EVIDENCE THAT SOME ~ T R I N S I C NEURONS OF THE ~ T E S T I N E CONTAIN SOMATOSTATIN
M. COSTA*, Y. PATEL, J.B. FURNESS and A. ARIMURA
School o f Medicine, Flinders Unit~rsity of 8outh Australia, Bedford Park and Medical Research Centre, Prince Henry's Hospital, Melbourne qAustralia) and Tulane University School o f Medicine, New Orleans, La. (U.Sd~.)
(Received August 3rd, 1977) (Accepted August 5th, 1977)
SUMMARY
Somatostatin-like immunoreactiviW was demonstrated in the external musculature, which includes the myenteric nerve plexus, and in the mucosa- submucosa, which includes the submucous plexus, of the guinea pig ileum using mdioimmunoassay. Extrinsic denervation for 22 to 24 days decreased somato- statin concentrations by one third in the external muscle, but no change occurred in the mucosa-submucosa. Somatostatin was demonstrated immuno- histochemically in nerve ceil bodies and axons of the myenteric and submucous plexuses. Varicose immunoreactive axons were closely related to nerve cell bodies in both plexuses. It is suggested that the somatostatin~ontaining neurons are intemeurons within the enteric nervous system.
Pharmacological investigations of neurotransmission in the intestine and of the reflexes subserving perista~is have clearly shown that there are intrinsic intestinal neurons releasing transmitters other than noradrenaline or ace#yl- choline [2,5,6,15]. Histochemical studies to characterize neurons in the small intestine that might release the unknown transmitters have revealed the enzymes monoamine oxidase and aromatic 1-amino acid decarboxylase in populations of intrinsic neurons [8,11] although no intrinsic neurons of the small intestine normally contain detectable levels of the commonly occurririg amines noradrenaline, adrenaline, dopamine or 5-hydroxytryptamine [6,8]. Some peptide secreting endocrine ceils have properties in common with cells which normally handle aromatic amines, including the presence of aromatic 1-amino acid decarboxylase [10,22] .,It is therefore of great interest to know if
*Reprint requests to: Dr. M. Costa, School of Medicine, F|inders University of South Australia, Bedford Park 5042, South Australia, Aus~alia.
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axon terminals in the small intestine showing immunoreactivity for the tetra- decapeptide somatostatin [16,18], arise from intrinsic neurons and might therefore be associated with the previously demonstrated amine handling neurons.
In the present work, the distribution of somatostatin immunoreactivity has been examined in the small intestine of guinea pigs and the concentrations of the peptide in different layers of the intestine have been estimated by radio- immunoassay before and after denervation of the intestine.
Adult guinea pigs, both male and female, weight 150--300 g were used. In some animals segments of small intestine were extrinsically denervated by crushing the mesenteric nerves between fine forceps 22--24 days before sacrifice [12]. Because noradrenergic nerves to the intestine are of extrinsic origin, the completeness of the denervation was assessed by localizing noradrenaline with a glyoxylic acid fluorescence method for catecholamines [13] applied to the two ends of the denervated segment, the piece in between being used for radio- immunoassay of somatostatin. The somatostatin antisera employed i n these studies were raised in rabbits against synthetic somatostatin bound to thyro- globulin and human serum globulin by Y. Patel and A. Arimura, respectively. Both antisera exhibit negligible cross-reactivity with a wide range of brain, pancreatic and gastrointestinal peptides, but react with some analogues of somatostatin [ 3,19,23]. The antigenic determinant site of Arimura's antiserum appears to reside at Asn s, Phe7-Lys 9 and Phe ~1, and that of Patel's antiserum a~; AsnS-Trp s and Phe 11 [23].
Guinea pigs were killed by being stunned and bled out and segments of small intestine were removed and placed in cold isotonic saline. Tissue for radio- immunoassay was dissected into layers at ice temperature and samples placed in i ml aliquots of I N acetic acid, boiled for 5 rain and then stored at -70°C until they were assayed by the method previously described [19]. For immunohistochemistry the layers were separated and the isolated external muscle, with the adhering myenteric plexus, and the isolated submucosa were stretched on glass slides. The edges were allowed to dry and adhere to the slides, while the central parts of the preparations were kept moist w~th saline. The tissue was men fixed in 4% formaldehyde in 0.1 M phosphate buffer at pH 7.0 for 2 h at 4°C, washed in 5% sucrose in the same buffer for 6--24 h at 4°C, rinsed in 0.3% triton in phosphate buffered saline (buffer strength 0.01 M, pH 7.4, at 36°C) for 0.5--2 h and then incubated in antiserum diluted I : 25 to I . 100 in phosphate buffered saline for 8.12 h at 36°C. The tissue was then rinsed in the phosphate buffered saline and incubated in fluorescein isothio- cyanate labelled sheep antirabbit 7-globulin antiserum (Wellcome Diagnostics) diluted I • 50 for I h at 36°C. The preparations were mounted in glycerinated phosphate-buffered saline and examined with a Leitz Ortholux fluorescence microscope.
By radioimmunoassay, the whole wall of the guinea pig ileum was found to contain 2.1 pg of somatostatin pe~ ~g of protein. This compares with 1.6 to 1.94 pg/~g protein in the small intestine of the rat [4,20]. When the layers
TABLE I
SOMATOSTAIN (pg PER #g PROTEIN) IN NORMAL/END DENERVATED SEGMENTS OF ILEUM
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Normal ileum Denervated ileum Significance of difference
External muscle
Mucosa plus submucosa
3.34 • 0.27 2.28 -+ 0.35 0.05 < P < 0.10 n = 6 n = 6 1.14 ± 0.15 1.21 +- 0.15 Not significant n = 3 n = 3
were separated, it was found that the external musculature contained 3.3 pg/pg protein and the mucosa plus submucosa contained 1.1 pg//~g protein (Table I). Extrinsic denervation of the ileum resulted in a decrease of about one-third in the concentration in the external musculature but in no significant change in the mucosa plus submucosa (Table I). There was some hypertrophy of the external musculature in the denervated areas of intestine and it is likely that some, if not all, of the decrease in the concentration of somatostatin was due to an increase in muscle protein in the samples rather than a decrease in somatostatin in the myenteric plexus. Of the 6 denervation experinie~ts, in 5 cases no noradrenergic axons could be detected in segments adjacent to that taken for assay. In the other, less than 5% of the normal number of noradren- ergic axons was present on one side and none were present on the other side of the assayed segment.
Somatostatin was localized immunohistochemically in axons and nerve cell bodies of both the myenteric and submucous plexuses (Figs. 1--11). Most of the axons were varicose (Figs. 1--3), the diameters of the varicosities being in the range 1---5/~m. There was often little or no immunoreactivity in the inter- varicose segments (Fig. 1).
Varicose and non-varicose axons were found in the nerve strands which run between intestinal ganglia (Fig. 3). The axons also ran through the ganglia (Fig. 5) and in some cases were arranged around non-reactive nervecell bodies (Fig, 4). In the ganglia of the myenteric plexus some groups of nerve cell bodies were associated with somatostatin reactive axons whereas other groups seemed to have no axons in close proximity. No innervation of the longitudinal or circular muscle was detected. Nerve cell bodies with immunoreactivity for somatostatin showed reaction product, which was usually granular, in the cytoplasm but not in the nucleus (Figs. 7--11). There was insufficient intensity of reaction in the dendrites or initial segments of the axons to trace the processes of the cells. In the submucosa the reaction was usually strong in the cell bodies and the number of positive neurons could be counted reliably. On average, there were two positive cells per ganglion (in 156 ganglia counted, there were 319 nerve cells). This represents 21% of all nerve cell-bodies in the submucosa. It was most usual to find I or 2 somatostatin positive cells per
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ganglion, bu t in about 10% of ganglia be tween 5 and 7 cells were seen. Nerve cell bodies were not always well demons t ra ted i~ .+,he myenter ic plexus and it was not possible to est imate their number accurately. Cell counts based on the best preparat ions suggest tha t there was abou t 2--3 immunoreact ive nerve cell bodies out of a total of, on average, 40 neurons per ganglion.
The specificity of the immunohis tochemica l reaction was tested with anti- serum from which antibodies to somatosta t in had been absorbed by authent ic somatosta t in (Beckman). For the absorpt ion, 1 ml of a 1 : 50 dilution of anti- serum was incubated with 120 ~g of somatos ta t in for 6 h at room t e m p e r a t u r e The absorbed antiserum did no t react with axons or nerv'e cell bodies in the intestine. No reaction was seen with non- immune rabbit serum.
The recent demonstra t ions of somatosta t in in nerve cell bodies and axons of the hypotha lamus and in some primary sensory neurons [ 1 ,9 ,16 ,17] , and of its calc ium-dependent release f rom isolated poster ior pi tui tary lobes by elevating potassium levels in the bathing medium [ 21 ] , suggest that this peptide might be a neurotransmit ter . The present work implies that somatosta t in could also be a t ransmit ter for intrinsic intestinal neurons, but the condit ions governing its release from these neurons is unknown. At present, acetylcholine is the only substance tha t can be conf ident ly postu!ated as a t ransmit ter in enteric neurons, while t.here is evidence for the presence of at least 4 other, unidentif ied, trans- mitters. Three of these form parts of the reflex pathways underlying ?eristalsis [ 7] . Of these, one is that of enteric inhibi tory nerves involved in the descend- ing inhibi tory componen t of the reflex, one is released by non-cholinergic exci ta tory nerves and the o ther is that of sensory nerves which initiate peri- stalsis. Moreover, a fourth unknown t ransmi t te r is released from ~xons forming inhibitory connections in the submucosa [15] and there may be additional transmitters released from interneurons. Somatosta t in has been shown to
Figs. 1 and 2. Examples of varicose axons showing immunoreac t iv i ty for somatos ta t in in the myenter ic plexus. Note tha t the react ion is s t rong in the varicosities bu t weak or absent in the intervaricose por t ions of the axons. Scale marke r in these and subsequen t micrographs. 10urn .
Fig. 3. Axons in one of the nerve strands which connec t the ganglia of the myenter ic plexus. In addi t ion to the varicose axons, a weak react ion can be seen in some of the non-varicose axons of the nerve strand.
Fig. 4. Part of the ganglion of the myenter ic plexus showing a basket like a r rangement of somatos ta t in positive axons which surrounds a non-react ive nerve cell body {arrow).
Fig. 5. Immunoreac t ive axons transversing one of the ganglia of the myente r ic plexus. Note tha t the axons are varicose bo th in the nerve s t rands and where they transverse the ganglion. The neuron cell bodies in the ganglion are not immunoreac t ive , but the ganglion can be recognized by non-specific p ro te in fluorescence.
Fig. 6. Varicose axons in a ganglion of the myen te r i c plexus. Note the large number ot varicose somatos ta t in conta ining axons which ramify amongst the non-reactive nerve cell
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inhibit the release of ace~ylcholine from nerves within the small intestine L_4 ] , so it is possible that the somatostat in ~ :ons observed in the present work could form inhibitory connections with enteric cholinergic neurons. The distribution of the varicose axons containing somatosta t in within the intestinal ganglia suggests that the somatostat in neurons are interneurons in intrinsic pathways of the enteric nervous system.
As mentioned above, cells which synthesize and release peptides often also contain the machinery to handle amines. However, in the submucosa of the small intestine, about 20% of the nerve cell bodies contain somatostatin, whereas only about 10% contain aromatic 1-amino acid decarboxylase [8, unpubl ished] . It is possible that only some of the somatosta t in neurons contain the decarboxylase or that the concentrat ions of the decarboxylase in about half the somatostat in containing neurons are less than could be detected. However, it seems more likely that the somatostat in-containing neurons and the neurons containing aromatic 1-amino acid decarboxylase belong to separate populations.
ACKNOWLEDGEMENTS
This work was supported by grants to Marcello Costa and John Furness from the National Health and Medical Research Council of Australia and the Australian Research Grants Commit tec and by U.S.P.H. Research Grant AM09094 to A. AriIaura.
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Figs. 7--11. Examples of nerve cell bodies which show immunoreactivity for somatostatin in the submucous plexus. The ganglia also contain'somatostatin-positive axon varicosities and some non-reactive neurons. The nuclei of the cells are unreactive (arrow, Fig. 10).
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