the eyecup was incubated in 10% succrose solution (1.5 ml/well) at room temperature for approximately 30 min until the eye was saturated and fell down to the bottom of the well. Then, the 10% succrose solution was replaced by 20% succrose solution and incubated at room temperature for approximately 2 h. Finally, the 20% succrose solution was replaced by 30% succrose solution and the eyecup was incubated over night at 4 ◦ C. Then, the eyes were embedded with O.C.T. compound freezing medium (Tissue Tek), frozen in liquid nitrogen and stored at −70 ◦ C. 6–15 m thick sections were cut from the specimens on a cryostat (Reichert Jung; Leica-Reichert, Vienna, Austria) and mounted on Superfrost Plus slides (Thermo Scientific, Braunschweig, Germany). The slides were dried for 30 min in an incubator and rinsed in PBS. Then, the slides were washed for 30 min in preheated Tris–EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0, 60 ◦ C) in an incubator at 60 ◦ C. The sections were then preincubated for 30 min with 0.25% normal goat serum/0.5% bovine serum albumin in PBS with 0.1% Triton X100, afterwards rinsed in PBS and subsequently incubated over night in disposable immunostaining chambers at room temper-
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Table 1 Concentration of PE-11-LI in particular tissues of the rat eye both in untreated controls and after capsaicin-pretreatment. Values represent means ± S.E.M. (fmol/mg wet weight; n = 10) and statistical calculation of differences between controls and capsaicin values was performed with the Mann Whitney U test (*** p < 0.001). Controls Cornea Iris/ciliary body Choroid/sclera Retina
0.77 3.53 3.05 5.48
± ± ± ±
0.41 0.40 0.24 0.34
Capsaicin 0.092 ± 0.029*** 0.0*** 1.09 ± 0.034*** 5.91 ± 0.14
ature with a rabbit PE-11 antiserum at a dilution of 1:1000 in PBS containing 0.1% Triton X-100. After a few washes with TBS and PBS, sections were incubated with the secondary antibody (Cy3-conjugated goat anti-rabbit IgG; Linaris, Wertheim-Bettingen, Germany) diluted 1:500 with PBS for 4 h in immunostaining chambers at room temperature. Stained sections were washed with PBS and TBS, mounted with Vectashield containing diamidin2-phenylindol (Vector, Burlingame, CA, USA) and coverslipped. Sections were visualized with a Nikon TE 2000 microscope (Nikon, Düsseldorf, Germany) equipped with a high sensitive cooled black and white CCD camera and Nikonˇıs Lucia G/F software. Furthermore, sections were processed for double immunofluorescence with the PE-11- and a glial fibrillary acidic protein (GFAP) antibody. The GFAP was immunostained via mouse anti-GFAP antibody (Linaris, ready to use) and fluorescein–isothiocyanate labeled goat anti-mouse IgG (GenWay, San Diego, CA, USA) diluted 1:400 in PBS. Specificity of the staining was evaluated by two further procedures, in particular both by incubating sections without the primary PE11 antiserum and by incubating sections with a PE-11-preadsorbed primary antibody. 2.5. Immunofluorescence for PE-11 in the rat trigeminal ganglion Two male rats (200 g) were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg) and perfused transcardially with saline followed by 4% paraformaldehyde. The trigeminal ganglia were removed and paraffinized to a paraffine tissue block. 10 m sections were cut on a microtome and mounted on Superfrost Plus slides. The slides were dried in an incubator at 60 ◦ C for 30 min and then deparaffinized/rehydrated. For this purpose, sections were incubated in three washes of xylene for 5 min each, incubated in two washes of 100% ethanol for 10 min each, incubated in two washes of 95% ethanol for 10 min each, incubated in one wash of 70% ethanol for 5 min and rinsed twice in dH2 O for 5 min each. The next steps inclusive antigen unmasking, blocking, incubating with the primary and secondary antiserum and visualizing were the same as described above. 3. Results 3.1. Concentration of PE-11-LI in various ocular tissues of the rat and the effect of capsaicin The concentration of PE-11-LI was measured by RIA in various ocular tissues of the rat and the results inclusive effect of capsaicin-pretreatment are summarized in Table 1. PE-11-LI was found to be present in each tissue of the rat eye, in particular in the cornea, the iris/ciliary body complex, the choroid/sclera and the retina. The highest levels were found in the retina, but the iris/ciliary body complex and choroid/sclera contained the peptide also in significant amounts and the peptide was even detectable in the cornea. Capsaicin-pretreatment led to a significant decrease of the concentration of PE-11-LI in each tissue except of the retina. In particular, an 88.05% (±7.07) decrease was observed in the cornea, a complete loss in the
Fig. 1. Characterization of PE-11-IR by reversed phase HPLC followed by RIA. One hundred microliters of a tissue extract of the rat eye were loaded into a reversed phase HPLC column and eluted with 0.1% trifluoroacetic acid–water. Horizontal line: gradient profile (percent acetonitrile, right ordinate). One milliliter-per-minute fractions were collected, lyophilized, reconstituted in assay buffer, and analyzed for PE-11 by RIA. The elution position of synthetic PE-11 is indicated by the arrow above the peak. The results revealed the presence of a major peak in the fractions 17–24 which coeluted with synthetic PE-11 and which amounted 71.84%. Furthermore, four minor peaks in fractions 8–10, 11–13, 25–28 and 29–32 were present which amounted 2.99%, 7.19%, 7.71% and 10.27%, respectively, but were not further characterized.
iris/ciliary body complex and a 64.26% (±14.17) decrease in the choroid/sclera. 3.2. Characterization of PE-11-LI in an extract of the rat eye Reversed phase-HPLC served to receive the molecular form of PE-11-LI in an extract of the rat eye. The result is illustrated in Fig. 1. Separation of immunoreactivities on a HPLC column and subsequent measurement of PE-11-LI in the fractions by RIA revealed one major peak in fractions 17–24 which coeluted with synthetic PE-11 and four minor peaks in positions 8–10, 11–13, 25–28, 29–32 which were not further characterized. The major peak in the position of synthetic PE-11 amounted 71.84%, the minor peaks in fractions 8–10, 11–13, 25–28 and 29–32 amounted 2.99%, 7.19%, 7.71% and 10.27%, respectively. These results indicate that approximately 70% of the immunoreactivities detected by the PE-11 antiserum represent authentic PE-11. 3.3. Visualization of PE-11-IR in the rat eye PE-11-IR was found to be distinctly distributed throughout the rat eye and the distribution pattern is illustrated in Fig. 2. In the cornea, sparse nerve fibers were found in the stroma and an immunoreactive band was observed in the innermost part of this tissue which confers to the endothelium (Fig. 2A). The bright immunofluorescence in the epithelium is autofluorescene since this immunoreactivity remained after removal of the primary antiserum. At the limbus, a prominent immunoreactivity was observed in association with blood vessels (Fig. 2B). In the iris, an immunofluorescent band was detected in clear association with the dilator muscle (Fig. 2C), but sparse nerve fibers were also present in the stroma (not shown) and there was a very dense network of nerve fibers in the sphincter muscle (Fig. 2D). In the ciliary body, nerve fibers again surrounded blood vessels (Fig. 2E) and immunoreactivity could be visualized in the stroma at the base of the processes and these nerves reached the stroma of the processes (Fig. 2F). In the choroid, nerve fibers were found again in association with blood vessels (Fig. 2G and H) but sparse fibers were also present in the stroma (Fig. 2H). In the retina, a dense network of immunoreactivity
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Fig. 2. Visualization of PE-11-IR in tissues of the rat eye. Particular tissues of the rat eye were dissected and processed by immunofluorescence according to a standard protocol. In brief, sections were incubated with a specific PE-11 antiserum at a dilution of 1:1000 over night and then for 4 h with a secondary antibody followed by visualization of immunoreactivities in the tissues. In the cornea (A), sparse nerve fibers were observed in the stroma (arrow) and an immunoreactive band in the endothelium (arrowheads). At the limbus (B), nerve fibers were found around blood vessels (arrows; v = vessel). In the iris, an association of immunoreactivity was found at the dilator muscle (C, arrows), but there were also sparse nerve fibers in the stroma (not shown) and a dense network of fibers in the sphincter muscle (D). In the ciliary body, nerve fibers were found again surrounding blood vessels (E, arrows; v = vessel), but sparse fibers were also observed in the stroma at the base of the ciliary processes reaching the stroma of the ciliary processes (F, arrows). In the choroid (G and H), there was an association of immunoreactivity with blood vessels (arrows, v = vessel), but nerve fibers were also observed in the stroma (arrowheads). In the retina (I), a dense network of immunoreactivity was observed in the innermost part which confers to glia (arrows) and glia was also labeled in the optic nerve (J, green fluorescent).
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Fig. 3. Demonstration of PE-11-IR by means of immunofluorescence in the rat trigeminal ganglion. Fixed rat trigeminal ganglia were paraffinized, sections were cut and processed by immunofluorescence as described in Section 2. In brief, the sections were again incubated with a specific primary PE-11 antibody over night and then for 4 h with a secondary antibody followed by visualization of PE-11-positive ganglion cells. Numerous ganglion cells were labeled and the cells were of small and medium size with a diameter of up to 30 m (A and B). PE-11-immunoreactive cells appeared mainly scattered (A), but occasionally, clusters were also observed (B).
was observed in the innermost part which showed colocalization with glial fibrillary protein (GFAP, not shown) and therefore confers to Müller glia (Fig. 2I). In the optic nerve, dense immunoreactivity was seen in glia (Fig. 2J). 3.4. Visualization of PE-11-IR in the rat trigeminal ganglion PE-11-IR was visualized by immunofluorescence in the rat trigeminal ganglion and with this procedure using a specific antiserum, numerous ganglion cells were labeled within the ganglion (Fig. 3A and B). In particular, the labeled cells were of small and medium size with a diameter of up to 30 m and occurred in scattered locations (Fig. 3A) but occasionally, small clusters or rows of cells were also observed (Fig. 3B). Nerve fibers or nonneuronal cells were not labeled. 4. Discussion For the first time, this study provides evidence that a chromogranin B-derived peptide is present in the eye. There are several novel findings. Firstly, PE-11 is a constituent of capsaicin-sensitive sensory nerves innervating the rat eye. Secondly, it could be detected in small and medium sized cells of the rat trigeminal ganglion and finally, it is distinctly distributed throughout the rat eye. There is no doubt that PE-11 is a sensory peptide innervating the eye since capsaicin pretreatment significantly lowered the levels in ocular tissues except of the retina and since this peptide was found to be present in the trigeminal ganglion which represents the sensory ganglion for cranial tissues (review see [20]). With the treatment regimen of this study, capsaicin destroys predominantly small neurons giving rise to unmyelinated C-fibers [11–13] and indeed, PE-11 was detected in the trigeminal ganglion in small cells. The complete loss of the peptide in the iris/ciliary body complex indicates an exclusive presence of PE11 in C-fiber afferents in this tissue and the significant drop in the cornea and choroid/sclera on a predominant presence in these unmyelinated fibers. In the cornea and especially in the choroid/sclera, however, PE-11 may be present also in some myelinated A␦ fibers which arise from small-sized cells as well and which are known to be less sensitive to capsaicin treatment. Alternatively, nerve fibers of other origin than the sensory one may contribute to the innervation of these tissues. Nevertheless, PE11 can now be added to the growing number of typical sensory peptides in the eye, in particular besides substance P, calcitonin gene-related peptide, neurokinin A, secretoneurin, galanin in rat
and pig, somatostatin, pituitary adenylate cyclase-activating peptide (PACAP)-27- and -38 in rabbits or cholecystokinin (review see [33]). The distribution pattern of PE-11 in the rat eye is typically peptidergic, namely the presence of sparse fibers in nearly all ocular tissues, the sparse presence of sensory nerve fibers in the stroma of the cornea, the iris, the ciliary body and the choroid and the dual innervation of both the sphincter and dilator muscle. However, there are two remarkable findings. Firstly, there is a clear association of nerve fibers with blood vessels not only at the limbus but also in the ciliary body and the choroid. This is similar to another granin-derived peptide, SN [32,33]. And then, there is immunoreactivity present in the endothelium of the cornea. This is similar to vasoactive intestinal peptide (VIP) which has also been found to be expressed in the human and bovine endothelium [16]. On the other hand, in the rat retina, PE-11 immunoreactivity is mainly concentrated in glial cells which is rather atypical for neuropeptides. Neuropeptides are in general mainly expressed in amacrine cells in the proximal inner nuclear layer and in displaced amacrine cells in the ganglion cell layer and partially also in sparse ganglion cells. Hence, the presence of this peptide in glial cells in the retina is unique. The functional significance of this peptide remains to be discussed. First of all, although there is no direct evidence, the prominent innervation of blood vessels indicates that PE-11 exerts a vasoregulatory effect. Then the peptide is obviously involved in an afferent transmission of sensory impulses because of the presence of PE-11 in sensory nerves. And finally, the peptide may also exert local effector functions in the iris/ciliary body complex, in particular it may be involved in neurogenic inflammation, since PE-11 is present in C-fibers there. This neurogenic inflammation in the eye is represented by an irritative response and consists of miosis, vasodilation, enhanced permeability of blood vessels, aqueous flare and increased intraocular pressure (for reviews see [33,34]). There is evidence, that substance P mediates miosis and calcitonin gene-related peptide the vascular effects at least in rabbits. Furthermore, the PACAPs participate as well as nitric oxide (NO) both of which stimulate the release of sensory peptides whereas NO furthermore mediates some of the ocular effects of calcitonin generelated peptide and PACAP (reviews see [33,34]). There are clear indices that PE-11 may also be integrated in the pathogenesis of this neurogenic inflammation, in particular in miosis because of the prominent innervation of the sphincter muscle and in the vascular effects because of the clear association of the peptide with blood vessels in the iris/ciliary body complex. However, this hypothesis remains to be confirmed.
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The significance of the presence of PE-11-IR in the corneal endothelium and in glia cells in the retina is also not clear yet. However, VIP, another peptide which is present in the corneal endothelial cells, is a trophic factor which protects the cells as an autocrine against oxidative stress [16] and through its autocrine VIP, these cells play an active role in maintaining the differentiated state and suppressing apoptosis [17]. It remains to be elucidated whether PE-11 features a similar role. And then, glial cells in the retina have been found to be important for the maintenance of a healthy tissue environment and a clear role for Müller glial cells in maintaining retinal homeostasis and trophic support for the neurons has been established [4,14]. PE-11 may exert such functions as well. But it must be emphasized that no biological effects have been found for this peptide so far, hence its role in the pathophysiology of the eye is only speculative. References [1] Aardal S, Helle KB. The vasoinhibitory activity of bovine chromogranin A fragment (vasostatin) and its independence of extracellular calcium in isolated segments of human blood vessels. Regul Pept 1992;41:9–18. [2] Bitsche M, Schrott-Fischer A, Hinterhölzl J, Fischer-Colbrie R, Sergi C, Glückert R, et al. First localization and biochemical identification of chromogranin B- and secretoneurin-like immunoreactivity in the fetal human vagal/nucleus solitary complex. Regul Pept 2006;134:97–104. [3] Blaschko H, Comline RS, Schneider FH, Silver M, Smith AD. Secretion of a chromaffin granule protein, chromogranin, from the adrenal gland after splanchnic stimulation. Nature 1967;215:58–9. [4] Bringmann A, Pannike T, Grosche J, Francke M, Wiedemann P, Skatchkov SN, et al. Müller cells in the healthy and diseased retina. Prog Retin Eye Res 2006;25:397–424. [5] Chanat E, Huttner WB. Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network. J Cell Biol 1991;115: 1505–19. [6] Curry WJ, McCollum AP, Brockbank S, Gardiner TA, Maule AG, Stitt AW. Characterization of WE-14 in porcine ocular tissue. Regul Pept 2003;113: 41–7. [7] Efendic A, Tatemoto K, Mutt V, Quan C, Chang D, Östenson C-G. Pancreastatin and islet hormone release. Proc Natl Acad Sci U S A 1987;84:7257–60. [8] Fischer-Colbrie R, Frischenschlager I. Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranin B and enkephalin-containing peptides. J Neurochem 1985;44:1854–61. [9] Fischer-Colbrie R, Laslop A, Kirchmair R. Secretogranin II: molecular properties, regulation of biosynthesis and processing to the neuropeptide secretoneurin. Prog Neurobiol 1995;46:49–70. [10] Helle KB. Chromogranins A and B and secretogranin II as prohormones for regulatory peptides from the diffuse neuroendocrine system. Results Probl Cell Differ 2010;50:21–44. [11] Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24:739–68. [12] Holzer P. Capsaicin as a tool to study sensory neuron functions. Adv Exp Med Biol 1991;298:3–16. [13] Holzer P. Capsaic: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev 1991;43:143–201. [14] Jadhav AP, Roesch K, Cepko CL. Development and neurogenic potential of Müller glia cells in the vertebrate retina. Prog Retin Eye Res 2009;28: 249–62. [15] Kato A, Kammen-Jolly K, Fischer-Colbrie R, Humpel C, Schrott-Fischer A, Marksteiner J. Co-distribution patterns of chromogranin B-like immunoreactivity with chromogranin A and secretoneurin within the human brainstem. Brain Res 2000;852:444–52.
[16] Koh S-WM, Waschek JA. Corneal endothelial cell survival in organ cultures under acute oxidative stress: effect of VIP. Invest Ophthalmol Vis Sci 2000;41:4085–92. [17] Koh S-WM, Chandrasekara K, Abbondandolo CJ, Coll TJ, Rutzen AR. VIP and VIP gene silencing modulation of differentiation marker N-cadherin and cell shape of corneal endothelium in human corneas ex vivo. Invest Ophthalmol Vis Sci 2008;49:3491–8. [18] Kralinger MT, Hinterhölzl J, Troger J, Nguyen QA, Kremser B, Fischer-Colbrie R, et al. Elevated levels of secretoneurin in the rabbit aqueous humor in response to formaldehyde irritation. Graefes Arch Clin Exp Ophthalmol 2003;241:577–81. [19] Kroesen S, Marksteiner J, Leitner B, Hogue-Angeletti R, Fischer-Colbrie R, Winkler H. Rat brain: distribution of immunoreactivity of PE-11, a peptide derived from chromogranin B. Eur J Neurosci 1996;8:2679–89. [20] Lazarov NE. Comparative analysis of the chemical neuroanatomy of the mammalian trigeminal ganglion and mesencephalic trigeminal nucleus. Prog Neurobiol 2002;66:19–59. [21] Li JY, Leitner B, Winkler H, Dahlström A. Distribution of chromogranins A and B and secretogranin II (secretoneurin) in rat pelvic neurons and vas deferens. Neuroscience 1998;84:281–94. [22] Li J-Y, Leitner B, Lovisetti-Scamihorn P, Winkler H, Dahlström A. Proteolytic processing, axonal transport and differential distribution of chromogranins A and B, and secretogranin II (secretoneurin) in rat sciatic nerve and spinal cord. Eur J Neurosci 1999;11:528–44. [23] Lorenz K, Troger J, Fischer-Colbrie R, Kremser B, Schmid E, Kralinger M, et al. Substance P and secretoneurin in vitreous aspirates of patients with various vitreoretinal diseases. Peptides 2008;29:1561–5. [24] Mahata SK, OˇıConnor DT, Mahata M, You SH, Taupenot L, Wu H, et al. Novel autocrine feedback control of catecholamine release. A descrete chromogranin A fragment is a noncompetitive nicotinic cholinergic antagonist. J Clin Invest 1997;100:1623–33. [25] Marksteiner J, Bauer R, Kaufmann WA, Weiss E, Barnas U, Maier H. PE-11, a peptide derived from chromogranin B, in the human brain. Neuroscience 1999;91:1155–70. [26] Marksteiner J, Lechner T, Kaufmann WA, Gurka P, Humpel C, Nowakowski C, et al. Distribution of chromogranin B-like immunoreactivity in the human hippocampus and its changes in Alzheimerˇıs disease. Acta Neuropathol 2000;100:205–12. [27] Montero-Hadjadje M, Vaingankar S, Elias S, Tostivint H, Mahata SK, Anouar Y. Chromogranins A and B and secretogranin II: evolutionary and functional aspects. Acta Physiol 2008;192:309–24. [28] Overdick B, Kirchmair R, Marksteiner J, Fischer-Colbrie R, Troger J, Winkler H, et al. Presence and distribution of a new neuropeptide, secretoneurin, in human retina. Peptides 1996;17:1–4. [29] Rosa P, Hille A, Lee RW, Zanini A, De Camili P, Huttner WB. Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway. J Cell Biol 1985;101:1999–2011. [30] Stemberger K, Pallhuber J, Doblinger A, Troger J, Kirchmair R, Kralinger M, et al. Secretoneurin in the human aqueous humor and the absence of an effect of frequently used eye drops on the levels. Peptides 2004;25:2115–8. [31] Strub JM, Garcia-Sablone P, Lonning K, Taupenot L, Hubert P, Van Dorsselaer A, et al. Processing of chromogranin B in bovine adrenal medulla: identification of secretolytin, the endogenous C-terminal fragment of residues 614–626 with antibacterial activity. Eur J Biochem 1995;229:356–68. [32] Troger J, Doblinger A, Leierer J, Laslop A, Schmid E, Teuchner B, et al. Secretoneurin in the peripheral ocular innervation. Invest Ophthalmol Vis Sci 2005;46:647–54. [33] Troger J, Kieselbach G, Teuchner B, Kralinger M, Nguyen QA, Haas G, et al. Peptidergic nerves in the eye, their source and potential pathophysiological relevance. Brain Res Rev 2007;53:39–62. [34] Troger J, Bechrakis N, Fischer-Colbrie R, Kralinger M, Schmid E, Teuchner B, et al. Neurogenic inflammation in the eye. In: Troger J, Kieselbach G, Bechrakis N, editors. Neuropeptides in the eye. Research Signpost; 2009. p. 69–78. [35] Wiedermann CJ. Secretoneurin: a functional neuropeptide in health and disease. Peptides 2000;21:1289–98. [36] Winkler H, Fischer-Colbrie R. The chromogranin A and B: The first 25 years and future perspectives. Neuroscience 1992;49:497–528.
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PE-11, a peptide derived from chromogranin B, in the rat eye Katrin Lorenz a , Josef Troger b,∗ , Oliver Gramlich a , Franz Grus a , Rosa Hattmannstorfer c , Reiner Fischer-Colbrie d , Stephanie Joachim e , Eduard Schmid b , Barbara Teuchner b , Gertrud Haas b , Nikolaos Bechrakis b a
Experimental Ophthalmology, Department of Ophthalmology, University Medical Center, Johannes Gutenberg-University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany Department of Ophthalmology, Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria c Clinical Department of Restorative and Prosthetic Dentistry, Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria d Department of Pharmacology, Medical University of Innsbruck, Peter Mayrstraße 1a, 6020 Innsbruck, Austria e Center for Vision Science, Ruhr University Eye Hospital, In der Schornau 23–25, 44892 Bochum, Germany b
a r t i c l e
i n f o
Article history: Received 16 December 2010 Received in revised form 14 March 2011 Accepted 14 March 2011 Available online 23 March 2011 Keywords: PE-11 Chromogranin B Rat Eye Capsaicin
a b s t r a c t The aim of the study was to investigate the presence and distribution of PE-11, a peptide derived from chromogranin B, in the rat eye. For this purpose, newborn rats were injected with a single dosage of 50 mg/kg capsaicin subcutaneously under the neck fold and after three months, particular eye tissues were dissected and the concentration of PE-11-like immunoreactivity was determined by radioimmunoassay. Furthermore, PE-11-like immunoreactivities were characterized in an extract of the rat eye by reversed phase HPLC. Then, the distribution pattern of PE-11 was investigated in the rat eye and rat trigeminal ganglion by immunofluorescence. As a result, PE-11 was present in each tissue of the rat eye and capsaicin pretreatment led to a 88.05% (±7.07) and a 64.26% (±14.17) decrease of the levels of PE-11 in the cornea and choroid/sclera, respectively, and to a complete loss in the iris/ciliary body complex. Approximately 70% of immunoreactivities detected by the PE-11 antiserum have been found to represent authentic PE-11. Sparse nerve fibers were visualized in the corneal and uveal stroma, surrounding blood vessels at the limbus, ciliary body and choroid and in association with the dilator and sphincter muscle. Furthermore, immunoreactivity was present in the corneal endothelium. In the retina and optic nerve, glia was labeled. In the rat trigeminal ganglion, PE-11-immunoreactivity was visualized in small and medium sized ganglion cells with a diameter of up to 30 m. In conclusion, there is unequivocal evidence that PE-11 is a constituent of capsaicin-sensitive sensory neurons innervating the rat eye and the distribution pattern is typically peptidergic in the peripheral innervation but in the retina completely atypical for neuropeptides and unique. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The chromogranins are the acidic proteins of secretory granules and comprise at least three subtypes: chromogranins A and B (for review see [36]) and secretogranin II (for review see [9]). Whereas secretogranin II has been discovered in the anterior pituitary [29], the other granins have been first found in the chromaffin granules of the adrenal medulla where they are coreleased with catecholamines [3,8]. Subsequent studies revealed that the granins are widely distributed throughout the neuroendocrine system where they are stored in large dense core vesicles [9,36]. The function is still not completely clear but it has been proposed that they may play a fundamental role in secretory granule formation and neurotransmitter/hormone release [5,27]. Furthermore, the granins
∗ Corresponding author. Tel.: +43 512 504 23758; fax: +43 512 504 23768. E-mail address: [email protected] (J. Troger). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.03.011
are characterized by numerous pairs of basic amino acids in the primary sequence as potential sites for intra- and extragranular processing indicating that they are precursors of functional active neuropeptides [10]. And indeed, chromogranin-derived peptides are generated in vivo and are functionally active. In particular, the chromogranin-A-derived peptide pancreastatin can inhibit glucose-stimulated insulin release from perfused rat pancreas or isolated acini [7]. Vasostatin, a peptide also derived from chromogranin A, exerts effects on vascular smooth muscle [1], and another chromogranin A-derived peptide, catestatin, was shown to be an effective inhibitor of catecholamine secretion from chromaffin cells [24]. A peptide derived from chromogranin B, secretolytin (CgB 614–626), was found to display antibacterial properties [31] and the secretogranin II-derived peptide secretoneurin (SN) exerts a variety of functional effects (review see [35]). PE-11 is another granin-derived peptide, in particular it is generated in vivo by proteolytical processing of chromogranin B [19]. Although no biological effect has been described for this peptide
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so far, it is widely distributed throughout the rat [19] and human brain [25], human brainstem [15], human hippocampus [26] and its presence in the fetal human vagal/nucleus solitary complex has been well documented [2]. Furthermore, PE-11 is present in the peripheral nervous system, in particular it has been found in large sensory axons in the dorsal roots of the spinal cord indicating that it is a sensory transmitter [22]. And it is also present in rat pelvic ganglia and vas deferens [21]. There is evidence that the processing of chromogranin B is quite extensive in the nervous system, in the rat brain and fetal human vagal/nucleus solitary complex it is completely processed [2,19], in the sciatic nerve and vas deferens to a high degree [21,22] and to varying degrees in particular tissues of the human brain [25]. With respect to the eye, the secretogranin II-derived peptide SN has been extensively studied. It is present in the human retina in amacrine and displaced amacrine cells [28] and it is a typical sensory transmitter with nerve fibers deriving from the trigeminal ganglion [32]. Furthermore, it is present in high concentrations in the human [30] and rabbit aqueous humor [18] and also in significant levels in the human vitreous of patients with various vitreoretinal diseases [23]. On the other hand, the presence and distribution of WE-14, a peptide derived from chromogranin A, has been investigated in porcine ocular tissues [6], but chromogranin B has not been studied in the eye so far. The present study concentrates on examinations on chromogranin B using an antibody raised against PE-11. It aims to find out whether it is present in capsaicin-sensitive sensory nerves of the rat eye and since this was found to be the case, to detect the peptide in the rat trigeminal ganglion. Capsaicin is the pungent ingredient of red pepper and when it is injected subcutaneously into newborn rats it destroys more than a half of the sensory neurons which makes it an ideal tool to investigate these neurons [11–13]. And then, the presence and distribution of PE-11 has been investigated in the rat eye in detail. 2. Materials and methods 2.1. Animal experiments In one set of experiments, capsaicin pretreatment in rats has been performed. For this purpose, newborn Sprague–Dawley rats were injected subcutaneously under the neck fold with a single dosage of 50 mg/kg capsaicin (obtained from Sigma–Aldrich, Vienna, Austria) on the first day after birth. Capsaicin was dissolved in saline containing 10% ethanol and 10% Tween 80. Animals without treatment served as controls. The animals were housed in cages with a dark–light cycle of 12 h each (lights on at 7 AM and off at 7 PM, in 23 ± 1 ◦ C) and fed a commercial chow and water ad libitum. The animals were allowed to grow for 3 months and were then sacrificed by an overdose of CO2 . All experimental and animal care procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the animal experiments were approved by the Ministry of Science in Austria. 2.2. Radioimmunoassay The radioimmunoassay (RIA) was performed with a specific antiserum and the generation of the antiserum was described previously [19]. In brief, it was generated against a synthetic peptide, PE-11, corresponding to rat chromogranin B 552–562. The antiserum only reacts with the free C-terminal part of PE-11 since an elongated peptide (chromogranin B 552–574) reacts exclusively within the RIA when it is first subjected to trypsin digestion. No cross-reactivity was found with peptides derived from chromo-
granin A and secretogranin II nor with the following neuropeptides: galanin, substance P, neuropeptide Y, neurotensin and calcitonin gene-related peptide. For determination of the concentration by RIA, rat eye tissues were prepared. For this purpose, the eyes were immediately removed after the rats were killed. A small incision was made at the limbus and the cornea inclusive limbus was circumferentially excised. Then, the iris/ciliary body-complex was removed and finally, the retina was detached from the underlying choroid/scleracomplex. The tissues were weighed and immediately frozen and kept frozen at −70 ◦ C. The tissues were then processed by RIA as described previously in detail [19]. In brief, tissues were sonicated in 300 L distilled water for 10 s and then immediately boiled for 10 min. After centrifugation (20 min at 12,000 g) the supernatant was analyzed for PE-11-immunoreactivity. For the RIA, samples, RIA buffer and antiserum (final dilution: 1:4500) were incubated for 24 h at 4 ◦ C. Then, the tracer (specific activity 77,000 d.p.m./ng) obtained by iodination of PE-11 with the chloramine T method was added and samples were incubated for a further 24 h at 4 ◦ C. Bound/free separation was performed with dextran-coated charcoal. 2.3. Characterization of PE-11-like immunoreactivities (LI) by reversed phase-HPLC followed by RIA The eyes of two Sprague–Dawley rats were enucleated, the lens was removed and the extraction from the residual tissues was performed by sonication as described above. For the reversed phase-HPLC, an aliquot of the extract was loaded into a reversed phase-HPLC column (LiChrospherWP300RP-18.5 m; Merck, Darmstadt, Germany) and eluted with a gradient ranging from 0% to 80% acetonitrile in 0.1% trifluoracetic acid–water over 60 min at a flow rate of 1 ml/min. Fractions (1.0 ml) were collected, lyophilized, reconstituted in assay buffer and analyzed for PE-11 by RIA as just described. The elution position of PE-11 was determined in a separate run with synthetic PE-11 as standard. 2.4. Immunofluorescence for PE-11 in the rat eye Nerve fibers in the rat eye were visualized by immunofluorescence. For this purpose, the eyes were enucleated and under a dissecting microscope, an incision was made in the cornea. Then, the eye ball was immersed in 4% paraformaldehyde for 1 h at room temperature followed by preparation of particular tissues inclusive removal of the lens and vitreous. Subsequently, the eyes were washed with PBS for a few times and then cryoprotection was performed. In particular, the eyecup was incubated in 10% succrose solution (1.5 ml/well) at room temperature for approximately 30 min until the eye was saturated and fell down to the bottom of the well. Then, the 10% succrose solution was replaced by 20% succrose solution and incubated at room temperature for approximately 2 h. Finally, the 20% succrose solution was replaced by 30% succrose solution and the eyecup was incubated over night at 4 ◦ C. Then, the eyes were embedded with O.C.T. compound freezing medium (Tissue Tek), frozen in liquid nitrogen and stored at −70 ◦ C. 6–15 m thick sections were cut from the specimens on a cryostat (Reichert Jung; Leica-Reichert, Vienna, Austria) and mounted on Superfrost Plus slides (Thermo Scientific, Braunschweig, Germany). The slides were dried for 30 min in an incubator and rinsed in PBS. Then, the slides were washed for 30 min in preheated Tris–EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0, 60 ◦ C) in an incubator at 60 ◦ C. The sections were then preincubated for 30 min with 0.25% normal goat serum/0.5% bovine serum albumin in PBS with 0.1% Triton X100, afterwards rinsed in PBS and subsequently incubated over night in disposable immunostaining chambers at room temper-
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Table 1 Concentration of PE-11-LI in particular tissues of the rat eye both in untreated controls and after capsaicin-pretreatment. Values represent means ± S.E.M. (fmol/mg wet weight; n = 10) and statistical calculation of differences between controls and capsaicin values was performed with the Mann Whitney U test (*** p < 0.001). Controls Cornea Iris/ciliary body Choroid/sclera Retina
0.77 3.53 3.05 5.48
± ± ± ±
0.41 0.40 0.24 0.34
Capsaicin 0.092 ± 0.029*** 0.0*** 1.09 ± 0.034*** 5.91 ± 0.14
ature with a rabbit PE-11 antiserum at a dilution of 1:1000 in PBS containing 0.1% Triton X-100. After a few washes with TBS and PBS, sections were incubated with the secondary antibody (Cy3-conjugated goat anti-rabbit IgG; Linaris, Wertheim-Bettingen, Germany) diluted 1:500 with PBS for 4 h in immunostaining chambers at room temperature. Stained sections were washed with PBS and TBS, mounted with Vectashield containing diamidin2-phenylindol (Vector, Burlingame, CA, USA) and coverslipped. Sections were visualized with a Nikon TE 2000 microscope (Nikon, Düsseldorf, Germany) equipped with a high sensitive cooled black and white CCD camera and Nikonˇıs Lucia G/F software. Furthermore, sections were processed for double immunofluorescence with the PE-11- and a glial fibrillary acidic protein (GFAP) antibody. The GFAP was immunostained via mouse anti-GFAP antibody (Linaris, ready to use) and fluorescein–isothiocyanate labeled goat anti-mouse IgG (GenWay, San Diego, CA, USA) diluted 1:400 in PBS. Specificity of the staining was evaluated by two further procedures, in particular both by incubating sections without the primary PE11 antiserum and by incubating sections with a PE-11-preadsorbed primary antibody. 2.5. Immunofluorescence for PE-11 in the rat trigeminal ganglion Two male rats (200 g) were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg) and perfused transcardially with saline followed by 4% paraformaldehyde. The trigeminal ganglia were removed and paraffinized to a paraffine tissue block. 10 m sections were cut on a microtome and mounted on Superfrost Plus slides. The slides were dried in an incubator at 60 ◦ C for 30 min and then deparaffinized/rehydrated. For this purpose, sections were incubated in three washes of xylene for 5 min each, incubated in two washes of 100% ethanol for 10 min each, incubated in two washes of 95% ethanol for 10 min each, incubated in one wash of 70% ethanol for 5 min and rinsed twice in dH2 O for 5 min each. The next steps inclusive antigen unmasking, blocking, incubating with the primary and secondary antiserum and visualizing were the same as described above. 3. Results 3.1. Concentration of PE-11-LI in various ocular tissues of the rat and the effect of capsaicin The concentration of PE-11-LI was measured by RIA in various ocular tissues of the rat and the results inclusive effect of capsaicin-pretreatment are summarized in Table 1. PE-11-LI was found to be present in each tissue of the rat eye, in particular in the cornea, the iris/ciliary body complex, the choroid/sclera and the retina. The highest levels were found in the retina, but the iris/ciliary body complex and choroid/sclera contained the peptide also in significant amounts and the peptide was even detectable in the cornea. Capsaicin-pretreatment led to a significant decrease of the concentration of PE-11-LI in each tissue except of the retina. In particular, an 88.05% (±7.07) decrease was observed in the cornea, a complete loss in the
Fig. 1. Characterization of PE-11-IR by reversed phase HPLC followed by RIA. One hundred microliters of a tissue extract of the rat eye were loaded into a reversed phase HPLC column and eluted with 0.1% trifluoroacetic acid–water. Horizontal line: gradient profile (percent acetonitrile, right ordinate). One milliliter-per-minute fractions were collected, lyophilized, reconstituted in assay buffer, and analyzed for PE-11 by RIA. The elution position of synthetic PE-11 is indicated by the arrow above the peak. The results revealed the presence of a major peak in the fractions 17–24 which coeluted with synthetic PE-11 and which amounted 71.84%. Furthermore, four minor peaks in fractions 8–10, 11–13, 25–28 and 29–32 were present which amounted 2.99%, 7.19%, 7.71% and 10.27%, respectively, but were not further characterized.
iris/ciliary body complex and a 64.26% (±14.17) decrease in the choroid/sclera. 3.2. Characterization of PE-11-LI in an extract of the rat eye Reversed phase-HPLC served to receive the molecular form of PE-11-LI in an extract of the rat eye. The result is illustrated in Fig. 1. Separation of immunoreactivities on a HPLC column and subsequent measurement of PE-11-LI in the fractions by RIA revealed one major peak in fractions 17–24 which coeluted with synthetic PE-11 and four minor peaks in positions 8–10, 11–13, 25–28, 29–32 which were not further characterized. The major peak in the position of synthetic PE-11 amounted 71.84%, the minor peaks in fractions 8–10, 11–13, 25–28 and 29–32 amounted 2.99%, 7.19%, 7.71% and 10.27%, respectively. These results indicate that approximately 70% of the immunoreactivities detected by the PE-11 antiserum represent authentic PE-11. 3.3. Visualization of PE-11-IR in the rat eye PE-11-IR was found to be distinctly distributed throughout the rat eye and the distribution pattern is illustrated in Fig. 2. In the cornea, sparse nerve fibers were found in the stroma and an immunoreactive band was observed in the innermost part of this tissue which confers to the endothelium (Fig. 2A). The bright immunofluorescence in the epithelium is autofluorescene since this immunoreactivity remained after removal of the primary antiserum. At the limbus, a prominent immunoreactivity was observed in association with blood vessels (Fig. 2B). In the iris, an immunofluorescent band was detected in clear association with the dilator muscle (Fig. 2C), but sparse nerve fibers were also present in the stroma (not shown) and there was a very dense network of nerve fibers in the sphincter muscle (Fig. 2D). In the ciliary body, nerve fibers again surrounded blood vessels (Fig. 2E) and immunoreactivity could be visualized in the stroma at the base of the processes and these nerves reached the stroma of the processes (Fig. 2F). In the choroid, nerve fibers were found again in association with blood vessels (Fig. 2G and H) but sparse fibers were also present in the stroma (Fig. 2H). In the retina, a dense network of immunoreactivity
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Fig. 2. Visualization of PE-11-IR in tissues of the rat eye. Particular tissues of the rat eye were dissected and processed by immunofluorescence according to a standard protocol. In brief, sections were incubated with a specific PE-11 antiserum at a dilution of 1:1000 over night and then for 4 h with a secondary antibody followed by visualization of immunoreactivities in the tissues. In the cornea (A), sparse nerve fibers were observed in the stroma (arrow) and an immunoreactive band in the endothelium (arrowheads). At the limbus (B), nerve fibers were found around blood vessels (arrows; v = vessel). In the iris, an association of immunoreactivity was found at the dilator muscle (C, arrows), but there were also sparse nerve fibers in the stroma (not shown) and a dense network of fibers in the sphincter muscle (D). In the ciliary body, nerve fibers were found again surrounding blood vessels (E, arrows; v = vessel), but sparse fibers were also observed in the stroma at the base of the ciliary processes reaching the stroma of the ciliary processes (F, arrows). In the choroid (G and H), there was an association of immunoreactivity with blood vessels (arrows, v = vessel), but nerve fibers were also observed in the stroma (arrowheads). In the retina (I), a dense network of immunoreactivity was observed in the innermost part which confers to glia (arrows) and glia was also labeled in the optic nerve (J, green fluorescent).
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Fig. 3. Demonstration of PE-11-IR by means of immunofluorescence in the rat trigeminal ganglion. Fixed rat trigeminal ganglia were paraffinized, sections were cut and processed by immunofluorescence as described in Section 2. In brief, the sections were again incubated with a specific primary PE-11 antibody over night and then for 4 h with a secondary antibody followed by visualization of PE-11-positive ganglion cells. Numerous ganglion cells were labeled and the cells were of small and medium size with a diameter of up to 30 m (A and B). PE-11-immunoreactive cells appeared mainly scattered (A), but occasionally, clusters were also observed (B).
was observed in the innermost part which showed colocalization with glial fibrillary protein (GFAP, not shown) and therefore confers to Müller glia (Fig. 2I). In the optic nerve, dense immunoreactivity was seen in glia (Fig. 2J). 3.4. Visualization of PE-11-IR in the rat trigeminal ganglion PE-11-IR was visualized by immunofluorescence in the rat trigeminal ganglion and with this procedure using a specific antiserum, numerous ganglion cells were labeled within the ganglion (Fig. 3A and B). In particular, the labeled cells were of small and medium size with a diameter of up to 30 m and occurred in scattered locations (Fig. 3A) but occasionally, small clusters or rows of cells were also observed (Fig. 3B). Nerve fibers or nonneuronal cells were not labeled. 4. Discussion For the first time, this study provides evidence that a chromogranin B-derived peptide is present in the eye. There are several novel findings. Firstly, PE-11 is a constituent of capsaicin-sensitive sensory nerves innervating the rat eye. Secondly, it could be detected in small and medium sized cells of the rat trigeminal ganglion and finally, it is distinctly distributed throughout the rat eye. There is no doubt that PE-11 is a sensory peptide innervating the eye since capsaicin pretreatment significantly lowered the levels in ocular tissues except of the retina and since this peptide was found to be present in the trigeminal ganglion which represents the sensory ganglion for cranial tissues (review see [20]). With the treatment regimen of this study, capsaicin destroys predominantly small neurons giving rise to unmyelinated C-fibers [11–13] and indeed, PE-11 was detected in the trigeminal ganglion in small cells. The complete loss of the peptide in the iris/ciliary body complex indicates an exclusive presence of PE11 in C-fiber afferents in this tissue and the significant drop in the cornea and choroid/sclera on a predominant presence in these unmyelinated fibers. In the cornea and especially in the choroid/sclera, however, PE-11 may be present also in some myelinated A␦ fibers which arise from small-sized cells as well and which are known to be less sensitive to capsaicin treatment. Alternatively, nerve fibers of other origin than the sensory one may contribute to the innervation of these tissues. Nevertheless, PE11 can now be added to the growing number of typical sensory peptides in the eye, in particular besides substance P, calcitonin gene-related peptide, neurokinin A, secretoneurin, galanin in rat
and pig, somatostatin, pituitary adenylate cyclase-activating peptide (PACAP)-27- and -38 in rabbits or cholecystokinin (review see [33]). The distribution pattern of PE-11 in the rat eye is typically peptidergic, namely the presence of sparse fibers in nearly all ocular tissues, the sparse presence of sensory nerve fibers in the stroma of the cornea, the iris, the ciliary body and the choroid and the dual innervation of both the sphincter and dilator muscle. However, there are two remarkable findings. Firstly, there is a clear association of nerve fibers with blood vessels not only at the limbus but also in the ciliary body and the choroid. This is similar to another granin-derived peptide, SN [32,33]. And then, there is immunoreactivity present in the endothelium of the cornea. This is similar to vasoactive intestinal peptide (VIP) which has also been found to be expressed in the human and bovine endothelium [16]. On the other hand, in the rat retina, PE-11 immunoreactivity is mainly concentrated in glial cells which is rather atypical for neuropeptides. Neuropeptides are in general mainly expressed in amacrine cells in the proximal inner nuclear layer and in displaced amacrine cells in the ganglion cell layer and partially also in sparse ganglion cells. Hence, the presence of this peptide in glial cells in the retina is unique. The functional significance of this peptide remains to be discussed. First of all, although there is no direct evidence, the prominent innervation of blood vessels indicates that PE-11 exerts a vasoregulatory effect. Then the peptide is obviously involved in an afferent transmission of sensory impulses because of the presence of PE-11 in sensory nerves. And finally, the peptide may also exert local effector functions in the iris/ciliary body complex, in particular it may be involved in neurogenic inflammation, since PE-11 is present in C-fibers there. This neurogenic inflammation in the eye is represented by an irritative response and consists of miosis, vasodilation, enhanced permeability of blood vessels, aqueous flare and increased intraocular pressure (for reviews see [33,34]). There is evidence, that substance P mediates miosis and calcitonin gene-related peptide the vascular effects at least in rabbits. Furthermore, the PACAPs participate as well as nitric oxide (NO) both of which stimulate the release of sensory peptides whereas NO furthermore mediates some of the ocular effects of calcitonin generelated peptide and PACAP (reviews see [33,34]). There are clear indices that PE-11 may also be integrated in the pathogenesis of this neurogenic inflammation, in particular in miosis because of the prominent innervation of the sphincter muscle and in the vascular effects because of the clear association of the peptide with blood vessels in the iris/ciliary body complex. However, this hypothesis remains to be confirmed.
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