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Distribution of Met-enkephalyl-Arg-Gly-Leu in rat larynx: Partial coexistence with vasoactive intestinal polypeptide, peptide histidine isoleucine and neuropeptide Y
Neuroscience Letters, 119 (1990) 64457
64
Elsevier Scientific Publishers Ireland Ltd. NSL 07253
Distribution of Met-enkephalyl-Arg-Gly-Leu in rat larynx: partial coexistence with vasoactive intestinal polypeptide, peptide histidine isoleucine and neuropeptide Y J. K r e k e l l, E. W e i h e l, D. N o h r I, N . Y a n a i h a r a 2 a n d E. W e b e r 3 ~Department of Anatomy, Johannes Gutenberg-University, Mainz (F.R.G.), 2Laboratoryof Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka (Japan) and 3Institutefor Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR 97201 (U.S.A.)
(Received 9 May 1990; Revisedversion received 5 July 1990; Accepted 11 July 1990) Key words: Larynx; Met-enkephalyl-Arg-Gly-Leu;Vasoactive intestinal polypeptide; Peptide histidine isoleucine; Neuropeptide Y; Calcitonin
gene-related peptide; Innervation; Coexistence; Rat Using light microscopic (LM) enzyme-immunohistochemistryon deparaffinized adjacent sections Met-enkephalyl-Arg-Gly-Leu(ME-RGL) immunoreactivitywas found to partially coexist with immunoreactiveneuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in intrinsic laryngeal neurons of the rat. Further ME-RGL-immunoreactive(ir) fibres were found around glands in the subepithelium, in connectivetissue of striated muscleand in the perichondrium, as well as around arterial and venous blood vessels.They frequently contacted mast cells and macrophages. The presence of ME-RGL indicates pro-enkephalin-related origin of this novel laryngeal opioid system. From the specifictarget relations and close interrelations of fibres staining for opioids with those staining for the other peptides - which are known to be more or less characteristicof the sympathetic(NPY), parasympathetic(VIP, PHI) and sensory(calcitoningene-relatedpeptide; CGRP) subdivisions of the peripheral nervous system- we deduce that opioid/non-opioid interactions might co-control various laryngeal functions, e.g. glandular secretion, blood flow, immune and inflammatoryresponses and/or might be of relevance in trophic mechanisms.
The chemical neuroanatomy of the respiratory tract has been subject of m a n y morphological and functional studies (cf. refs. 1, 3, 13, 14, 16). However, these investigations do not focus on the larynx, consequently little is known about the neuropeptidergic content of laryngeal autonomic and sensory nerves [2, 8, 18]. Since the larynx is affected by m a n y illnesses in which neuropeptides might be crucially involved, e.g. hyperreactivity reactions (laryngeal edema) or inflammatory processes, we found it worthwhile to study the laryngeal peptidergic innervation more precisely. In the light of the recently reported presence of opioid peptides in the lung [15, 17, 19] we wanted to clarify the unelucidated question as to whether the laryngeal part of the respiratory tract receives such an opioid innervation, too. Adult rats (n = 9) were deeply anesthesized by sodium pentobarbital (120 mg/kg b.wt., i.p.) and were sacrificed by decapitation. The larynx was dissected within 5 min post mortem and fixed in Bouin's solution, acid-free Bouin's solution or Bouin Hollande. Sequential deparaffinized sections (5-7/~m thick) were processed for light microscopic (LM) immunohistochemistry using biotinyCorrespondence: E. Weihe, Department of Anatomy, Johannes Gutenberg-Universit/it, Saarstrasse 19-21, D-6500 Mainz, F.R.G.
0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
lated species-specific secondary antisera and streptavidin-horseradish peroxidase complexes (Amersham). The diaminobenzidine (DAB) reaction was performed in 50 m M phosphate buffer (pH 7.4) and enhanced by 0.08 % a m m o n i u m nickel sulfate (Fluka). Primary polyclonal antisera against the pro-enkephalin (PRO-ENK)-specific sequence M E - R G L (Weber, R3-3; 1:20,000), M E - R G L (Yanaihara, R-0171; 1:8000), P H I (Yanaihara, R-8403; 1:30,000), VIP (CRB, 1:20,000), VIP (Yanaihara, R-502; 1:15000, N P Y (Amersham, R P N 1702; 1:8000) and C G R P (Peninsula, RAS 6012 N; 1:6000) were used in working dilutions as indicated in brackets. There was no evidence for mutual cross-reactivity. In order to work out coexistence patterns adjacent sections and mirrored adjacent sections were stained with the primary antisera of interest. With all fixation solutions similar results were obtained as summarized in Table I. M E - R G L - i r fibres were present throughout the different compartments of the larynx. They occurred in the subepithelium, in connective tissue of striated muscle, around glands and frequently surrounded arteries, veins and microvasculature. The perichondrium received a relatively dense octapeptidergic innervation, which was
65 TABLE I RELATIVE DENSITY AND TARGET DISTRIBUTION OF VARIOUS NEUROPEPTIDE-IR FIBRES IN RAT LARYNX; SUBJECTIVE RATING + , low; + + , moderate, + + + , high, + + + + , very high density.
ME-RGL NPY VIP PHI CGRP
Sub-epithelium
Striated muscle
Glands
Arteries
Veins
Micro-vasculature
Perichondrium
+++ + ++ ++ ++++
+++ ++ ++++ ++++ +++
++++ ++ ++++ ++++ ++
+++ ++++ +++ +++ ++++
+++ ++++ +++ +++ +++
+++ ++++ +++ +++ +++
+++ + ++ ++ +++
similarly dense as compared to CGRP innervation and exceeded that with fibres containing other neuropeptides. ME-RGL-ir fibres contacted mast cells and macrophages which were mainly located next to blood vessels and in submucosal areas. A subpopulation of ganglionic cell bodies of local intrinsic ganglia located in striated muscle and in iuxtaposition to cartilage stained for ME-RGL, too. By incubating adjacent and mirrored sections with the different antisera, partial coexistence of ME-RGL, NPY, VIP and PHI in such laryngeal microganglia was demonstrated (Figs. 1 and 2).
The distribution patterns of NPY-, VIP- and PHI-ir fibres partly overlapped with ME-RGL-ir fibres. The relative density of fibres staining for the different peptides showed histotopographical variations. ME-RGL-ir fibres were more frequent in the subepithelium and the perichondrium, whereas the connective tissue of striated muscle received a denser VIP/PHI than opioidergic innervation. The ME-RGL and VIP/PHI innervation of laryngeal glands was striking, similarly dense and overlapped almost completely. Apart from the supply to blood vessels, the ME-RGL innervation was denser than that with NPY. There were no ir-CGRP ganglionic cell
VIP
o#"
I;
a
25~m b
c
Fig. 1. Adjacent sections (a vs b vs c) showing partial coexistence of VIP, ME-RGL and PHI in a subpopulation of ganglionic cell bodies in an intrinsic ganglion in rat larynx (magnification marked by bar).
66
| ; !~
Fig. 2. Adjacent sections (a-d) of neurons in striated muscle of rat larynx demonstrating partial coexistence of NPY, ME-RGL, PHI. Note absence of ir-CGRP from neurons, but presence in ir-fibers surrounding non-ir perikarya (magnification marked by bar).
bodies, but CGRP-ir fibres surrounded non-ir neuronal perikarya. Apart from glands there was a very dense ubiquitous network of CGRP-ir fibers as reported by others [18]. This study provides evidence for an opioidergic system in the innervation of the rat larynx which was found to be similar in other species (unpublished; Krekel and Weihe). The presence of ir-ME-RGL indicates that this novel laryngeal opioid system is derived from the PROENK-opioid precursor. Indirect evidence for laryngeal opioid innervation without further characterization of molecular species involved was obtained by Dahlqvist and Forsgren [2] who revealed ir-enkephalin in ganglia associated with the superior and recurrent laryngeal nerve, but did not investigate the presence and distribution of opioid innervation in the larynx itself. The co-occurrence of ME-RGL-ir with VIP/PHI-ir in intrinsic ganglionic cells in conjunction with its absence from or paucity in sensory neurons may indicate that the major part of laryngeal PRO-ENK-octapeptide innervation is of intrinsic (postganglionic parasympathetic?) origin [19]. However, from the known presence of ME-
RGL-ir in postganglionic sympathetic neurons a sympathetic opioid component in the larynx could also be deduced [9, 10]. Further, the possibility of a pro-dynorphin (PRO-DYN)-related postganglionic sympathetic or primary sensory innervation of the larynx has to be considered [5, 11, 19]. Since antibodies against ME-RGL, VIP, PHI and NPY raised in different species were not available to us, we could not show coexistence of these neuropeptides in nerve fibers directly by employing double-fluorescence on identical sections. However, preliminary doubleimmunofluorescence investigations with pan-opioid antibody 3E7 (Gramsch) suggested coexistence of panopioid and VIP/PHI immunoreactivity in laryngeal nerve fibres, though without characterizing whether the coexisting opioids were derived from PRO-ENK, PRODYN or even from pro-opiomelanocortin (POMC) [19]. As there is a close interrelation between the fibres staining for PRO-ENK-octapeptide and those staining for non-opioid peptides (which are known to be more or less characteristic for the sympathetic (NPY), parasympathetic (VIP/PHI) and sensory (CGRP) subdivision of
67 the PNS) and have been shown to partially coexist in various tissues [7, 8, 12, 19] we draw the conclusion that there is a mutual o p i o i d / n o n - o p i o i d interaction in the neural control o f various laryngeal functions like glandular secretion, vasodilation and vasopermeability. There m a y be even m o r e complex opioid-non-opioid peptidergic interactions [12, 19]. Nevertheless, we assume that M E - R G L might be a n o t h e r i m p o r t a n t factor in controlling vascular and glandular functions and - like other peptides - - could be involved in i m m u n e responses and trophic mechanisms. A n opioid influence on c h o n d r o c y t e function is conceivable. The presence o f opioid peptides in intrinsic ganglia o f rat larynx - - which are t h o u g h t to be o f postganglionic parasympathetic origin - - implicates different concepts: firstly M E - R G L might be i m p o r t a n t for the autoregulation o f the neurons it is contained in by influencing the release o f its presumed classical (acetylcholine) and peptide (in particular V I P / P H I ) co-transmitters [6, 19]. Further laryngeal opioids might influence local effector functions o f spinal and vagal-sensory afferents which are e n d o w e d with multiple opioid receptors (cf. ref. 19). A n open question remains, whether laryngeal opioid peptides exert a postsynaptic effect. Since peripherally acting opioids are beneficial in respiratory distress (e.g. coughing) [4] we feel tempted to deduce that appropriately designed opioids might be o f therapeutic value in laryngeal pathophysiology. Parts o f this study have been reported at the N e u r o a n a t o m y and N e u r o b i o l o g y S y m p o s i u m N a n c y , 25. / 26. 09. 1989 and are contained in the thesis o f J o a c h i m Krekel, w h o received a fellowship f r o m the J o h a n n e s Gutenberg-University, M a i n z (Titel 681 02). The study was supported by the Deutsche Forschungsgemeinschaft ( D F G G r a n t We 910/1-2 and 2-2) and by Naturwissenschaftliches Medizinisches F o r s c h u n g s z e n t r u m ( N M F Z ) o f the University o f Mainz. 1 Barnes, P.J., Regulatory peptides in the respiratory system, Experientia, 43 (1987) 832-839. 2 Dahlqvist, A. and Forsgren, S., Networks of peptide-containing nerve fibres in laryngeal nerve paraganglia. An immunohistochemical study, Acta Otolaryngol., 107 (1989) 289-295. 3 Dey, R.D., Hoffpauir, J. and Said, S.I., Co-localization of vasoactive intestinal peptide- and substance P-containing nerves in cat bronchi, Neuroscience, 24, 1 (1988) 275-281. 4 Fuller, R.W., Karlsson, J.A., Choudry, N.B. and Pride, N.B., Effect of inhaled and systemic opiates on responses to inhaled capsaicin in humans, J. Appl. Physiol., 65 (1988) 1125-1130.
5 Gibbins, I.L. and Morris, J.L., Sympathetic noradrenergic neurons containing dynorphin but not neuropeptide Y innervate small cutaneous blood vessels of guinea-pigs, J. Auton. Nerv. Syst., 29 (1990) 137-150. 6 Gray, D.B., Pilar, G.R. and Ford, M.J., Opiate and peptide inhibition of transmitter release in parasympathetic nerve terminals, J. Neurosci., 9 (1989) 1683-1692. 7 Grunditz, T., Ekman, R., Hakanson, R., Sundler, F. and Uddman, R., Neuropeptide Y and vasoactive intestinal peptide coexist in rat thyroid nerve fibers emanating from the thyroid ganglion, Regul. Pept., 23 (1988) 193-208. 8 Grunditz, T., Hakanson, R., Hedge, G., Rerup, C., Sundler, F. and Uddman, R., Peptide histidine isoleucine amide stimulates thyroid hormone secretion an coexists with vasoactive intestinal polypeptide in intrathyroid nerve fibers from laryngeal ganglia, Endocrinology, 118 (1986) 783-790. 9 H/ippSla, O., Soinila, S., P/iivirinta, H. and Panula, P., (Met)Enkephalin-Arg-Phe- and (Me0Enkephalin-Arg-Gly-Leu-immunoreactive nerve fibers and neurons in the superior cervical ganglion of the rat, Neuroscience, 21 (1987) 283-295. 10 Kondo, H., Yamamoto, M., Yanaihara, N. and Nagatsu, I., Transient involvement of enkephalins in both the sympathetic and parasympathetic innervations of the submandibular gland of rats, Cell Tissue Res., 253 (1988) 529-537. 11 Kummer, W., Heym, C., Colombo, M. and Lang, R., Immunohistochemical evidence for extrinsic and intrinsic opioid systems in the guinea pig superior cervical ganglion, Anat. Embryol., 174 (1986) 401--405. 12 Lindh, B., Lundberg, J.M., H6kfelt, T., Elfvin, L.G., Fahrenkrug, J., Fischer, J., Coexistence of CGRP- and VIP-like immunoreactivities in a population of neurons in the cat stellate ganglia, Acta Physiol. Stand., 131 (1987) 475--476. 13 Lundberg, J.M., Fahrenkrug, J., Hrkfelt, T., Martling, C.-R., Larsson, O., Tatemoto, K. and .~nggard, A., Coexistence of PHI and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man, Peptides, 5 (1984) 593-605. 14 Lundberg, J.M. and Tatemoto, K., Pancreatic polypeptide family (APP, BPP, NPY, and PYY) in relation to sympathetic vasoconstriction resistant to ,,-adrenoceptor blockade, Acta Physiol. Scand., 116 (1982) 393-402. 15 Nohr, D., Krekel, J. and Weibe, E., Opioid peptides are present in nerve fibres of the respiratory tract, Regul. Pept., 22 (1988) 424. 16 Nohr, D. and Weihe, E., Light microscopic immunohistochemistry reveals species-dependent presence of tachykinins in intrinsic neurons in the mammalian respiratory tract, Regul. Pept., 22 (1988) 425. 17 Shimosegawa, T., Foda, H.D. and Said, S., Immunohistochemical demonstration of enkephalin-containing nerve fibers in guinea pig and rat lungs, Am. Rev. Resp. Dis., 140 (1989) 441--448. 18 Terenghi, G., Polak, J.M., Rodrigo, J., Muiderry, P.K. and Bloom, S.R., Calcitonin gene-related peptide-immunoreactive nerve fibers in the tongue, epiglottis and pharynx of the rat: occurrence, distribution and origin, Brain Res., 365 (1986) 1-14. 19 Weihe, E., Neuropeptides in primary sensory neurons. In W. Zenker and W.L. Neuhuber (Eds.), The Primary Sensory Neuron - - A Survey of Recent Morpho-Functional Aspects, Plenum, New York, 1990, pp. 127-159.
64
Elsevier Scientific Publishers Ireland Ltd. NSL 07253
Distribution of Met-enkephalyl-Arg-Gly-Leu in rat larynx: partial coexistence with vasoactive intestinal polypeptide, peptide histidine isoleucine and neuropeptide Y J. K r e k e l l, E. W e i h e l, D. N o h r I, N . Y a n a i h a r a 2 a n d E. W e b e r 3 ~Department of Anatomy, Johannes Gutenberg-University, Mainz (F.R.G.), 2Laboratoryof Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka (Japan) and 3Institutefor Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR 97201 (U.S.A.)
(Received 9 May 1990; Revisedversion received 5 July 1990; Accepted 11 July 1990) Key words: Larynx; Met-enkephalyl-Arg-Gly-Leu;Vasoactive intestinal polypeptide; Peptide histidine isoleucine; Neuropeptide Y; Calcitonin
gene-related peptide; Innervation; Coexistence; Rat Using light microscopic (LM) enzyme-immunohistochemistryon deparaffinized adjacent sections Met-enkephalyl-Arg-Gly-Leu(ME-RGL) immunoreactivitywas found to partially coexist with immunoreactiveneuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in intrinsic laryngeal neurons of the rat. Further ME-RGL-immunoreactive(ir) fibres were found around glands in the subepithelium, in connectivetissue of striated muscleand in the perichondrium, as well as around arterial and venous blood vessels.They frequently contacted mast cells and macrophages. The presence of ME-RGL indicates pro-enkephalin-related origin of this novel laryngeal opioid system. From the specifictarget relations and close interrelations of fibres staining for opioids with those staining for the other peptides - which are known to be more or less characteristicof the sympathetic(NPY), parasympathetic(VIP, PHI) and sensory(calcitoningene-relatedpeptide; CGRP) subdivisions of the peripheral nervous system- we deduce that opioid/non-opioid interactions might co-control various laryngeal functions, e.g. glandular secretion, blood flow, immune and inflammatoryresponses and/or might be of relevance in trophic mechanisms.
The chemical neuroanatomy of the respiratory tract has been subject of m a n y morphological and functional studies (cf. refs. 1, 3, 13, 14, 16). However, these investigations do not focus on the larynx, consequently little is known about the neuropeptidergic content of laryngeal autonomic and sensory nerves [2, 8, 18]. Since the larynx is affected by m a n y illnesses in which neuropeptides might be crucially involved, e.g. hyperreactivity reactions (laryngeal edema) or inflammatory processes, we found it worthwhile to study the laryngeal peptidergic innervation more precisely. In the light of the recently reported presence of opioid peptides in the lung [15, 17, 19] we wanted to clarify the unelucidated question as to whether the laryngeal part of the respiratory tract receives such an opioid innervation, too. Adult rats (n = 9) were deeply anesthesized by sodium pentobarbital (120 mg/kg b.wt., i.p.) and were sacrificed by decapitation. The larynx was dissected within 5 min post mortem and fixed in Bouin's solution, acid-free Bouin's solution or Bouin Hollande. Sequential deparaffinized sections (5-7/~m thick) were processed for light microscopic (LM) immunohistochemistry using biotinyCorrespondence: E. Weihe, Department of Anatomy, Johannes Gutenberg-Universit/it, Saarstrasse 19-21, D-6500 Mainz, F.R.G.
0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
lated species-specific secondary antisera and streptavidin-horseradish peroxidase complexes (Amersham). The diaminobenzidine (DAB) reaction was performed in 50 m M phosphate buffer (pH 7.4) and enhanced by 0.08 % a m m o n i u m nickel sulfate (Fluka). Primary polyclonal antisera against the pro-enkephalin (PRO-ENK)-specific sequence M E - R G L (Weber, R3-3; 1:20,000), M E - R G L (Yanaihara, R-0171; 1:8000), P H I (Yanaihara, R-8403; 1:30,000), VIP (CRB, 1:20,000), VIP (Yanaihara, R-502; 1:15000, N P Y (Amersham, R P N 1702; 1:8000) and C G R P (Peninsula, RAS 6012 N; 1:6000) were used in working dilutions as indicated in brackets. There was no evidence for mutual cross-reactivity. In order to work out coexistence patterns adjacent sections and mirrored adjacent sections were stained with the primary antisera of interest. With all fixation solutions similar results were obtained as summarized in Table I. M E - R G L - i r fibres were present throughout the different compartments of the larynx. They occurred in the subepithelium, in connective tissue of striated muscle, around glands and frequently surrounded arteries, veins and microvasculature. The perichondrium received a relatively dense octapeptidergic innervation, which was
65 TABLE I RELATIVE DENSITY AND TARGET DISTRIBUTION OF VARIOUS NEUROPEPTIDE-IR FIBRES IN RAT LARYNX; SUBJECTIVE RATING + , low; + + , moderate, + + + , high, + + + + , very high density.
ME-RGL NPY VIP PHI CGRP
Sub-epithelium
Striated muscle
Glands
Arteries
Veins
Micro-vasculature
Perichondrium
+++ + ++ ++ ++++
+++ ++ ++++ ++++ +++
++++ ++ ++++ ++++ ++
+++ ++++ +++ +++ ++++
+++ ++++ +++ +++ +++
+++ ++++ +++ +++ +++
+++ + ++ ++ +++
similarly dense as compared to CGRP innervation and exceeded that with fibres containing other neuropeptides. ME-RGL-ir fibres contacted mast cells and macrophages which were mainly located next to blood vessels and in submucosal areas. A subpopulation of ganglionic cell bodies of local intrinsic ganglia located in striated muscle and in iuxtaposition to cartilage stained for ME-RGL, too. By incubating adjacent and mirrored sections with the different antisera, partial coexistence of ME-RGL, NPY, VIP and PHI in such laryngeal microganglia was demonstrated (Figs. 1 and 2).
The distribution patterns of NPY-, VIP- and PHI-ir fibres partly overlapped with ME-RGL-ir fibres. The relative density of fibres staining for the different peptides showed histotopographical variations. ME-RGL-ir fibres were more frequent in the subepithelium and the perichondrium, whereas the connective tissue of striated muscle received a denser VIP/PHI than opioidergic innervation. The ME-RGL and VIP/PHI innervation of laryngeal glands was striking, similarly dense and overlapped almost completely. Apart from the supply to blood vessels, the ME-RGL innervation was denser than that with NPY. There were no ir-CGRP ganglionic cell
VIP
o#"
I;
a
25~m b
c
Fig. 1. Adjacent sections (a vs b vs c) showing partial coexistence of VIP, ME-RGL and PHI in a subpopulation of ganglionic cell bodies in an intrinsic ganglion in rat larynx (magnification marked by bar).
66
| ; !~
Fig. 2. Adjacent sections (a-d) of neurons in striated muscle of rat larynx demonstrating partial coexistence of NPY, ME-RGL, PHI. Note absence of ir-CGRP from neurons, but presence in ir-fibers surrounding non-ir perikarya (magnification marked by bar).
bodies, but CGRP-ir fibres surrounded non-ir neuronal perikarya. Apart from glands there was a very dense ubiquitous network of CGRP-ir fibers as reported by others [18]. This study provides evidence for an opioidergic system in the innervation of the rat larynx which was found to be similar in other species (unpublished; Krekel and Weihe). The presence of ir-ME-RGL indicates that this novel laryngeal opioid system is derived from the PROENK-opioid precursor. Indirect evidence for laryngeal opioid innervation without further characterization of molecular species involved was obtained by Dahlqvist and Forsgren [2] who revealed ir-enkephalin in ganglia associated with the superior and recurrent laryngeal nerve, but did not investigate the presence and distribution of opioid innervation in the larynx itself. The co-occurrence of ME-RGL-ir with VIP/PHI-ir in intrinsic ganglionic cells in conjunction with its absence from or paucity in sensory neurons may indicate that the major part of laryngeal PRO-ENK-octapeptide innervation is of intrinsic (postganglionic parasympathetic?) origin [19]. However, from the known presence of ME-
RGL-ir in postganglionic sympathetic neurons a sympathetic opioid component in the larynx could also be deduced [9, 10]. Further, the possibility of a pro-dynorphin (PRO-DYN)-related postganglionic sympathetic or primary sensory innervation of the larynx has to be considered [5, 11, 19]. Since antibodies against ME-RGL, VIP, PHI and NPY raised in different species were not available to us, we could not show coexistence of these neuropeptides in nerve fibers directly by employing double-fluorescence on identical sections. However, preliminary doubleimmunofluorescence investigations with pan-opioid antibody 3E7 (Gramsch) suggested coexistence of panopioid and VIP/PHI immunoreactivity in laryngeal nerve fibres, though without characterizing whether the coexisting opioids were derived from PRO-ENK, PRODYN or even from pro-opiomelanocortin (POMC) [19]. As there is a close interrelation between the fibres staining for PRO-ENK-octapeptide and those staining for non-opioid peptides (which are known to be more or less characteristic for the sympathetic (NPY), parasympathetic (VIP/PHI) and sensory (CGRP) subdivision of
67 the PNS) and have been shown to partially coexist in various tissues [7, 8, 12, 19] we draw the conclusion that there is a mutual o p i o i d / n o n - o p i o i d interaction in the neural control o f various laryngeal functions like glandular secretion, vasodilation and vasopermeability. There m a y be even m o r e complex opioid-non-opioid peptidergic interactions [12, 19]. Nevertheless, we assume that M E - R G L might be a n o t h e r i m p o r t a n t factor in controlling vascular and glandular functions and - like other peptides - - could be involved in i m m u n e responses and trophic mechanisms. A n opioid influence on c h o n d r o c y t e function is conceivable. The presence o f opioid peptides in intrinsic ganglia o f rat larynx - - which are t h o u g h t to be o f postganglionic parasympathetic origin - - implicates different concepts: firstly M E - R G L might be i m p o r t a n t for the autoregulation o f the neurons it is contained in by influencing the release o f its presumed classical (acetylcholine) and peptide (in particular V I P / P H I ) co-transmitters [6, 19]. Further laryngeal opioids might influence local effector functions o f spinal and vagal-sensory afferents which are e n d o w e d with multiple opioid receptors (cf. ref. 19). A n open question remains, whether laryngeal opioid peptides exert a postsynaptic effect. Since peripherally acting opioids are beneficial in respiratory distress (e.g. coughing) [4] we feel tempted to deduce that appropriately designed opioids might be o f therapeutic value in laryngeal pathophysiology. Parts o f this study have been reported at the N e u r o a n a t o m y and N e u r o b i o l o g y S y m p o s i u m N a n c y , 25. / 26. 09. 1989 and are contained in the thesis o f J o a c h i m Krekel, w h o received a fellowship f r o m the J o h a n n e s Gutenberg-University, M a i n z (Titel 681 02). The study was supported by the Deutsche Forschungsgemeinschaft ( D F G G r a n t We 910/1-2 and 2-2) and by Naturwissenschaftliches Medizinisches F o r s c h u n g s z e n t r u m ( N M F Z ) o f the University o f Mainz. 1 Barnes, P.J., Regulatory peptides in the respiratory system, Experientia, 43 (1987) 832-839. 2 Dahlqvist, A. and Forsgren, S., Networks of peptide-containing nerve fibres in laryngeal nerve paraganglia. An immunohistochemical study, Acta Otolaryngol., 107 (1989) 289-295. 3 Dey, R.D., Hoffpauir, J. and Said, S.I., Co-localization of vasoactive intestinal peptide- and substance P-containing nerves in cat bronchi, Neuroscience, 24, 1 (1988) 275-281. 4 Fuller, R.W., Karlsson, J.A., Choudry, N.B. and Pride, N.B., Effect of inhaled and systemic opiates on responses to inhaled capsaicin in humans, J. Appl. Physiol., 65 (1988) 1125-1130.
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