ABSENCE OF TYROSINE HYDROXYLAS~ ACTIVITY AND DOPAMINE fl-HYDROXYLASE IMMUNOREACTIVITY

IN INTRINSIC NERVES OF THE GUINEA-PIG ILEUM

J. B. FURNESS, M. COSTA and C. G. FREEMAN Centre for Neuroscience, Departments of Human Morphology and Human Physiology, School of Medicine, Flinders University, Bedford Park, 5042, South Australia, Australia

Abstract-The activity of tyrosine hydroxylase and the localization of dopamine /3-hydroxylase were determined in the myenteric and submucous plexuses of the normal guinea-pig ileum and in these plexuses after extrinsic denervation. In the normal ileum, the distribution of axons showing immuno- reactivity for dopamitte @-hydroxylase was not distinguishable from the distribution of noradrenergic axons determined by the fluorescence histochemical localization of catecholamines. The distribution of tyrosine hydroxylase in the different layers of the intestine correlated well with the distribution of dopamine j%hydroxylase and noradrenaline, tyrosine hydroxylase activity being most concentrated in the myenteric and submucous plexuses. Extrinsic denervation resulted in the complete disappearance of both biochemically detectable tyrosine hydroxylase and immunohistochemically demonstrable dopa- mine P-hydroxylase.

It is concluded that if the line-handling neurons which are known to be intrinsic to the intestine synthesize an aromatic amine, it is almost certainly not a catecholamine, and is probably an indole- amine.

IT IS well established that the cell bodies of nor- adrenergic neurons in sympathetic pathways to the small intestine are in extrinsic ganglia, and so when nerves running through the mesentery to the small intestine are interrupted, the nerve endings soon degenerate and noradrenaline can no longer be detected histochemically or biochemically within the small intestine (HAMBERGER & NORBERG, 1965; JAW BOWITZ, 1965; FURNE~~ & COSTA, 1971~; 1974; 1978; JUORIO & GABELLA, 1974). However, there is now convincing evidence for intrinsic neurons which might utilize an aromatic amine as a neurotransmitter. Three enzymes, monoamine oxidase (amine oxidase; EC 1.4.3.4), aromatic l-amino acid decarboxylase (EC 4.1.1.26) and tryptophan hydroxylase (tryptophan 5monooxygenase; EC 1.14.16.4), which could be in- volved in the metabolism and synthesis of aromatic amines have been demonstrated in intrinsic neurons of the guinea-pig ileum (FURNESS & COSTA, 1971b; COSTA, FURNESS & MCLEAN, 1976; DREYFUS, BORN- STEIN & GERSWON, 1977; GERSHON, DREYFUS, PICKEL, JOH & REIS, 1977). If all the neurons which contain aromatic l-amino acid decarboxylase and mono~ine oxidase also contain tryptophan hydroxylase, it would be expected that they synthesize an indole- amine. However, it has been shown that a substance pharmacologically similar to dopamine is an inhibi- tory transmitter released from intrinsic neurons in the guinea-pig small intestine (HIRST & MCKIRDY, 1975; Hnzsr & SILINSKY, 1975). If this substance is also chemically similar to dopamine then an explanation must be sought for the failure of histochemical methods to detect an endogenous store of dopamine or other fluorophore-forming amine in intrinsic

Abbreoiation: DBH, dopamine /Lhydroxylase.

nerves of the small intestine. Possible explanations are that dopamine is present in such small amounts or is bound in such a form that it cannot be detected histochemically or that the transmitter is an analogue of dopamine which does not form a fluorophore. If one of these alternatives is the explanation then intrinsic nerves would be expected to contain tyro- sine hydr~xyl~e (tyrosine 3-monooxygen~e; EC 1.14.16.2) (BLASCHKO, 1973; GEFWN & JARROI”T, 1977) and might also contain dopamine j?-hydroxylase, (DBH, dopamine /3-monooxygenase ; EC 1.14.17.1).

EXPERIMENTAL PROCEDURES

Tyrosine hydroxylase activity was determined by the radiometric method of HENDRY & IVERSEN (1971) in seg- ments of normal and extrinsically denervated ileum from five animals. The denervations were performed by crushing the mesenteric nerves supplying 6-8 cm segments of ileum 15-26 days before the tissue was taken (FURNESS & C&W, 19’78). The effectiveness of denervation was evaluated in OS-lcm lengths taken from each end of the denervated area, using the glyoxylic acid fluorescence histochemi~~ technique for noradrenaline (FURNESS & COSTA, 1975). For assay, the longitudinal muscle (with attached myenteric plexus) and the submucosa were separated from the other layers of the intestine by dissection in saline at 0°C. Each sample was divided into two parts, one being homogenized in Srn~ tris buffer at pH 6.0 and then dialyzed twice, for 1 h each time, against the same buffer, and the other being homogenized but not dialyzed before incubation. The pur- pose of the dialysis was to remove endogenous tyrosine which could interfere in the assay; tests using C3H]tyrosine added to homogenates showed that dialysis removed 8994% of tyrosine. The substrate, [3HJtyrosine (Amer- sham) with a specific activity of 15Ci/mmol, was used at a concentration of 5.5 x 10-s mmol/mg wet weight of tis- sue in the incubation mix. Incubation was for 15min at

306

FIG. 1. Immunoh&Aemical local&&on of doprmine fl-hydroxylase (DOH) in whok mount prep- arations from normal and extrinsically denervated segments of ikum. (la) DBH is assoeiatai with varicose axons in a ganglion of the myenteric pkxus from a normal segment of ileum. (lb) In a ganglion of the mycnteric plexus from a denervated area, there is no immunoreactivity for DBH. (Ic) The localization of DBH in the submucosa DBH is associated with axons of the submucous ganglia (0) and with the axons suppIying arterioka (a). (Id) In the submucosa alter extrit& danervation, no DBH immunoreactivity is associated either with ganglia (g) or with arteriolce (a). Calibration:

307

FIG. I

Absence of catecholamine enzymes in intrinsic intestinal neurons 309

37°C. Buffer blank values were determined by omitting the tissue from the assay. Values for boiled tissue homogenates were also determined; these did not differ significantly from buffer blanks.

Dopamine /3-hydroxylase was localized by immunohisto- chemistry in whole mounts of the myenteric plexus attached to the longitudinal muscle and in whole mounts of the submucosa. The unfixed layers of intestine were mounted on glass slides to which they adhered firmly after being air dried for 15-30min. The slides were then placed in 4”/, formaldehyde in 0.1 M phosphate buffer, pH 7.0, at room temperature for 1 h and then washed with several changes of the same buffer over a period of 1 h. The fixed whole mounts were incubated with rabbit anti-bovine anti- DBH for 1 h at 37”C, using a dilution of 1:20 to 1: 50. They were then washed in phosphate buffered saline, pH 7.5, for 30 min and incubated for 30 min at 37°C in a fluor- escein isothiocyanate conjugate of anti-rabbit y-globulin antiserum (Wellcome Diagnostics) diluted 1:SO. Antisera were diluted in phosphate buffered saline containing 0.3% Triton X-100. The antiserum to DBH was raised in rabbits against purified bovine DBH (RUSH, THOMAS, KINDLER & UDENFRIEND, 1974).

Fluorescence of noradrenaline and fluorescein was observed and photographed using a Leitz Ortholux II microscope with a high pressure mercury lamp as a source for incident light illumination.

RESULTS

Tyrosine hydroxylase

There were no differences in the assayed activities of tyrosine hydroxylase in homogenates which were dialyzed compared with nondialyzed homogenates. This indicates that the concentrations of endogenous tyrosine were insufficient to interfere in the assay. The determinations for the dialyzed and non-dialyzed homogenates have therefore been pooled. The greatest concentrations of tyrosine hydroxylase ac- tivity were found in the enteric plexuses, each of which had about the same activity. The average ac- tivity of tyrosine hydroxylase in the normal myenteric plexus was 1.93 + 0.34 (average + S.E.M., n = lo), pg DOPA formed per mg tissue per hour at 37°C and in the submucous plexus the activity was 1.98 + 0.34 (n = 10) pg DOPA/mg/h. These average values repre- sented counts per min of eight times the buffer or boiled tissue blanks. No tyrosine hydroxylase activity could be detected after denervation. In fact, the aver- age counts per min obtained were slightly below, although not significantly different from, the counts in the blanks.

The specific activity of the mucosa plus circular muscle which remains after removal of the plexuses was only 0.16 f 0.01 pg DOPA/mg/h (n = 5), about 8% of the concentration found in the enteric nerve plexuses.

Fluorescence histochemistry of amines

In normal segments of intestine, the majority of noradrenaline-containing axons were found in the enteric plexuses, as has been described previously

(COTTA & GABELLA, 1971). In the ends of the dener- vated segments which were taken for tyrosine hy- droxylase determination or for the localization of DBH, there was no reaction for catecholamines in the plexuses, which confirms previous observations in the denervated small intestine of the guinea-pig (FURNFSS & COSTA, 1978).

Dopamine b-hydroxylase

Immunoreactivity for DBH was located in varicose axons which formed networks around the ganglia of the myenteric and submucous plexuses (Fig. la and c) and around small arteries in the intestine. The distri- bution was indistinguishable from the arrangement of noradrenergic axons which was determined by the glyoxylic acid histochemical method for amines. There was no reaction in nerve cell bodies.

In segments taken after extrinsic denervation of the small intestine there was no reaction for DBH in any axons of the enteric plexuses (Fig. lb and d).

DISCUSSION

GERSHON et al. (1977) used immunohistochemical methods to show that certain neurons in the intestine contain tryptophan hydroxylase. It was further shown by DREYFUS et al. (1977) that some intestinal neurons could take up tryptophan and convert it to a fluoro- phore-forming product, most likely Shydroxytrypta- mine, indicating that they contain both tryptophan hydroxylase and aromatic l-amino acid decarboxy- lase. In the light of these observations, the present demonstration that there is no tyrosine hydroxylase or DBH associated with intrinsic neurons of the intes- tine implies that those neurons which contain aro- matic l-amino acid decarboxylase synthesize indole- amines. Therefore, the distribution of the cell bodies and axons of amine-handling neurons in the intestine which has been described recently (FURNESS & COSTA, 1978) probably applies to the distribution of indole- amine synthesizing neurons. On the other hand, it is also possible that some neurons containing aro- matic l-amino acid decarboxylase do not utilize any aromatic amine as a transmitter, but that they pro- duce and release peptides; it is known that peptide hormone secreting cells contain aromatic l-amino acid decarboxylase (PEARSE, 1968; FUJITA & KOBAYASHI, 1974) and that a number of peptides are contained within neurons in the intestine (see FUR-

NESS & COSTA, 1978). There are some suggestions from the pharmacologi-

cal analysis of neurotransmission in the gastrointes- tinal tract that indoleamines are released as transmit- ters (B~~LBRING & GERSHON, 1967; FURNET, & COSTA,

1973; COSTA & FURNESS, 1976) which would be con- sistent with the presence of tryptophan hydroxylase, aromatic l-amino acid decarboxylase and monoamine oxidase in intestinal nerves. There is also a suggestion that dopamine might be an intestinal neurotrans- mitter (HJRST & MCKIRDY, 1975; HIRST & SILINSKY,

310 J. H. F~XN~SS. M. COSTA and C‘. G. FR~~Y.IA\

1975). The latter authors have shown that dopamine transmitter from intrinsic neurons of the small mte>-

hyperpolarizes neurons of the submucous plexus in tine.

which inhibitory post-synaptic potentials can be

recorded and that both the dopamine effect and the .4c~knw/edyement~ We would like to thank Dr R :\

inhibitory potentials are blocked by methysergide; S- RI:SH for supplying the antiserum 10 DBH and for 1115

hydroxytryptamine depolarizes these neurons. These helpful advice. WC would also like to thank VI ~b~1.t

ESS~N and PAT VILIMAS for their valued assistancr Thl\ results suggest that dopamine and the inhibitory work was supported by grants from the Australian transmitter activate the same receptors but the Research Grants Committee and the NatIonal Health and

present results imply that dopamine itself is not a Medical Research Council of Australia

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