Neuro~cience Lciwr~', 143 (1992) 60 64 '~' 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ ()5.01~
60
NSL 08854
Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut A. Belai a, H . H . H . W . Schmidt b, C.H.V. Hoyle a, C.J.S. Hassall a, M.J. Saffrey~, J. Moss a, U. F r r s t e r m a n n b'~, F. M u r a d b'c and G. Burnstock a °Department of Anatomy and Developmental Biology, and Centrefor Neuroscience, University College London, London (UK), bDepartment of Pharmacology, Northwestern University, Chicago, 1L 60611 (USA) and CAbbott Laboratories, Abbott Park, IL 60064 (USA) (Received 3 March 1992; Revised version received 5 May 1992; Accepted 6 May 1992)
Key words: Nitric oxide synthase; NADPH-diaphorase; Myenteric plexus The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of wholemount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and iluem, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.
Following the identification of nitric oxide (NO) as an agent responsible for endothelium-derived relaxing factor activity [7, 12, 18], its possible role as mediator of cellular activities in non-vascular tissues, including the gastrointestinal tract, has also been investigated. It has been previously suggested that an inhibitory substance in addition to, or other than ATP [11, 15, 19] and VIP [9, 14] may participate in enteric non-adrenergic noncholinergic (NANC) inhibitory neurotransmission of the gastrointestinal tract [5, 17], and recently several investigators have provided pharmacological evidence implicating NO as another NANC inhibitory neurotransmitter in some of its regions [3, 13, 21, 23]. Morphological studies to identify neuronal structures that may utilise NO as a mediator of cellular activities have been performed by localising enzymes involved in the synthesis of NO [4, 10, 22]. The aim of the present study was to investigate the pattern of distribution and coexistence of nitric oxidesynthase (NOS) and NADPH-diaphorase in neuronal structures of the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proxCorrespondence: G. Burnstock, Department of Anatomy and Developmental Biology and Centre for Neurocience, University College London, Gower Street, London WCIE 6BT, UK.
imal colon and distal colon of the rat. The colocalization was achieved by simultaneously labelling NOS and NADPH-diaphorase in the same segment of tissue, with the immunolabelling of NOS carried out prior to the histochemical staining of NADPH-diaphorase. Adult male Wistar rats were killed by an overdose of CO2 and exsanguination and the stomach, duodenum, ileum, caecum, proximal and distal colon were dissected out and washed with Hank's balanced salt solution (Life Sciences Ltd.). Small segments of these tissues were then cut out and stretched with mucosal side downwards on a piece of Sylgard silicon rubber, and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2 h at 4°C. The longitudinal and circular muscle coats together with the myenteric plexus were then peeled away from the rest of the gut and used as myenteric plexus preparations. The tissues were then processed as described previously [1, 2]. Briefly, the stretched preparations were washed with 80% alcohol, dehydrated, rehydrated, and washed 3 times each for 5 min with PBS containing 0.1% Triton X-100. For immunohistochemical localisation of NOS, the tissues were incubated with antisera raised in rabbits against purified soluble NOS extracted from rat cerebellum [20], at a dilution of 1:1000 inPBS in a humid chamber for 18 h at room temperature. The NOS antibody was then visualised with biotinylated donkey anti-
61
:T
Fig. 1. Micrographs showing the pattern of distribution and colocalisation of NOS and NADPH-diaphorase in the myenteric neurons and nerve fibres of whole mount preparations of the antrum (a, b); duodenum (c, d) and ileum (e, f) of the rat (n=6). Almost all NOS positive neurons in all the above regions of the gut (a, c, e) were also stained for NADPH-diaphorase (b, d and f respectively). Some of the neurons that stain for both NOS and NADPH-diaphorase show similar intensity of staining (arrows) where as others do not (arrowheads). Bars 30/~m. rabbit a n t i b o d y ( A m e r s h a m , 1:250) followed by streptavidin fluorescein ( A m e r s h a m , 1:100). T h e tissues were s u b s e q u e n t l y washed a n d m o u n t e d with citifluor (City University, L o n d o n , U K ) . C o n t r o l e x p e r i m e n t s to determine the specificity o f the a n t i s e r u m used were c a r r i e d o u t by p r e i n c u b a t i n g the a n t i s e r u m with purified soluble N O S from rat c e r e b e l l u m (see Fig. 3). To investigate the possible coexistence o f N O S a n d
N A D P H - d i a p h o r a s e , the N O S - l a b e l l e d tissues were w a s h e d with PBS a n d N A D P H - d i a p h o r a s e staining was p e r f o r m e d by i n c u b a t i n g the tissues with 1.2 m M o f f l N A D P H , 0.24 m M n i t r o b l u e t e t r a z o l i u m , 15.2 m M Lmalic acid a n d 0.1% T r i t o n X-100 in 0.1 M Tris HC1 ( p H 7.4) for 2 h at 37°C. In all instances the i m m u n o l a b e l l i n g o f N O S was p e r f o r m e d before the N A D P H - d i a p h o r a s e staining.
62
Fig. 2. Micrographs showing the pattern of distribution and colocalisation of NOS and NADPH-diaphorase in the myenteric neurons and nerve fibres of whole mount preparations of the caecum (a, b); proximal colon (c, d) and distal colon (e, f) of the rat (n=6). Almost all N OS-positive neurons in all parts of the large intestine (a, c, e) were also stained tbr NADPH-diaphorase (b, d and f, respectively). Arrowheads show neurons that stain for NOS and NADPH-diaphorase but have ~ different intensity of staining. Bars = 30/lm.
A substantial number of neuronal cell bodies and nerve fibres in the myenteric plexus of the rat antrum, duodenum, ileum, caecum, proximal and distal colon were stained for NOS (Figs. 1 and 2). Almost all NOS immunoreactive neurons in the myenteric plexus of all regions of the gut examined were also stained for NADPH-diaphorase (Fig. l a - f and 2a-f). However the
distribution of nerve fibres that stained for both NOS and NADPH-diaphorase in the secondary and tertiary plexuses and the circular muscle layer varied from one region of the gut to the other. In the stomach (Fig. la), duodenum (Fig. lc) and ileum (Fig. le), only a few of the NOS-positive nerve fibres were stained for NADPH-diaphorase (Fig. lb, c and f, respectively), whereas in the
63
Fig. 3. Micrographs showing NOS immunoreactivity in the myenteric plexus and circular muscle layer of rat. A: NOS immunoreactivity using non-absorbed (1:1000) NOS antiserum: B: after preadsorbtion of NOS antiserum (1:1000) with purified soluble NOS (20 ,ug/ml) from rat cerebellum. 1 m, longitudinal muscle: g, myenteric ganglia: cm, circular muscle. Bars - 25 #m.
caecum (Fig. 2a) and distal colon (Fig. 2e), almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase (Fig. 2b and 2t", respectively). The size and shape of both NOS and NADPH-diaphorasepositive neuron; varied from one region of the gut to the other and within the ganglia of the same region of the gut
(Figs. lc,d and 2c,d). Some of the intensely stained NOSpositive neurons were also intensely stained for NADPH-diaphorase while others were not. We have shown for the first time the coexistence of NOS and NADPH-diaphorase in neurons and nerve fibres of the myenteric plexus in whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal and distal colon of the rat using a double labelling technique. The presence of NO synthesising enzymes in the enteric neurons and nerve fibres, and the pharmacological evidence of NO-mediated relaxation of the smooth muscle of the gut [3, 13, 21,23] strongly suggest that NO may have a role in N A N C inhibitory neurotransmission in some regions of the gastrointestinal tract, perhaps as a co-transmitter together with ATP and VIP [11]. NOS has been reported to exist in several isoforms, and generally two main types, the cytosolic and particulate NOS, have been identified [8]. In the brain for exampie, the presence of soluble but not particulate NOS has been shown, whereas, the vascular endothelial cells contain only the particulate isoform of NOS [8, 20]. So far little has been done to purify and characterise NOS enzymes in the peripheral nervous system. Mitchell and colleagues [16] have recently demonstrated the presence of both soluble and particulate isoforms of NOS in N A N C nerves of the rat anococcygeus muscle. However, it has been noted that unlike the brain, considerable enzymatic activity of NOS in NANC tissues is associated with the particulate fraction [16]. In the present study, it was possible to localize NOScontaining neurons and nerve fibres using antisera raised against soluble NOS extracted from the rat cerebellum [20]. However. the exact form of NOS and whether there is more than one isoform of NOS in the enteric nervous system, as has been reported in the rat anococcygeus muscle, awaits further investigation. The presence of NADPH-diaphorase activity in all myenteric neurons that contain NOS immunoreactivity further confirms the suggestion that NOS and neuronal NADPH-diaphorase are identical [6. 10]. Hope and colleagues [10] have demonstrated the presence ot" NADPHdiaphorase activity that could be separated from NOS by column chromatography and not recognized by the NADPH-diaphorase-specific antibody. A similar observation had also been reported from another laboratory [6]. Such findings [6, 10] may account for the lack of correspondence in the intensity of NOS immunoreactivity and NADPH-diaphorase activity in some myenteric neurons observed in the present study. In the light of the above reports it will be of great interest to establish the isoform types of NOS in the enteric nervous system. Further studies to investigate the
64
presence of NOS and NADPH-diaphorase activities in neurons of the other enteric ganglia (submucous ganglia, in gall bladder and pancreas), and their similarities and/ or differences to those found in the myenteric ganglia are currently being carried out. 1 Belai, A. and Burnstock, G., Selective damage of intrinsic calcitonin gene-related peptide-like immunoreactive enteric nerve fibres in streptozotocin-induced diabetic rats, Gastroenterology, 92 (1987) 730-734. 2 Belai, A. and Burnstock, G., Changes in adrenergic and peptidergic nerves in the submucous plexus of streptozotocin-diabetic rat ileum, Gastroenterology, 98 (1990) 1427-1436. 3 Boeckxsteans, G.E., Pelckmans, P.A., Bult, H., De Man, J.G., Herman, A.G. and Van Maercke, Y.M., Non-adrenergic non-cholinergic relaxation mediated by nitric oxide in the canine ileocolonic junction, Eur. J. Pharmacol., 190 (1990) 239--246. 4 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature, 347 (1990) 768-770. 5 Costa, M., Furness, J.B. and Humphreys, C.M.S., Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract, Naunyn-Schmiedebergs Arch. Pharmacol., 332 (1986) 79 88. 6 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. Sci. USA, 88 (1991) 7797-7801. 7 Furchgott, R.F. and Zawadzki, J.V., The obligatory role of endothelial cells in the relaxation arterial smooth muscle by acetylcholine, Nature, 288 (1980) 373-376. 8 F6rstermann, U., Schmidt, H.H.H.W., Pollock, J.S., Sheng, H., Mitchell, J.A., Warner, T.D., Nakane, M. and Murad, F., Isoforms of nitric oxide synthase: characterisation and purification from different cell types, Biochem. Pharmacol., 42 (1991) 1849-1857. 9 Grider, J.R., Cable, M.B., Said, S.I. and Makhlouf, G.M., Vasoactive intestinal peptide (VIP) as neural mediator of gastric relaxation, Am. J. Physiol., 248 (1985) G73 G78. 10 Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811--2814. 11 Hoyle, C.H.V. and Burnstock, G., Neuromuscular transmission in the gastrointestinal tract. In G.M, Makhlouf (Ed.), Handbook of Physiology, Sect. 6: The Gastrointestinal System, Vol. I, American Physiological Society, Washington, DC, 1989, pp. 435~J,64.
12 lgnarro, L.J., Buga, G.M., Wood. K.S.. Byrns, R.E. and Chaudhury, G., Endothetium-derived relaxing l:actor produced and released from artery and vein is nitric oxide. Proc. Natl, Sci. tSA. 84 (1987) 9265-9269. 13 Li, C.G. and Rand, M.J., Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundu~. [iur..!. Pharmacol., 191 (1990)303-309. 14 Makhlouf. G.M., Grider, J.R, and Schubert, M.L., ldentilication of physiological function of gut peptides. In G.M. Makhlouf (Ed.}, Handbook of Physiology, Sect. 6: The Gastrointestinal System, Vol. 11, American Physiological Society. Washington, DC, 1989, pp. 123 13I. 15 Manzini, S., Maggi, C.A. and Meli. A., Pharmacological evidence that at least two different non-adrenergic non-cholinergic inhibitory systems are present in the rat small intestine, Eur. J. Pharmacol,, 123 (1986) 229 238. 16 Mitchell, J.A., Sheng, H., FOrstermann, U. and Murad, F.. Characterisation of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle, Br. J. Pharmacol., 104 (1991)289 291. 17 Niel, J.P., Bywater, R.A.R. and Taylor, G.S., Apamin resistant poststimulus hyperpolarization in the circular muscle of the guineapig ileum, J. Auton. Nerx. Syst.. 9 (1983) 565 569. 18 Palmer, R.M.J., Ferrige, A.S. and Moncada, S.. Nitric oxide release accounts for the biological activity ofendothelium-derived relaxing factor, Nature, 327 (1987) 524 526. 19 Satchell, D.G., Nucleotide pyrophosphatase antagonizes responses to adenosine 5'-triphosphate and non-adrenergic, non-cholinergic inhibitory nerve-stimulation in the guinea-pig isolated taenia toll, Br. J. Pharmacol., 74 (1981) 319- 321. 20 Schmidt, H.H.H,W., Pollock, J.S., Nakane, M., Gorsky, I,.D., F6rstennann, U. and Murad, F., Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 365-369. 21 Shuttleworth, C.W.R., Murphy, R. and Furness, J.B., Evidence that nitric oxide participates in non-adrenergic inhibitory transmission to intestinal muscle in the guinea-pig, Neuroscience Lett., 130 (1991) 77 80. 22 Snyder, S.H. and Bredt, D.S., Nitric oxide as neuronal messenger, Trends Pharmacol. Sci,, 12 (1991) 125- 128. 23 Toda, N., Baba, H. and Okamura, T., Role of nitric oxide in mmadrenergic, non-cholinergic nerve-mediated relaxation in dog duodenal longitudinal muscle strips, Jpn. J. Pharmacol., 53 ~1990) 28 284.
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NSL 08854
Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut A. Belai a, H . H . H . W . Schmidt b, C.H.V. Hoyle a, C.J.S. Hassall a, M.J. Saffrey~, J. Moss a, U. F r r s t e r m a n n b'~, F. M u r a d b'c and G. Burnstock a °Department of Anatomy and Developmental Biology, and Centrefor Neuroscience, University College London, London (UK), bDepartment of Pharmacology, Northwestern University, Chicago, 1L 60611 (USA) and CAbbott Laboratories, Abbott Park, IL 60064 (USA) (Received 3 March 1992; Revised version received 5 May 1992; Accepted 6 May 1992)
Key words: Nitric oxide synthase; NADPH-diaphorase; Myenteric plexus The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of wholemount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and iluem, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.
Following the identification of nitric oxide (NO) as an agent responsible for endothelium-derived relaxing factor activity [7, 12, 18], its possible role as mediator of cellular activities in non-vascular tissues, including the gastrointestinal tract, has also been investigated. It has been previously suggested that an inhibitory substance in addition to, or other than ATP [11, 15, 19] and VIP [9, 14] may participate in enteric non-adrenergic noncholinergic (NANC) inhibitory neurotransmission of the gastrointestinal tract [5, 17], and recently several investigators have provided pharmacological evidence implicating NO as another NANC inhibitory neurotransmitter in some of its regions [3, 13, 21, 23]. Morphological studies to identify neuronal structures that may utilise NO as a mediator of cellular activities have been performed by localising enzymes involved in the synthesis of NO [4, 10, 22]. The aim of the present study was to investigate the pattern of distribution and coexistence of nitric oxidesynthase (NOS) and NADPH-diaphorase in neuronal structures of the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proxCorrespondence: G. Burnstock, Department of Anatomy and Developmental Biology and Centre for Neurocience, University College London, Gower Street, London WCIE 6BT, UK.
imal colon and distal colon of the rat. The colocalization was achieved by simultaneously labelling NOS and NADPH-diaphorase in the same segment of tissue, with the immunolabelling of NOS carried out prior to the histochemical staining of NADPH-diaphorase. Adult male Wistar rats were killed by an overdose of CO2 and exsanguination and the stomach, duodenum, ileum, caecum, proximal and distal colon were dissected out and washed with Hank's balanced salt solution (Life Sciences Ltd.). Small segments of these tissues were then cut out and stretched with mucosal side downwards on a piece of Sylgard silicon rubber, and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2 h at 4°C. The longitudinal and circular muscle coats together with the myenteric plexus were then peeled away from the rest of the gut and used as myenteric plexus preparations. The tissues were then processed as described previously [1, 2]. Briefly, the stretched preparations were washed with 80% alcohol, dehydrated, rehydrated, and washed 3 times each for 5 min with PBS containing 0.1% Triton X-100. For immunohistochemical localisation of NOS, the tissues were incubated with antisera raised in rabbits against purified soluble NOS extracted from rat cerebellum [20], at a dilution of 1:1000 inPBS in a humid chamber for 18 h at room temperature. The NOS antibody was then visualised with biotinylated donkey anti-
61
:T
Fig. 1. Micrographs showing the pattern of distribution and colocalisation of NOS and NADPH-diaphorase in the myenteric neurons and nerve fibres of whole mount preparations of the antrum (a, b); duodenum (c, d) and ileum (e, f) of the rat (n=6). Almost all NOS positive neurons in all the above regions of the gut (a, c, e) were also stained for NADPH-diaphorase (b, d and f respectively). Some of the neurons that stain for both NOS and NADPH-diaphorase show similar intensity of staining (arrows) where as others do not (arrowheads). Bars 30/~m. rabbit a n t i b o d y ( A m e r s h a m , 1:250) followed by streptavidin fluorescein ( A m e r s h a m , 1:100). T h e tissues were s u b s e q u e n t l y washed a n d m o u n t e d with citifluor (City University, L o n d o n , U K ) . C o n t r o l e x p e r i m e n t s to determine the specificity o f the a n t i s e r u m used were c a r r i e d o u t by p r e i n c u b a t i n g the a n t i s e r u m with purified soluble N O S from rat c e r e b e l l u m (see Fig. 3). To investigate the possible coexistence o f N O S a n d
N A D P H - d i a p h o r a s e , the N O S - l a b e l l e d tissues were w a s h e d with PBS a n d N A D P H - d i a p h o r a s e staining was p e r f o r m e d by i n c u b a t i n g the tissues with 1.2 m M o f f l N A D P H , 0.24 m M n i t r o b l u e t e t r a z o l i u m , 15.2 m M Lmalic acid a n d 0.1% T r i t o n X-100 in 0.1 M Tris HC1 ( p H 7.4) for 2 h at 37°C. In all instances the i m m u n o l a b e l l i n g o f N O S was p e r f o r m e d before the N A D P H - d i a p h o r a s e staining.
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Fig. 2. Micrographs showing the pattern of distribution and colocalisation of NOS and NADPH-diaphorase in the myenteric neurons and nerve fibres of whole mount preparations of the caecum (a, b); proximal colon (c, d) and distal colon (e, f) of the rat (n=6). Almost all N OS-positive neurons in all parts of the large intestine (a, c, e) were also stained tbr NADPH-diaphorase (b, d and f, respectively). Arrowheads show neurons that stain for NOS and NADPH-diaphorase but have ~ different intensity of staining. Bars = 30/lm.
A substantial number of neuronal cell bodies and nerve fibres in the myenteric plexus of the rat antrum, duodenum, ileum, caecum, proximal and distal colon were stained for NOS (Figs. 1 and 2). Almost all NOS immunoreactive neurons in the myenteric plexus of all regions of the gut examined were also stained for NADPH-diaphorase (Fig. l a - f and 2a-f). However the
distribution of nerve fibres that stained for both NOS and NADPH-diaphorase in the secondary and tertiary plexuses and the circular muscle layer varied from one region of the gut to the other. In the stomach (Fig. la), duodenum (Fig. lc) and ileum (Fig. le), only a few of the NOS-positive nerve fibres were stained for NADPH-diaphorase (Fig. lb, c and f, respectively), whereas in the
63
Fig. 3. Micrographs showing NOS immunoreactivity in the myenteric plexus and circular muscle layer of rat. A: NOS immunoreactivity using non-absorbed (1:1000) NOS antiserum: B: after preadsorbtion of NOS antiserum (1:1000) with purified soluble NOS (20 ,ug/ml) from rat cerebellum. 1 m, longitudinal muscle: g, myenteric ganglia: cm, circular muscle. Bars - 25 #m.
caecum (Fig. 2a) and distal colon (Fig. 2e), almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase (Fig. 2b and 2t", respectively). The size and shape of both NOS and NADPH-diaphorasepositive neuron; varied from one region of the gut to the other and within the ganglia of the same region of the gut
(Figs. lc,d and 2c,d). Some of the intensely stained NOSpositive neurons were also intensely stained for NADPH-diaphorase while others were not. We have shown for the first time the coexistence of NOS and NADPH-diaphorase in neurons and nerve fibres of the myenteric plexus in whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal and distal colon of the rat using a double labelling technique. The presence of NO synthesising enzymes in the enteric neurons and nerve fibres, and the pharmacological evidence of NO-mediated relaxation of the smooth muscle of the gut [3, 13, 21,23] strongly suggest that NO may have a role in N A N C inhibitory neurotransmission in some regions of the gastrointestinal tract, perhaps as a co-transmitter together with ATP and VIP [11]. NOS has been reported to exist in several isoforms, and generally two main types, the cytosolic and particulate NOS, have been identified [8]. In the brain for exampie, the presence of soluble but not particulate NOS has been shown, whereas, the vascular endothelial cells contain only the particulate isoform of NOS [8, 20]. So far little has been done to purify and characterise NOS enzymes in the peripheral nervous system. Mitchell and colleagues [16] have recently demonstrated the presence of both soluble and particulate isoforms of NOS in N A N C nerves of the rat anococcygeus muscle. However, it has been noted that unlike the brain, considerable enzymatic activity of NOS in NANC tissues is associated with the particulate fraction [16]. In the present study, it was possible to localize NOScontaining neurons and nerve fibres using antisera raised against soluble NOS extracted from the rat cerebellum [20]. However. the exact form of NOS and whether there is more than one isoform of NOS in the enteric nervous system, as has been reported in the rat anococcygeus muscle, awaits further investigation. The presence of NADPH-diaphorase activity in all myenteric neurons that contain NOS immunoreactivity further confirms the suggestion that NOS and neuronal NADPH-diaphorase are identical [6. 10]. Hope and colleagues [10] have demonstrated the presence ot" NADPHdiaphorase activity that could be separated from NOS by column chromatography and not recognized by the NADPH-diaphorase-specific antibody. A similar observation had also been reported from another laboratory [6]. Such findings [6, 10] may account for the lack of correspondence in the intensity of NOS immunoreactivity and NADPH-diaphorase activity in some myenteric neurons observed in the present study. In the light of the above reports it will be of great interest to establish the isoform types of NOS in the enteric nervous system. Further studies to investigate the
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presence of NOS and NADPH-diaphorase activities in neurons of the other enteric ganglia (submucous ganglia, in gall bladder and pancreas), and their similarities and/ or differences to those found in the myenteric ganglia are currently being carried out. 1 Belai, A. and Burnstock, G., Selective damage of intrinsic calcitonin gene-related peptide-like immunoreactive enteric nerve fibres in streptozotocin-induced diabetic rats, Gastroenterology, 92 (1987) 730-734. 2 Belai, A. and Burnstock, G., Changes in adrenergic and peptidergic nerves in the submucous plexus of streptozotocin-diabetic rat ileum, Gastroenterology, 98 (1990) 1427-1436. 3 Boeckxsteans, G.E., Pelckmans, P.A., Bult, H., De Man, J.G., Herman, A.G. and Van Maercke, Y.M., Non-adrenergic non-cholinergic relaxation mediated by nitric oxide in the canine ileocolonic junction, Eur. J. Pharmacol., 190 (1990) 239--246. 4 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature, 347 (1990) 768-770. 5 Costa, M., Furness, J.B. and Humphreys, C.M.S., Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract, Naunyn-Schmiedebergs Arch. Pharmacol., 332 (1986) 79 88. 6 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. Sci. USA, 88 (1991) 7797-7801. 7 Furchgott, R.F. and Zawadzki, J.V., The obligatory role of endothelial cells in the relaxation arterial smooth muscle by acetylcholine, Nature, 288 (1980) 373-376. 8 F6rstermann, U., Schmidt, H.H.H.W., Pollock, J.S., Sheng, H., Mitchell, J.A., Warner, T.D., Nakane, M. and Murad, F., Isoforms of nitric oxide synthase: characterisation and purification from different cell types, Biochem. Pharmacol., 42 (1991) 1849-1857. 9 Grider, J.R., Cable, M.B., Said, S.I. and Makhlouf, G.M., Vasoactive intestinal peptide (VIP) as neural mediator of gastric relaxation, Am. J. Physiol., 248 (1985) G73 G78. 10 Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811--2814. 11 Hoyle, C.H.V. and Burnstock, G., Neuromuscular transmission in the gastrointestinal tract. In G.M, Makhlouf (Ed.), Handbook of Physiology, Sect. 6: The Gastrointestinal System, Vol. I, American Physiological Society, Washington, DC, 1989, pp. 435~J,64.
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