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Ultrastructural localization of nerve terminals containing nitric oxide synthase in rat adrenal gland
ELSEVIER
Neuroscience Letters 204 (1996) 153-156
HtOROSCItHC[ I[IT[RS
Ultrastructural localization of nerve terminals containing nitric oxide synthase in rat adrenal gland Koichi Tanaka*, Tanemichi Chiba The Third Department of Anatomy, Chiba University School of'Medicine, 1-8-1 lnohana, Cyuo-Ku, Chiba 260, Japan
Received 8 December 1995; revised version received 28 December 1995; accepted 28 December 1995
Abstract
Immunoelectron microscopy was performed to localize immunoreactivity for nitric oxide synthase (NOS) in rat adrenal gland. NOS-immunoreactive (NOS-ir) nerve fibers were observed around blood vessels and endocrine cells in the zona glomerulosa of the cortex. Electron microscopy showed that NOS-ir axon varicosities were in close contact with the smooth muscles of blood vessels and with the cytolemma of endocrine cells. In the adrenal medulla, several NOS-ir ganglion cells were found. Synaptic contacts between non-immunoreactive axons and dendrites of NOS-ir neurons were observed. NOS-ir nerve fibers were distributed among chromaffin cells. Positive axon varicosities were in close contact with the catecholamine-storing chromaffin cells. These results suggest that NOSir nerve cells control directly the secretion of corticosteroid and catecholamine in addition to the vascular tone.
Keywords: Nitric oxide synthase; Adrenal gland; Innervation; Rat; Immunohistochemistry; Chromaffin cells; Adrenal cortex
Nitric oxide (NO) is a free radical gas that has attracted great interest as a novel signaling molecule in the nervous system [6,10]. In blood vessels, NO mediates endothelium-derived relaxing factor activity, and when produced by macrophages, neutrophils, and microglial cells, it mediates cytotoxic effects [14,15]. NO is formed from Larginine by the action of nitric oxide synthase (NOS), and the presence o f this enzyme and its m R N A has been used to identify cells that synthesize NO [6]. In addition, there is evidence that N A D P H - d i a p h o r a s e activity can be used as a marker for N O S in many cell types [9,11 ]. The presence o f NOS has also been proved in the nervous elements of the adrenal gland [2-5]. In the rat, NOS-immunoreactive (NOS-ir) nerve fibers were observed among the adrenal chromaffin cells in the medulla, and in the zona glomerulosa o f the cortex, as well as near the intra-adrenal blood vessels [2,5]. However, these studies did not clarify whether NOS-ir nerve fibers exert influences directly on chromaffin and cortical cells or indirectly through neural control of blood vessels. The present study was performed to observe whether NOS-ir
* Corresponding author. Tel.: +81 43 2262024; fax: +81 43 2262025;
e-mail: [email protected].
axons have direct contacts with these endocrine cells in the adrenal gland. Studies were carried out on three male S p r a g u e Dawley rats weighing 3 0 0 - 3 5 0 g . The animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg), perfused via the ascending aorta with 20 ml aqueous 0.9% NaCI, and then fixed by perfusion with 500 ml of fixative composed of 4.0% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Adrenal glands were postfixed with 4.0% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 1 h before being sectioned at a thickness of 5 0 f f m with a Vibratome. The sections were immersed in 0.1 M phosphate buffered saline (PBS) containing 20% succharose for 1 h and freeze-thawed with liquid nitrogen. Pre-embedding immunohistochemistry was performed on these section using a NOS antibody (a gift of Dr. H. Esumi, National Cancer Research Institute, East). The specificity of NOS antibody used in the present study was tested and found to react only with neuronal NOS but neither with endothelial nor macrophage NOS as described previously [ 16,18]. Free-floating sections of the adrenal gland were preincubated in PBS containing 1% normal goat serum, 0.2% bovine serum albumin and
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S0304-3940(96) 12345-2
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K. Tanaka, T. Chiba / Neuroscience Letters 204 (1996) 153-156
Fig. 1. Light micrographs of NOS immunoreactivityin 50-/~m-thick sections of adrenal gland embedded in resin which were processed for electron microscopicstudies as shown in Figs. 2 and 3. (a) Adrenalcortex. NOS-ir nerve fibers (arrows) are distributed in subcapsularregion, zona glomerulosa (ZG) and around blood vessels (asterisk). AF, zona fascicalata. (b) Adrenal medulla. NOS-ir ganglion cells (G) are seen. Positive varicosities are observed between the chromaffin cells (C). Bars = 20/~m. 0.05% Triton X-100 for 1 h at room temperature. The antiserum against NOS was applied at a dilution of 1:200 in PBS containing 0.2% bovine serum albumin and 0.05% Triton X-100 for 2 days at 4 ° C . After rinsing with PBS, sections were incubated with biotinylated anti-rabbit IgG produced in goat (Vector) diluted 1:100 in PBS containing 0.05% Triton X-100 for 1 h at room temperature. After rinsing with PBS, sections were incubated with avidinbiotin complex (Vector) at 1:100 dilution in PBS for 1 h at room temperature. They were then transferred to 0.05 M Tris-HC1 buffer (pH 7.2) and incubated with 0.02% diaminobenzidine, 0.003% H202 in 0.05 M T r i s HC1 buffer for 10 min. The sections were fixed with 2.5% glutaraldehyde in PBS for 1 h and 1% osmium tetroxide and 1.5% potassium ferrocyanide in phosphate buffer for 1 h. They were then dehydrated with ethanol and flatly
embedded in Spurr's resin. These sections were observed and photographed with a light microscope. Ultra-thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and observed with a JEOL 1200EX electron microscope. Examination of the sections immunostained with NOS antibody revealed that several ganglion cells in the adrenal medulla and many nerve fibers were positively immunoreactive, while adreno-medullary chromaffin and cortical endocrine cells were non-reactive (Fig. 1a,b). NOS-ir fibers formed a plexus in the subcapsular region and zona glomerulosa of the cortex as reported previously (Fig. l a) [2]. Electron microscopy was used to investigate the fine structure and localization of these NOS-ir nerve fibers in greater detail. NOS-ir nerve fibers were found around arterioles located between the capsule and zona glomerulosa of the cortex. Labeled axon varicosities were observed in close apposition to the smooth muscle of the arteriole (Fig. 2a,b). NOS-ir varicosities in the cortex were observed among endocrine cells of the zona glomerulosa. Most axon profiles were ensheathed with the Schwann cell processes except for the synaptic region. Labeled nerve varicosities were found in close proximity to the endocrine cells (Fig. 2c,d), and were filled with synaptic vesicles and mitochondria. NOS-ir densities were observed in the axoplasm and large granules. In the adrenal medulla, several NOS-ir ganglion cells were found (Fig. lb). Although labeled neurons commonly formed clusters, a few solitary neurons were also observed. Most NOS-ir neurons were large and were round in shape. A small number of immunoreactive neurons appeared to contain two nuclei. NOS-ir ganglion cells were surrounded by chromaffin cells (Fig. 3a) and were often separated by thin Schwann cell processes. Synaptic contacts between non-immunoreactive axons and dendrite of NOS-ir neurons were observed (Fig. 3b). NOS-ir nerve fibers were distributed among chromaffin cells (Fig. lb). Immunoreactive nerve varicosities were in close contact with the catecholamine-storing chromaffin cells (Fig. 3c,d). Postsynaptic density was not seen. We have demonstrated the presence of NOS-ir nerve fibers in the rat adrenal gland. These results were in good agreement with previous studies. Functional studies suggest a role for NO in the control of adrenal blood flow [7, 8], and evidence also exists which suggests its involvement in catecholamine [17,20] and corticosteroid [1,8] release from the gland. Afework et al. [2--4] reported the presence of NOS in the zona glomerulosa and the
Fig. 2. Electron micrographs of NOS-ir nerve terminals in the adrenal cortex of the rat. (a,b) NOS-ir nerve varicosities(arrows) are found to contact with smooth muscles (M) around a blood vessel. L, lumen of the vessel. (c,d) A NOS-iraxon (arrows) is located in close proximity to a secretory cell in the zona glomerulosa(ZG). Bars in (a--c)= 1/zm. Bar in (d) = 0.5/~m. Fig. 3. Electron micrographs of NOS-immunoreactivityin the adrenal medulla. (a) A process of a NOS-ir ganglion cell is surrounded by chromaffin cells (C). (b) A synaptic contact between a non-immunoreactiveaxon and the NOS-ir process (arrowheads). (c,d) A NOS-ir varicosity (arrow) is located in close contact with catecholamine-storingchromaffincells (c). Bars in (a-c) = 1/tm. Bar in (d) = 0.5/~m.
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K. Tanaka, T. Chiba / Neuroscience Letters 204 (1996)153-156
adrenal medulla. N O S - i r axons w e r e c o m m o n l y distributed around b l o o d vessels in close c o n t a c t with s m o o t h muscles in s o m e tissues [12,13]. In the present study, we o b s e r v e d direct c o n t a c t o f N O S - i r varicosities with cortical and m e d u l l a r y cells in addition to s m o o t h muscles o f the b l o o d vessels. N O S - i r n e r v e fibers exert influences through direct contacts with m e d u l l a r y and cortical endocrine cells in addition to vasodilating activity to the blood vessels. T h e p r e s e n c e o f N O S - i r varicosities in close p r o x i m i t y to s m o o t h m u s c l e cells was reported [12,13]. W e h a v e p r e v i o u s l y s h o w n that N O S - i r n e r v e fibers w e r e in close contact with the specialized cardiac m u s c l e o f g u i n e a pig heart [19] and the c y t o l e m m a o f respiratory epithelial cells and acinar cells o f s e r o m u c o u s glands in the rat nasal m u c o s a [ 12]. The adrenal cortical cells and c h r o m a f f i n cells are n e w l y identified cell types which r e c e i v e direct inputs f r o m N O S neurons, N O is k n o w n as a neurotransmitter which affects an intracellular target by t r a n s m e m brane diffusion and is e f f e c t i v e o v e r a long distance and in a limited t i m e c o u r s e [6,10]. In this respect, close apposition o f N O S - i r axon varicosities with adrenal cortical and c h r o m a f f i n cells suggests direct influence o f N O alone or in c o - o p e r a t i o n with c o - e x i s t i n g transmitters to the e n d o c r i n e cells. W e thank Dr. H i r o y a s u E s u m i for g e n e r o u s l y supplying a n t i - N O S serum. [1] Adams, M.L., Nock, B., Truong, R. and Cicero, T.J., Nitric oxide control of steroidogenesis: endocrine effects of NG-nitro-Larginine and comparisons to alcohol, Life Sci., 50 (1992) PL35PL40. [2] Afework, M., Tomlinson, A., Belai, A. and Burnstock, G., Colocalization of nitric oxide synthase and NADPH-diaphorase in rat adrenal gland, NeuroReport, 3 (1992) 893-896. [3] Afework, M., Tomlinson, A. and Burnstock, G., Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing, adult and aging Sprague-Dawley rats, Cell Tissue Res., 276 (1994) 133-141. [4] Afework, M., Ralevic, V. and Burnstock, G., The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat, Neurosci. Lett., 190 (1995) 109-112. [5] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neuronal role for nitric oxide, Nature, 347 (1990) 768-770.
[6] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3-11. [7] Breslow, M.J., Tobin, J.R., Bredt, D.S., Ferris, C.D., Snyder, S.H. and Traystman, R.J., Role of nitric oxide in adrenal medullary vasodilation during catecholamine secretion, Eur. J. Pharmacol., 210 (1992) 105-106. [8] Cameron, L.A. and Hinson, J.P., The role of nitric oxide derived from L-arginine in the control of steroidogenesis, and perfusion medium flow rate in the isolated perfused rat adrenal gland, J. Endocrinol., 139 (1993) 415-423. [9] Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neuronal NADPH-diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sei. USA, 88 (1991) 7797-7801. [10] Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-67. [11] Hanazawa, T., Motosugi, H., Konno, A., Kaneko, T., Tanaka, K. and Chiba, T., Distribution of NADPH-diaphorase positive nerve fibers in the rat nasal mucosa, Neurosci. Lett., 159 (1993) 71-74. [12] Hanazawa, T., Konno, A., Kaneko, T., Tanaka, K., Ohshima, H., Esumi, H. and Chiba, T., Nitric oxide synthase-immunoreactive nerve fibers in the nasal mucosa of the rat, Brain Res., 657 (1994) 7-13. [13] Llewellyn-Smith, 1.J., Song, Z.-M., Costa, M., Bredt, D.S. and Snyder, S.H., Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons, Brain Res., 577 (1992) 337-342. [14] Moncada, S. and Higgs, A., The L-arginine-nitric oxide pathway, J. Med., 329 (1993) 2002-2012. [15] Nathan, C., Nitric oxide as a secretory product of mammalian cells, FASEB J., 6 (1992) 3051-3064. [16] Ohshima, H., Oguchi, S., Adachi, H., lida, S., Suzuki, H., Sugimura, T. and Esumi, H., Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution, Biochem. Biophys. Res. Commun., 183 (1992) 238-244. [17] Oset-Gasque, M.J., Parram6n, M., Hortelano, S., Bosc~i, L. and Gonzfilez, M.P., Nitric oxide implication in the control of neurosecretion by chromaffin cells, J. Neurochem., 63 (1994) 16931700. [18] Tanaka, K., Ohshima, H., Esumi, H. and Chiba, T., Direct synaptic contacts of nitric oxide synthase-immunoreactive nerve terminals on the neurons of the intracardiac ganglia of the guinea pig, Neurosci. Lett., 158 (1993) 67-70. [19] Tanaka, K. and Chiba, T., Nitric oxide synthase-containing nerves in the atrioventricular node of the guinea pig heart, J. Autonomic Nerv. Syst., 51 (1995) 245-253. [20] Uchiyama, Y., Morita, K., Kitayama, S., Suemitsu, T., Minami, N., Miyasako, T. and Dohi, T., Possible involvement of nitric oxide in acetylcholine-induced increase of intracellular Ca2+ concentration and catecholamine release in bovine adrenal chromaffin cells, Jpn. J. Pharmacol., 65 (1994) 73-77.
Neuroscience Letters 204 (1996) 153-156
HtOROSCItHC[ I[IT[RS
Ultrastructural localization of nerve terminals containing nitric oxide synthase in rat adrenal gland Koichi Tanaka*, Tanemichi Chiba The Third Department of Anatomy, Chiba University School of'Medicine, 1-8-1 lnohana, Cyuo-Ku, Chiba 260, Japan
Received 8 December 1995; revised version received 28 December 1995; accepted 28 December 1995
Abstract
Immunoelectron microscopy was performed to localize immunoreactivity for nitric oxide synthase (NOS) in rat adrenal gland. NOS-immunoreactive (NOS-ir) nerve fibers were observed around blood vessels and endocrine cells in the zona glomerulosa of the cortex. Electron microscopy showed that NOS-ir axon varicosities were in close contact with the smooth muscles of blood vessels and with the cytolemma of endocrine cells. In the adrenal medulla, several NOS-ir ganglion cells were found. Synaptic contacts between non-immunoreactive axons and dendrites of NOS-ir neurons were observed. NOS-ir nerve fibers were distributed among chromaffin cells. Positive axon varicosities were in close contact with the catecholamine-storing chromaffin cells. These results suggest that NOSir nerve cells control directly the secretion of corticosteroid and catecholamine in addition to the vascular tone.
Keywords: Nitric oxide synthase; Adrenal gland; Innervation; Rat; Immunohistochemistry; Chromaffin cells; Adrenal cortex
Nitric oxide (NO) is a free radical gas that has attracted great interest as a novel signaling molecule in the nervous system [6,10]. In blood vessels, NO mediates endothelium-derived relaxing factor activity, and when produced by macrophages, neutrophils, and microglial cells, it mediates cytotoxic effects [14,15]. NO is formed from Larginine by the action of nitric oxide synthase (NOS), and the presence o f this enzyme and its m R N A has been used to identify cells that synthesize NO [6]. In addition, there is evidence that N A D P H - d i a p h o r a s e activity can be used as a marker for N O S in many cell types [9,11 ]. The presence o f NOS has also been proved in the nervous elements of the adrenal gland [2-5]. In the rat, NOS-immunoreactive (NOS-ir) nerve fibers were observed among the adrenal chromaffin cells in the medulla, and in the zona glomerulosa o f the cortex, as well as near the intra-adrenal blood vessels [2,5]. However, these studies did not clarify whether NOS-ir nerve fibers exert influences directly on chromaffin and cortical cells or indirectly through neural control of blood vessels. The present study was performed to observe whether NOS-ir
* Corresponding author. Tel.: +81 43 2262024; fax: +81 43 2262025;
e-mail: [email protected].
axons have direct contacts with these endocrine cells in the adrenal gland. Studies were carried out on three male S p r a g u e Dawley rats weighing 3 0 0 - 3 5 0 g . The animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg), perfused via the ascending aorta with 20 ml aqueous 0.9% NaCI, and then fixed by perfusion with 500 ml of fixative composed of 4.0% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Adrenal glands were postfixed with 4.0% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 1 h before being sectioned at a thickness of 5 0 f f m with a Vibratome. The sections were immersed in 0.1 M phosphate buffered saline (PBS) containing 20% succharose for 1 h and freeze-thawed with liquid nitrogen. Pre-embedding immunohistochemistry was performed on these section using a NOS antibody (a gift of Dr. H. Esumi, National Cancer Research Institute, East). The specificity of NOS antibody used in the present study was tested and found to react only with neuronal NOS but neither with endothelial nor macrophage NOS as described previously [ 16,18]. Free-floating sections of the adrenal gland were preincubated in PBS containing 1% normal goat serum, 0.2% bovine serum albumin and
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S0304-3940(96) 12345-2
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K. Tanaka, T. Chiba / Neuroscience Letters 204 (1996) 153-156
Fig. 1. Light micrographs of NOS immunoreactivityin 50-/~m-thick sections of adrenal gland embedded in resin which were processed for electron microscopicstudies as shown in Figs. 2 and 3. (a) Adrenalcortex. NOS-ir nerve fibers (arrows) are distributed in subcapsularregion, zona glomerulosa (ZG) and around blood vessels (asterisk). AF, zona fascicalata. (b) Adrenal medulla. NOS-ir ganglion cells (G) are seen. Positive varicosities are observed between the chromaffin cells (C). Bars = 20/~m. 0.05% Triton X-100 for 1 h at room temperature. The antiserum against NOS was applied at a dilution of 1:200 in PBS containing 0.2% bovine serum albumin and 0.05% Triton X-100 for 2 days at 4 ° C . After rinsing with PBS, sections were incubated with biotinylated anti-rabbit IgG produced in goat (Vector) diluted 1:100 in PBS containing 0.05% Triton X-100 for 1 h at room temperature. After rinsing with PBS, sections were incubated with avidinbiotin complex (Vector) at 1:100 dilution in PBS for 1 h at room temperature. They were then transferred to 0.05 M Tris-HC1 buffer (pH 7.2) and incubated with 0.02% diaminobenzidine, 0.003% H202 in 0.05 M T r i s HC1 buffer for 10 min. The sections were fixed with 2.5% glutaraldehyde in PBS for 1 h and 1% osmium tetroxide and 1.5% potassium ferrocyanide in phosphate buffer for 1 h. They were then dehydrated with ethanol and flatly
embedded in Spurr's resin. These sections were observed and photographed with a light microscope. Ultra-thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and observed with a JEOL 1200EX electron microscope. Examination of the sections immunostained with NOS antibody revealed that several ganglion cells in the adrenal medulla and many nerve fibers were positively immunoreactive, while adreno-medullary chromaffin and cortical endocrine cells were non-reactive (Fig. 1a,b). NOS-ir fibers formed a plexus in the subcapsular region and zona glomerulosa of the cortex as reported previously (Fig. l a) [2]. Electron microscopy was used to investigate the fine structure and localization of these NOS-ir nerve fibers in greater detail. NOS-ir nerve fibers were found around arterioles located between the capsule and zona glomerulosa of the cortex. Labeled axon varicosities were observed in close apposition to the smooth muscle of the arteriole (Fig. 2a,b). NOS-ir varicosities in the cortex were observed among endocrine cells of the zona glomerulosa. Most axon profiles were ensheathed with the Schwann cell processes except for the synaptic region. Labeled nerve varicosities were found in close proximity to the endocrine cells (Fig. 2c,d), and were filled with synaptic vesicles and mitochondria. NOS-ir densities were observed in the axoplasm and large granules. In the adrenal medulla, several NOS-ir ganglion cells were found (Fig. lb). Although labeled neurons commonly formed clusters, a few solitary neurons were also observed. Most NOS-ir neurons were large and were round in shape. A small number of immunoreactive neurons appeared to contain two nuclei. NOS-ir ganglion cells were surrounded by chromaffin cells (Fig. 3a) and were often separated by thin Schwann cell processes. Synaptic contacts between non-immunoreactive axons and dendrite of NOS-ir neurons were observed (Fig. 3b). NOS-ir nerve fibers were distributed among chromaffin cells (Fig. lb). Immunoreactive nerve varicosities were in close contact with the catecholamine-storing chromaffin cells (Fig. 3c,d). Postsynaptic density was not seen. We have demonstrated the presence of NOS-ir nerve fibers in the rat adrenal gland. These results were in good agreement with previous studies. Functional studies suggest a role for NO in the control of adrenal blood flow [7, 8], and evidence also exists which suggests its involvement in catecholamine [17,20] and corticosteroid [1,8] release from the gland. Afework et al. [2--4] reported the presence of NOS in the zona glomerulosa and the
Fig. 2. Electron micrographs of NOS-ir nerve terminals in the adrenal cortex of the rat. (a,b) NOS-ir nerve varicosities(arrows) are found to contact with smooth muscles (M) around a blood vessel. L, lumen of the vessel. (c,d) A NOS-iraxon (arrows) is located in close proximity to a secretory cell in the zona glomerulosa(ZG). Bars in (a--c)= 1/zm. Bar in (d) = 0.5/~m. Fig. 3. Electron micrographs of NOS-immunoreactivityin the adrenal medulla. (a) A process of a NOS-ir ganglion cell is surrounded by chromaffin cells (C). (b) A synaptic contact between a non-immunoreactiveaxon and the NOS-ir process (arrowheads). (c,d) A NOS-ir varicosity (arrow) is located in close contact with catecholamine-storingchromaffincells (c). Bars in (a-c) = 1/tm. Bar in (d) = 0.5/~m.
Ji
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K. Tanaka, T. Chiba / Neuroscience Letters 204 (1996)153-156
adrenal medulla. N O S - i r axons w e r e c o m m o n l y distributed around b l o o d vessels in close c o n t a c t with s m o o t h muscles in s o m e tissues [12,13]. In the present study, we o b s e r v e d direct c o n t a c t o f N O S - i r varicosities with cortical and m e d u l l a r y cells in addition to s m o o t h muscles o f the b l o o d vessels. N O S - i r n e r v e fibers exert influences through direct contacts with m e d u l l a r y and cortical endocrine cells in addition to vasodilating activity to the blood vessels. T h e p r e s e n c e o f N O S - i r varicosities in close p r o x i m i t y to s m o o t h m u s c l e cells was reported [12,13]. W e h a v e p r e v i o u s l y s h o w n that N O S - i r n e r v e fibers w e r e in close contact with the specialized cardiac m u s c l e o f g u i n e a pig heart [19] and the c y t o l e m m a o f respiratory epithelial cells and acinar cells o f s e r o m u c o u s glands in the rat nasal m u c o s a [ 12]. The adrenal cortical cells and c h r o m a f f i n cells are n e w l y identified cell types which r e c e i v e direct inputs f r o m N O S neurons, N O is k n o w n as a neurotransmitter which affects an intracellular target by t r a n s m e m brane diffusion and is e f f e c t i v e o v e r a long distance and in a limited t i m e c o u r s e [6,10]. In this respect, close apposition o f N O S - i r axon varicosities with adrenal cortical and c h r o m a f f i n cells suggests direct influence o f N O alone or in c o - o p e r a t i o n with c o - e x i s t i n g transmitters to the e n d o c r i n e cells. W e thank Dr. H i r o y a s u E s u m i for g e n e r o u s l y supplying a n t i - N O S serum. [1] Adams, M.L., Nock, B., Truong, R. and Cicero, T.J., Nitric oxide control of steroidogenesis: endocrine effects of NG-nitro-Larginine and comparisons to alcohol, Life Sci., 50 (1992) PL35PL40. [2] Afework, M., Tomlinson, A., Belai, A. and Burnstock, G., Colocalization of nitric oxide synthase and NADPH-diaphorase in rat adrenal gland, NeuroReport, 3 (1992) 893-896. [3] Afework, M., Tomlinson, A. and Burnstock, G., Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing, adult and aging Sprague-Dawley rats, Cell Tissue Res., 276 (1994) 133-141. [4] Afework, M., Ralevic, V. and Burnstock, G., The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat, Neurosci. Lett., 190 (1995) 109-112. [5] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neuronal role for nitric oxide, Nature, 347 (1990) 768-770.
[6] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3-11. [7] Breslow, M.J., Tobin, J.R., Bredt, D.S., Ferris, C.D., Snyder, S.H. and Traystman, R.J., Role of nitric oxide in adrenal medullary vasodilation during catecholamine secretion, Eur. J. Pharmacol., 210 (1992) 105-106. [8] Cameron, L.A. and Hinson, J.P., The role of nitric oxide derived from L-arginine in the control of steroidogenesis, and perfusion medium flow rate in the isolated perfused rat adrenal gland, J. Endocrinol., 139 (1993) 415-423. [9] Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neuronal NADPH-diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sei. USA, 88 (1991) 7797-7801. [10] Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-67. [11] Hanazawa, T., Motosugi, H., Konno, A., Kaneko, T., Tanaka, K. and Chiba, T., Distribution of NADPH-diaphorase positive nerve fibers in the rat nasal mucosa, Neurosci. Lett., 159 (1993) 71-74. [12] Hanazawa, T., Konno, A., Kaneko, T., Tanaka, K., Ohshima, H., Esumi, H. and Chiba, T., Nitric oxide synthase-immunoreactive nerve fibers in the nasal mucosa of the rat, Brain Res., 657 (1994) 7-13. [13] Llewellyn-Smith, 1.J., Song, Z.-M., Costa, M., Bredt, D.S. and Snyder, S.H., Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons, Brain Res., 577 (1992) 337-342. [14] Moncada, S. and Higgs, A., The L-arginine-nitric oxide pathway, J. Med., 329 (1993) 2002-2012. [15] Nathan, C., Nitric oxide as a secretory product of mammalian cells, FASEB J., 6 (1992) 3051-3064. [16] Ohshima, H., Oguchi, S., Adachi, H., lida, S., Suzuki, H., Sugimura, T. and Esumi, H., Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution, Biochem. Biophys. Res. Commun., 183 (1992) 238-244. [17] Oset-Gasque, M.J., Parram6n, M., Hortelano, S., Bosc~i, L. and Gonzfilez, M.P., Nitric oxide implication in the control of neurosecretion by chromaffin cells, J. Neurochem., 63 (1994) 16931700. [18] Tanaka, K., Ohshima, H., Esumi, H. and Chiba, T., Direct synaptic contacts of nitric oxide synthase-immunoreactive nerve terminals on the neurons of the intracardiac ganglia of the guinea pig, Neurosci. Lett., 158 (1993) 67-70. [19] Tanaka, K. and Chiba, T., Nitric oxide synthase-containing nerves in the atrioventricular node of the guinea pig heart, J. Autonomic Nerv. Syst., 51 (1995) 245-253. [20] Uchiyama, Y., Morita, K., Kitayama, S., Suemitsu, T., Minami, N., Miyasako, T. and Dohi, T., Possible involvement of nitric oxide in acetylcholine-induced increase of intracellular Ca2+ concentration and catecholamine release in bovine adrenal chromaffin cells, Jpn. J. Pharmacol., 65 (1994) 73-77.