Neuropeptide tyrosine (NPY)-like immunoreactivity in adrenal chromaffin cells and intraadrenal nerve fibers of rats.код для вставкиСкачать
THE ANATOMICAL RECORD 214:321-328 (1986) Neuropeptide Tyrosine (NPY)-Like lmmunoreactivity in Adrenal Chromaffin Cells and Intraadrenal Nerve Fibers of Rats HIROFUMI KURAMOTO, HISATAKE KONDO, AND TSUNEO FUJITA Department of Anatomy, School of Medicine, Niigata University, Asahimachi 1, Niigata 951, Japan ABSTRACT The present peroxidase-antiperoxidase imniunohistochemical study demonstrated that approximately 50%of the total chromaffin cells of the rat adrenal medulla exhibited NPY-like immunoreactivity. The immunoreactive material was localized in the core of the chromaffin granules as well as diffusely in the cytoplasm. By combination of immunohistochemistry with noradrenaline-fluorescence microscopy, all NPY-immunoreactive chromaffin cells are nonfluorescent, indicating that all NPY-chromaffin cells co-store adrenaline. A comparison of two consecutive sections, each of which was processed for the immunostaining with anti-NPY and antiMet-Enk-Arg-Gly-Leu antisera, respectively, indicated that NPY and preproenkephalin A and its derivatives coexist in approximately one-fifth of the total NPYimmunoreactive cells. In addition to the NPY-immunoreactive cells, a plexus of NPY-immunoreactive nerve fibers with varicosities was found in the subcapsular regions of the adrenal gland. The nerve fibers were often associated with small blood vessels and extended into the zona glomerulosa. Single NPY-immunoreactive fibers were sparsely distributed in the deeper regions of the cortex and in the medulla. Ganglion cells in the adrenal gland were not seen exhibiting intensely positive NPY-like immunoreactivity. The NPY-immunoreactive nerve fibers contained abundant small clear vesicles mixed with a few small and large granular vesicles. The immunoreactive material appeared on the granular cores as well as in the axoplasm. The NPY fibers were closely apposed to smooth muscle cells and pericytes of small blood vessels in the cortex. They were sometimes seen in close apposition to the fenestrated endothelial cells with a common basal lamina intervening. The NPY fibers also made synaptic contacts with both cortical and chromafin cells. The occurrence of several neuropeptides such as enkephalin (Kobayashi et al., 1983; Kondo et al., 1984; Livett et al., 1982; Schultzberg et al., 19781, substance P (Kuramoto et al., 1983, vasoactive intestinal polypeptide (VIP) (Holzwarth, 1984; Kondo et al., 19861, and neurotensin (Lundberg et al., 1982a) has been demonstrated in the adrenal chromaffin cells of various mammals in which catecholamines had been thought to be the dominant, if not exclusive, biologically active substances. Possible functional significances of these peptides have been proposed in several ways in relation to the secretion of catecholamines Civett et al., 1983; Schultzberg et al., 1978). In addition, a recent study has revealed the occurrence of neuropeptide tyrosine (NPY) (Tatemoto, 1982; Tatemoto et al., 1982)-like immunoreactivity in the chromaffin cells and nerve fibers of the adrenal gland of several mammals (Varndell et al., 1984). In the peripheral nervous system, NPY has been shown to be a potent vasoconstrictor and to inhibit pancreatic secretion (Lundberg and Tatemoto, 1982; Lundberg et al., 1982b). In order to understand the functional significance of NPY in the fulfillment of the adrenal hormone 0 1986 ALAN R. LISS, INC secretion, several morphological questions should be answered: Do NPY-like immunoreactive cells co-store noradrenaline or adrenaline? Do NPY cells co-store other peptides whose presence in the medulla is known? Where is NPY localized in the chromafin cells? What are the ultrastructural characteristics of NPY-immunoreactive nerve fibers, and what types of cells do the NPY fibers make synaptic contacts with? In the immunocytochemical study of Varndell et al. (1984), NPY was colocalized with noradrenaline and enkephalin in adrenal chromaffin cells, but the description was brief and no details concerning the fine structure of NPY nerve fibers were presented. In a series of recent papers detailing the localization of peptides in the rat adrenal gland, we have reported the ultrastructure of enkephalin-, substance P-, and VIPlike immunoreactive cells and nerve fibers (Kondo et al., 1984; Kondo et al., 1986; Kuramoto et al., 1985). The present study represents the fourth in this series and Received July 15, 1985; accepted October 18, 1985. 322 H. KURAMOTO, H. KONDO. AND T. FUJITA was undertaken to clarify the morphological characteristics of NPY-like immunoreactive cellular and neuronal elements and to answer those questions listed above. MATERIALS AND METHODS Five male adult rats weighing 180-200 gm were used in the present study. Under Nembutal(35 mgkg) anesthesia, the animals were perfused first with 200 ml of physiological saline through the heart, followed by 200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. The adrenal glands were rapidly removed and immersed in the same fixative for a n additional 2 hours. The tissues, after rinsing in 0.1 M phosphate buffer for 1hour, were immersed overnight in the phosphate buffer containing 30%sucrose; they were frozen in liquid nitrogen and 15-ym-thick sections were made on a cryostat. The sections were incubated for 12 hours a t room temperature with rabbit anti-NPY antiserum (Amersham International PLC, England) at a dilution of 1:1,600. The sites of antigen-antibody reaction were visualized by the peroxidase-antiperoxidase (PAP) method according to Sternberger (1979). For electron microscopy, the sections were further postfixed with 1% osmic acid in 0.1 M cacodylate buffer, pH 7.4, for 20 minutes, after completion of the PAP procedure. They were embedded in Epon 812 according to conventional procedure and ultrathin sections were examined after brief staining with uranyl acetate. For examination of the colocalization of NPY with enkephalin or catecholamine, sections were mounted on glass slides, covered with glycerin and subsequently viewed and photographed with a fluorescence microscope (Leitz Orthoplan) equipped with appropriate filter set. It is well established that fixation with 4% paraformaldehyde is adequate for demonstrating noradrenaline fluorescence in the adrenal medulla (Eranko, 1967; Falck and Torp, 1961). After the fluorescent cells were photographed, the sections were incubated with the antiNPY antiserum or with the rabbit anti-methionine-enkephaline-arginine-glycine-leucine(Met-Enk-Arg-GlyLeu) antiserum (N. Yanaihara, Shizuoka, Japan) at a dilution 1:3,000. It is generally agreed that the occurrence of Met-Enk-Arg-Gly-Leu-like immunoreactivity strongly suggests the presence of preproenkephalin A as a precursor, and of its derivatives, including Met- and Leu-Enk (Kilpatrick et al., 1981;Noda et al., 1982). For quantitative analyses of the coexistence of NPY and Met-Enk-Arg-Gly-Leu, ten sets of two consecutive sections with a thickness of 2.5 pm at intervals of 25 pm were made from the adrenal medulla on a cryostat. One of the consecutive sections in each set was immunostained with the NPY antiserum, and the other with Met-Enk-Arg-Gly-Leu antiserum. All NPY-immunoreactive cells containing nuclear rofiles in a square field with a size of 0.28 x 0.44 mm i?were counted first and cells in a n adjacent section which corresponded to the NPY-immunoreactive cells and simultaneously showed Met-Enk-Arg-Gly-Leu-likeimmunoreactivity were subsequently counted. The immunoreactive cells appeared in small groups or singly and were distributed in the medulla without any topographical relations to sinusoids or to the boundary region between the cortex and the medulla (Figs. 1, 3). The cells were polygonal in shape and lacked cytoplasmic processes. The intensity of the immunoreaction varied from cell to cell and the immunoreactive material within the cytoplasm appeared granular. All nuclei were free of immunoreaction. In fluorescence microscopy, approximately one-fifth of the total chromaffin cells emitted blue-white fluorescence specific for noradrenaline. The fluorescing noradrenaline cells occurred in small groups or singly and were distributed throughout the adrenal medulla (Fig. 2). After processing the same sections for immunohistochemistry, none of the fluorescent noradrenaline cells were immunoreactive for NPY (Figs. 1, 2). By comparing the two consecutive sections, each of which was processed for the immunostaining with antiNPY and anti-Met-Enk-Arg-Gly-Leuantiserum, respectively, approximately one-fifth (16.1 & 6.9%, n = 10) of the total NPY-immunoreactive chromaffin cells exhibited positive immunoreactivity to the Met-Enk-Arg-GlyLeu antiserum simultaneously (Fig. 4). In immuno-electron microscopy of the NPY-positive chromaffin cells, many of the cytoplasmic granules had rounded cores with high electron density due to the immunoreaction (Fig. 6). The intensity of the cellular immunoreaction in light microscopy depended on the number of the highly electron-dense granules in the cytoplasm. The cytoplasm of the cells showed a coarse appearance with moderate increase in electron density. NPY-like lrnrnunoreactivity in lntraadrenal Nerve Fibers A distinct plexus of NPY-like immunoreactive nerve fibers with varicosities encircled small blood vessels which penetrated the capsule and coursed in the subcapsular regions of the adrenal gland (Fig. 5). The immunoreactive nerve fibers extended into the zona glomerulosa where they surrounded blood vessels and cortical cells. Single varicose nerve fibers with the NPYlike immunoreactivity were distributed sparsely in the zona fasciculata and reticularis of the cortex and in the medulla. Ganglion cells exhibiting intensely positive NPY-like immunoreactivity were not seen in the medulla or cortex. With immuno-electron microscopy the NPY-positive nerve fibers were identified as electron-dense profiles, 0.3-0.8 pm in diameter, and enclosed by thin processes of Schwann cells (Figs. 7-12). The immunoreactive nerve fibers contained abundant small clear vesicles, 45-50 nm in diameter, mixed with a few small granular vesicles of similar size and with larger granular vesicles, 90-100 nm in diameter. The immunoreactive material was localized in the core of both small and large granular vesicles, and also in the axoplasm surrounding vesicles and mitochondria. However, the interior of the small clear vesicles was free of the immunoreaction. The NPY-immunoreactive nerve fibers often were closely apposed to smooth muscle cells and pericytes of small blood vessels in the cortex: a n interstitial space of RESULTS 100-200 nm intervened between the components (Fig. NPY-like Immunoreactivity in the Chromaffin Cells 9). The apposed surface of the nerve fibers was largely Approximately 50% of all chromaffin cells in the ad- denuded of Schwann cell sheaths. Denuded nerve fibers renal medulla exhibited NPY-like immunoreactivity. with NPY-like immunoreactivity sometimes were in NPY-LIKE IMMUNOREACTIVITY 323 Figs. 3 , 4 . Immuno-light micrographs showing NPY-immunoreactive Figs. 1 , 2. Immuno-light micrograph showing NPY-immunoreactive chromaffin cells (Fig. 1) and fluorescence micrograph showing fluores- chromaffin cells (Fig. 3) and Met-Enk-Arg-Gly-Leu-immunoreactive cent noradrenaline cells (Fig. 2) on the same section of the rat adrenal chromaffin cells (Fig. 4) on two consecutive sections of the rat adrenal medulla. Note all NPY-immunoreactivechromaffin cells (*) are nonflu- medulla. Note some chromaffin cells (*I immunoreactive both for the two antisera. x750. orescent. V: sinusoidal blood vessels. ~ 3 1 0 . H. KURAMOTO, H. KONDO, AND T. FUJITA 324 Fig. 5. Immuno-light micrograph showing NPY-immunoreactive nerve fibers with varicosities in the subcapsular region (C), the zona glomerulosa (G),and fasciculate (F) of the rat adrenal cortex. Note the close association of the immunoreactive nerve fibers with small blood vessels (V). ~ 2 8 0 . close apposition to the fenestrated endothelial cells of cortical capillaries. The nerve fibers and endothelium were separated by a 70-nm space, and a common basal lamina intervened (Fig. 10). Immunoreactive nerve fibers were directly apposed to both cortical and chromaffin cells, but were separated by a n intercellular space approximately 20 nm in width (Figs. 7, 8). Membrane specializations were not found at the apposition site. On several occasions, NPY-immunoreactive and nonreactive nerve fibers, or two NPY-immunoreactive nerve fibers, were seen to be in direct contact with each other, and simultaneously with cortical cells. At the contact sites between two immunoreactive nerve fibers, a membrane density was seen on one of the apposed membranes (Figs. 11,12). As controls for immunohistochemistry, sections of the adrenal medulla were incubated with anti-NPY and anti-Met-Enk-Arg-Gly-Leu antisera preabsorbed with NPY and Met-Enk-Arg-Gly-Leu, respectively (10 pgiml diluted at 1:1,600 and 1:3,000, respectively). Immunoreactive cells or fibers were not recognized in any portions of the adrenal gland. fined to the chromaffin granules of the immunoreactive cells. In previous studies, Met-Enk-Arg-Gly-Leu-likeimmunoreactivity also has been demonstrated to be localized within the chromaffin granules (Kobayashi et al., 1983; Kondo et al., 1984). Furthermore, the co-storage of serotonin in adrenaline cells recently has been demonstrated immunohistochemically (Verhofstad and Jonsson, 1983).These findings suggest that the four bioactive substances coexist in a substantial number of chromaffin cells, possibly in chromaffin granules. The coexistence of various combinations of bioactive substances in the adrenal chromaffin cells suggests the presence of several subpopulations of chromaffin cells in terms of bioactive substances contained, in addition to the classical adrenaline and noradrenaline cells. The present finding that NPY coexists with adrenaline in adrenal chromaffin cells of rats is in contradiction to the previous study of the adrenal medulla of horses by Varndell et al. (1984). They identified the NPY-immunoreactive cells as noradrenaline-chromaffin cells based on the fine structural differences in the chromaffin granules. According to the fine structural criteria for the identification of chromaffin granules in specimens fixed with glutaraldehyde and osmium tetroxide, noradrenaline granules are characterized by solid cores of high electron density which often are irregular in shape and located eccentrically. Adrenaline granules, on the other hand, have rounded cores with moderate electron density (Coupland et al., 1964). However, in the immuno-electron microscopic study by DISCUSSION By means of immunohistochemistry combined with fluorescent microscopy, the present study has demonstrated that all NPY-immunoreactive chromaffin cells also are adrenaline-containing cells, and that some of the NPY-chromaffin cells co-store preproenkephalin A and its derivatives. NPY -like immunoreactivity is con- NPY-LIKE IMMUNOREACTIVITY Fig. 6. Immuno-electron micrograph showing a NPY-immunoreactive chromafin cell (*) next to a nonimmunoreactive cell (Cn). Note the increase in electron density of most chromafin granules and the cytoplasm of the immunoreactive cell due to the immunoreaction. x23.000. Varndell et al. (19841, the specimens were fixed only with aldehydes and, therefore, the resulting images did not show any difference in electron density of two types of granules. Nevertheless, those investigators did report “noradrenaline granules” with irregularly shaped cores in the horse adrenal medulla. The identification of the 325 Figs. 7, 8. Fine structures of the NPY-immunoreactive nerve fibers synaptic contacts (dots) with nonimmunorective chromafin cells (Cn). Note small (short arrows) and large (long arrows) granular vesicles intermingled with numerous small clear vesicles in the immunoreactive nerve fibers. ~ 2 3 , 0 0 0 . (*) in two cell types in that study, however, seems less reliable than the noradrenaline fluorescence method employed in the present study. This is one of the plausible explanations for the discrepancy. An alternative explanation for the discrepancy is the species difference between rats and horses. It is well 326 H. KURAMOTO, H. KONDO, AND T. FUJITA Figs. 9, 10. Fine structure of the NPY-immunoreactive nerve fibers apposition t o pericyte (P)and smooth muscle cell (S)(Fig. 9) (*) in close and fenestrated endothelial cell (E) (Fig. 10) in the subcapsular region of the rat adrenal cortex, n: nonimmunoreactive nerve fiber, Arrows indicate endothelial fenestrae. x 23,000. Figs. 11, 12. A NPY-imrnunoreactive nerve fiber (*) and nonimrnunoreactive fibers (n) (Fig. 11) or two adjacent NPY imrnunoreactive fibers (*) (Fig. 12) in direct contact (arrows)with each other and simultaneously with cortica! cells (Co) containing lipid droplets 6). Note the membrane densification (double arrows) on one of the apposed plasma membrane. x23.000. 327 NPY-LIKE IMMUNOREACTIVITY known that the proportion of noradrenaline versus adrenaline-chromaffin cells and the occurrence of chromaffin cells containing a given peptide in the adrenal medulla varies in different species (Coupland, 1965; Schultzberg et a]., 1978). It is not unlikely that the combination of catecholamine and peptides co-stored in chromaffin cells might differ in different species. Further examinations of the adrenal medulla of various species by combined immunohistochemistry with fluorescence microscopy would seem necessary to understand more clearly the coexistence of NPY and catecholamines. Since no ganglion cells with intense NPY-like immunoreactivity were found within the adrenal gland, the intraadrenal NPY-immunoreactive nerve fibers are regarded as extrinsic in origin. A considerable number of postganglionic neurons in the celiac ganglion and a small population of neurons in the myenteric and submucous ganglion are known to exhibit intense NPY-like immunoreactivity (Furness et al., 1983; Lundberg et al., 1985). The neuronal connection between the adrenal gland and the celiac ganglion is well established, and serves as a route for nerve fibers to pass into and innervate the gland (Pick, 1970). In addition, conventional electron microscopy combined with histochemical techniques has demonstrated the presence of some postganglionic noradrenergic nerve fibers in the adrenal medulla (Prentice and Wood, 1975). It is, therefore, reasonable to assume that the intraadrenal NPY-immunoreactive nerve fibers are postganglionic, and that their cell somas are located in the celiac ganglion. In support of this assumption, the composition of vesicles in the NPY fibers is quite similar to that of the postganglionic noradrenergic nerve fibers; that is, the presence of small and large granular vesicle's mixed with abundant small clear vesicles. It is well established that noradrenaline is stored in the small and large granular vesicles of the postganglionic sympathetic nerve fibers (Fried, 1980; Smith, 1972). The occurrence of the immunoreactive material in small as well as large granular vesicles of the NPY fibers suggests the coexistence of NPY with noradrenaline in the vesicular compartments of the autonomic nerve fibers. This is in marked contrast to the coexistence of NPY with adrenaline in the chromaffin cells, which is homologous to the postganglionic neurons in ontogeny. The close apposition between NPY fibers and vascular smooth muscles suggests that NPY may exert its vasomotor effect as a cotransmitter with noradrenaline. Local intraarterial infusion of NPY is known to induce a dose-dependent vasoconstriction in the cat submandibular gland (Lundberg and Tatemoto, 1982; Lundberg et al., 198213)and a similar effect might be expected in the intraadrenal blood vessels. The close apposition between NPY fibers and cortical fenestrated endothelial cells, with a common basal lamina, is quite similar to the topographical relation of the neurohypophyseal axon terminals and fenestrated capillaries (Bloom and Fawcett, 1975).This similarity infers that NPY fibers in the adrenal cortex may exert their effect by way of endocrine secretion. The present study further disclosed the direct apposition of NPY fibers to certain cortical and medullary chromaffin cells. The direct apposition is functionally regarded as a synapse, i.e., the site where synaptic re- lease and regulation take place, although morphologically, it lacks such membrane specializations as seen in typical synapse in the central nervous system (Pappas and Waxman, 1972; Unsicker, 1971). Based upon the present findings, it might be possible to suggest several ways of action of NPY in the adrenal gland, i.e., 1) by being secreted from NPY-chromaffin cells and influencing the secretory activity of adjacent chromaffin cells via paracrine or hemocrine action, 2 ) by being released from the NPY nerve fibers and exerting its effect on postsynaptic chromaffin cells and cortical cells through synaptic and endocrine action, and 3) by being released from the NPY fibers and influencing the vascular smooth muscle cells to regulate the intraadrenal circulation. In addition, from the finding on the coexistence of more than two bioactive substances such as NPY and catecholamines in chromaffin cells and nerve fibers, it is also necessary to consider its own effect on NPY-secreting cells and nerves themselves by autoregulation. Physiological and pharmacological analyses of the effect of NPY in the secretory activity of the adrenal gland are necessary to examine each possible way of action of NPY proposed above and those analyses would be crucial to understand more precisely the nature of the adrenal gland. ACKNOWLEDGMENTS The authors wish to thank Dr. N. Yanaihara for a kind gift of anti-Met-Enk-Arg-Gly-Leuantiserum. They also acknowledge the technical assistance and secretarial help of Mr. M. Takeda and Miss H. Kato. LITERATURE CITED Bloom, W., and D.W. Fawcett (1975) A Textbook of Histology, 10th Edition. W.B. Saunders Co., Philadelphia. Coupland, R.E. (1965) Natural History of the Chromaffin Cell. Longmans, London. Coupland, R.E., A.S. 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