Localization of enkephalin-like immunoreactivity in the cat carotid and aortic body chemoreceptors.код для вставкиСкачать
THE ANATOMICAL RECORD 203:405-410 (1982) Localization of Enkephalin-likelmmunoreactivity in the Cat Carotid and Aortic Body Chemoreceptors JOHN T. HANSEN. JAMES BROKAW. DOUGLAS CHRISTIE, A N D MICHAL KARASEK Department of Anatomy, The University of Texas Health Science Center at San Antonio, San Antonio, T X 78284 ABSTRACT The purpose of this study was to determine if enkephalin-like immunoreactivity was present in the glomus cells of the carotid and aortic body peripheral arterial chemoreceptors. Cat carotid and aortic bodies were reacted with antisera to met- and leu-enkephalin using the indirect peroxidase-antiperoxidase immunocytochemical method of Sternberger (1979). Both the carotid and aortic bodies demonstrated clusters of immunoreactive cells for both met- and leuenkephalin. Additionally, met-enkephalin-like immunoreactivity was observed in many of the dense-core vesicles of the glomus cells of the carotid body. The glomus cells of these chemoreceptors are known to contain catecholamines which may modulate chemoreceptor activity. The presence of opioid peptide-like substances co-existing with the glomus cell catecholamines, perhaps in the same vesicles, may have important implications for a trophic influence of these peptides on glomus cell chemoreceptor modulation. The carotid and aortic bodies comprise several groups of specialized epithelioid cells which are known t o function as arterial chemoreceptors. These chemoreceptors are sensitive to changes in the PO>,pCO,, and pH of the arterial blood and form an important component in a reflex arc which initiates appropriate pulmonary and cardiovascular responses (Comroe, 1974). Both the carotid and aortic bodies contain an abundance of catecholamines (CAs),especially dopamine (DA)and norepinephrine (NE)(Hellstrom and Koslow, 1975; Hansen and Yates, 1975; Mills et al., 1978; Hansen and Christie, 1981).These CAs are stored in dense-core vesicles (Chen and Yates, 1969)which are the most striking characteristic of the major parenchymal cell type, the glomus (Type I) cell. Glomus cell CAs are thought to be involved in the initiation and/or modulation of the chemoreceptor response by acting either transsynaptically or in a paracrine fashion on adjacent glomus cells and afferent nerve endings (Osborne and Butler, 1975; McDonald and Mitchell, 1975; Krammer, 1978). In addition to CAs, recent immunocytochemical studies have demonstrated that enkephalin-like peptides are present in the cells of the carotid body (Lundberg et al., 1979; Wharton et al., 1980). This study was initiated 1)to confirm these previous results, 2) to determine where in the carotid body enkephalin-like 0003-276X/82/2033-0405$02.00- 1982 ALAN R. LISS. INC. immunoreactivity was localized, and 3) to determine if enkephalin-like immunoreactivity was present in the aortic body chemoreceptors as well. MATERIALS AND METHODS Light Microscopic Localization Four young adult female cats weighing 2-3 kg were anesthetized with sodium pentobarbital (Nembutal, 25 mgikg iv) and then sacrificed by an overdose just prior to removal of the carotid and aortic bodies. Following removal of the tissues, one half of the carotid body and all of the aortic body tissues were immersed in formol sublimate for 4 hours at room temperature. The tissue then was washed overnight in phosphate-buffered saline (PBS)at pH 7.2 and paraffin embedded. Five micron serial sections were collected on chrome-alum-coated slides, deparafinized, and stored in a freezer until processing for immunocytochemistry. A modification of the indirect immunocytochemical method of Sternberger (1979) was utilized. Staining of the tissue sections was done by placing the slides in a humid chamber with a moistened mat and applying the reagents directly onto the sections. Initially, sections were rinsed for 10 minutes in PBS and then incubated for 30 minutes in 4% normal ~~ - Received October 8 1981 accepted March 15 1982 406 J.T. H A N S E N ET AI, goat serum (Cappel Laboratories, Cochranville, PA) in PBS containing 0.3% Triton XlOO (PBS-Tx).Following this incubation, the sections were washed in PBS and then incubated in the presence of rabbit anti-met- or leu-enkephalin (Immuno Nuclear Corporation, Stillwater, MN) diluted 1:lOOO with PBS-Tx for 18 hours at 4°C. The sections then were washed in PBS and incubated for 30 minutes with goat antiserum to rabbit IgG (Cappel Laboratories, Cochranville, PA) diluted 1:20 with PBS-Tx. After another PBS wash, the sections were incubated in peroxidase-antiperoxidase diluted 1:40 with PBS-Tx for 30 minutes. Following a PBS wash, the sections were incubated in a solution of diaminobenzidine (DAB) containing H,O, (38 mg DAB in 50 ml of 0.05 M Tris buffer at pH 7.6 and 35 p1 of 30% H,O,) for 3 minutes. After a final PBS wash, the slides were dried, coverslipped, and viewed in the light microscope. Control incubations were performed by replacing the primary antisera with normal rabbit serum N YO) or by incubation in primary antisera which was preabsorbed to synthetic enkephalin prior to tissue incubation. The primary antisera were produced in a rabbit with glutaraldehyde bovine thyroglobulin conjugate. Met-enkephalin had a crossreactivity of 1.6% with leu-enkephalin and less than 0.002% with substance P and &endorphin.Leuenkephalin had a crossreactivity of 29% with met-enkephalin and less than 0.002% with substance P, @-endorphin,and somatostatin. Ultras tructural Localization One half of the carotid body tissue was immersed in phosphate-buffered 4% paraformaldehyde, 0.5% glutaraldehyde for 4 hours a t room temperature. The tissue then was washed overnight in PBS at pH 7.2 and cut into very thin slices with a razor blade. Staining of the tissue sections was performed in a small vial attached to a tissue-infiltrating rotor. Initially, sections were rinsed for 10 minutes in PBS and then incubated for 2 hours in 4% normal goat serum in PBS. Following this incubation, the tissue slices were washed in PBS and then incubated in the presence of rabbit anti-met-enkephalin diluted 1:500 with PBS for 18 hours at 4°C. The tissue then was washed in PBS and incubated for 30 minutes with goat antiserum to rabbit IgG diluted 1 2 0 with PBS. After another PBS wash, the tissue was incubated in peroxidase-antiperoxidase diluted 1:40 with PBS for 30 minutes. Following a PBS wash, the tissue was incubated in a solu- tion of DAB containing H,O, for 5 minutes. After a final PBS wash, the tissue was processed for electron microscopy by post-fixing the tissue for 20 minutes in 1.5% osmium tetroxide in PBS. The tissue then was dehydrated in a graded series of ethanols, passed through propylene oxide, and embedded in Epon-Araldite. Thin sections were collected on copper grids, either unstained or lightly stained with lead citrate, and examined in the electron microscope. Control incubations were performed by replacing the primary antisera with antisera which was preabsorbed to synthetic met-enkephalin (SigmaChemical Company, St. Louis, MO) prior to tissue incubation. RESULTS Following incubation in both met- and leuenkephalin antisera, a positive enkephalin-like immunoreactivity was observed in the glomus cells of the carotid body (Figs. 12).Immunoreactive cells often were organized in clusters which were scattered throughout the carotid body. The peroxidase-antiperoxidase reaction product was distributed throughout the glomus cell cytoplasm, thereby circumscribing the centrally located and unstained nucleus (Fig. 2 ) . A larger number of immunoreactive cell clusters were observed following metenkephalin incubation compared to leuenkephalin incubation. Glomus cells of the aortic body chemoreceptors also exhibited both met- and leu-enkephalin-likeimmunoreactivity (Figs. 3,4). Both the subclavian glomera (aortic body accumulations adjacent to the origins of the subclavian arteries) (Fig. 3) and aorticopulmonary glomera (accumulations adjacent to the pulmonary arterial trunk and aortic arch) (Fig. 4) possessed immunoreactive cells. None of the immunoreactive cells observed in the carotid and aortic bodies following incubation in primary antisera were observed in sections incubated in normal rabbit serum or preabsorbed antisera. Since met-enkephalin-likeimmunoreactivity was greatest in the carotid body, we attempted to localize met-enkephalinat the ultrastructural level. Peroxidase-antiperoxidase reaction product was observed in many of the cytoplasmic dense-core vesicles of the glomus cells (Figs. 5.6). However, not all of the dense-core vesicles were stained. Immunostaining was absent in other glomus cell organelles including the Golgi apparatus, endoplasmic reticulum, and the small (40-50 nm) electron-lucent vesicles (Fig. ENKEPHALINS, CAT CAROTID, AND AORTIC BODIES 407 P Fig. 1. Clusters of immunoreactive carotid body glomus cells following Incubation with met-enkephalin antiserum. x 210. Fig. 3. A small cluster of immunoreactive aortic (subclavian) body glomus cells following incubation with metenkephalin antiserum. x 525. Fig. 2. Several carotid body glomus cells demonstrating leu-enkephalin-like immunoreactivity. Note the peroxidaseantiperoxidase reaction product in the cytoplasm surrounding the central unstained nucleus. x 840. Fig. 4. Met-enkephalin-like immunoreactive glomus cells in the aorticopulmonary glomera. x 210. 408 J.T. HANSEN ET AL. Pig. 5. Ultrastructural localization of met-enkephalinlike imrnunoreactivity in some of the densecore vesicles of the carotid body glomus cells (arrows). Reaction product is absent in the smaller clear-core vesicles (arrowheads). the Golgi, adjacent nerve fibers (*), and processes of the sup .. ,, ,-_. .- ^^^ Fig. 6. Higher power view of the met-enkephalin-like immunoreactivity observed in the dense-core vesicles of the glomus cells. M, mitochondrion. x 84,000. ENKEPHALINS. CAT CAROTID. AND AOH'I'IC RODlES 5). Likewise, no reaction product was observed in adjacent nerve endings or in the supporting (Type 11)cells (Fig. 5). None of the immunoreactive sites observed in the dense-core vesicles of the carotid body glomus cells following incubation in met-enkephalin antisera were observed in tissue incubated in the preabsorbed antisera. DISCUSSION The results of this study confirm the findings of Lundberg et al. (1979) and Wharton et al. (1980) with regard to the presence of enkephalin-like immunoreactivity in the glomus cells of the cat carotid body. Additionally, the results of this study demonstrate for the first time that met- and leu-enkephalin-like immunoreactivity also exists in the glomus cells of the cat aortic body chemoreceptors. Finally, and perhaps most importantly, we have d e m onstrated met-enkephalin-like immunoreactivity in the dense-core vesicles of the carotid body glomus cells. These dense-core vesicles have been shown to contain the CAs which are present in the glomus cells (Chen and Yates, 1969). Therefore, a t least some of these CAcontaining dense-core vesicles also may contain an opioid peptide-like substance which coexists with the glomus cell CAs. Functionally, little is known about the role of opioid peptides in the glomus cells of the peripheral arterial chemoreceptors. However, opiates do depress respiration (Florez and Mediavilla, 1977) and both met- and leu-enkephalin decrease the spontaneous chemoreceptor discharge frequency in the carotid sinus nerve (McQueen and Ribeiro, 1981).The intensity and duration of the depression evoked by the enkephalins is reduced following naloxone, suggesting that these peptides act at opiate receptors in the carotid body (McQueen and Ribeiro, 1981). Moreover, opioid peptides affect the release of classical neurotransmitters (Eidelberg, 1976). In this regard, it is interesting that we have demonstrated the presence of met-enkephalin-like immunoreactivity in the same dense-core vesicles which are thought to contain CAs. The co-existence of CAs and the opioid-like peptides have been reported in the adrenal medulla, with the highest specific activity in the purified adrenomedullary chromaffin granule fraction (Viveros et al., 1979). Opioid peptides and NE also have been identified biochemically in sympathetic nerves (Wilson et d., 1980) and these investigators suggest that opiate-like peptides and NE probably co-exist in the large dense-core vesicles of the 409 nerve. Other paraneurons (Fujita and Kobayashi, 1979), such as the small intensely fluorescent (SIF) cells of autonomic ganglia, also contain opioid immunoreactivity in addition to a classical neurotransmitter such as a CA or acetylcholine (Hokfelt et al., 1980). Therefore, our demonstration of met-enkephalin-like immunoreactivity in dense-core vesicles which probably contain CAs strongly supports the hypothesis that this peptide may function with the CA as a co-transmitter. Perhaps the association of CAs and peptides in glomus cells is involved in the hypothesized modulatory role of these substances during chemoreception. For example, it has been suggested that peptides may act as co-transmitters while the amine acts as a primary transmitter (Livett et al., 1981). A co-transmitter could function as a trophic factor for the receptor of the primary transmitter, thereby modulating the sensitivity of the receptor (Costaet al., 1981).Certainly, further investigation into this intriguing possibility warrants consideration. ACKNOWLEDGMENTS The authors wish to thank Ms. Gwynne Duke for her excellent technical assistance and Ms. Nora Dimas for typing the manuscript. Dr. Hansen also wishes to thank Dr. T.H. Williams, The University of Iowa College of Medicine, for graciously permitting him to learn immunocytochemistry in his laboratory and Dr. D.C. Herbert for his helpful discussion. Dr. Karaseks permanent address is the Laboratory of Electron Microscopy, Department of Pathological Anatomy, Institute of Pathology, Medical Academy, Lodz, Poland. This study was supported by NIH grant H L 25508. Dr. Hansen is the recipient of a NIH Research Career Development Award KO4 H L 00680. LITERATURE C I TED Chen, I-Li, and K.D. Yates 11969) Electron microscopic radioatographic studies of the carotid body following injections of labeled biogenic amine precursors. J. Cell Biol., 42794-803. Comroe. J.H. 11974) Physiology of Respiration. Year Book Medical Publishers, Chicago, pp. 33-69. Costa, E.. A. Guidotti, I. Hanbauer. T. Hexum, I,. Saini. S. Stine. and H.-Y.T. Yang(1981) Regulationof acetylcholine receptors by endogenous co-transmitters: Studies of adrenal medulla. Fed. Proc.. 40160-165. Kidelberg, E. 11976) Possible actions of opiates upon synapses. Prog. Neurobiol., 6:81-102. Florez. J.. and A. 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