Storage, uptake and synthesis of catecholamines in the intrinsic adrenergic neurones in the proximal...

Z. Zenforsch. 120, 364-385 (1971) � 9 Springer-Ve~ag 1971

Storage, Uptake and Synthesis of Catecholamines in the Intrinsic Adrenergic Neurones

in the Proximal Colon of the Guinea-Pig*

M. COSTA a n d J . B. F U l l n e s s

Department of Zoology, University of Melbourne, Parkville, Victoria, Australia

Received May 18, 1971

Summary. In the present work, the effects of drugs on the storage, uptake and synthesis of catecholamines in intrinsic and extrinsic adrenergic neurones of the guinea-pig intestine are compared, using the fluorescence histoehemical technique for localising catecholamines. In respect to the properties examined in this work, the intrinsic adrenergic neurones of the proximal colon of the guinea-pig were found to be qualitatively similar to adrenergic neurones of the sympathetic chains: the intrinsic cells and their terminals are depleted by reserpine or guanethidine; they concentrate and retain catecholamines and this uptake is blocked by desmethylimipramine or phenoxybenzamine; after depletion by reserpine, the fluorescence can be restored by the dopamine and noradrenaline precursor, dopa and this restoration is prevented by blocking the decarboxylation of dopa to dopamine. However, there are clear quantitative differences: the terminals of intrinsic neurones are less susceptible than are extrinsic neurones to depletion by reserpine, guanethidine or 6-hydroxydopamine; the intrinsic neurones more readily retain noradrenaline after reserpinisation. I t is suggested that quantitative differences between extrinsic and intrinsic neurones of the intestine could involve a difference in the activity of monoamine oxidase.

Key-Words: Gastrointestinal tract - - Adrenergic neurones - - Adrenergic mechanisms --- Fluorescence histochemistry.

A d r e n e r g i c gang l ion cells a re f o u n d in t h e m y e n t e r i c p lexus of t h e p r o x i m a l colon, in c o n t r a s t to t h e s i t u a t i o n in all o t h e r pa r t s of t h e m a m m a l i a n gas t ro-

i n t e s t i n a l t r a c t wh ich h a v e been i n v e s t i g a t e d (Costa, Fu rnes s a n d Gabel la , 1971 ; F u r n e s s a n d Costa , 1971). I n t h e p re sen t work , t h e sens i t iv i t i es of these cells a n d t h e i r processes to t h e ac t i on of d rugs wh ich are k n o w n to a f fec t t h e up t ake ,

s t o r age a n d syn thes i s of c a t e c h o l a m i n e s are e x a m i n e d . These p rope r t i e s of t h e in t r in s i c a d r e n e r g i c neu rones of t h e p r o x i m a l colon are c o m p a r e d wi th those of a d r e n e r g i c neurones , w i t h cell bodies in p r e v e r t e b r a l gangl ia , whose t e r m i n a l s

s u p p l y o t h e r reg ions of t h e g a s t r o i n t e s t i n a l t r ac t .

Methods In all experiments guinea-pigs of both sexes were used. The animals weighed 150-600 g.

The tissue to be examined was dissected immediately after the animals were sacrificed by bleeding under ether anaesthesia. Between dissection and preparation the tissue was stored for up to 15 rain in saline at room temperature or for up to 2 hours in ice-cold saline. Storage did not alter the fluorescence reaction of the tissue. Lamina preparations of the myenteric

* This work was supported by grants from the Australian Research Grants Committee and the National Health and Medical Research Council. We thank Prof. G. Burnstock for his support and for his critical reading of the manuscript. We are particularly grateful to Dr. H. Corrodi for a gift of FLA 63.

Histochemistry of Drug Actions on CAs of Intrinsic Intestinal Neurones 365

plexus from duodenum, ileum, proximal colon and distal colon were prepared (see Furness and Costa, 1971). Similar preparations of the submucosa from the duodenum, ileum and proximal colon were made. For comparison, stretch preparations of the inferior mesenteric ganglia and, in some cases, of the right atrium were examined. The preparations were dried over P205 for 1-2 hours and then incubated in paraformaldehyde vapour at 80 ~ C for 1 hour. Control tissue from untreated animals was taken with each experimental group.

Drugs were applied both in vivo and in vitro. Injections were given by different routes depending on the drug (see below) to guinea-pigs which were liberally supplied with food and water. Drugs were applied in vitro to tissue immersed in modified Krebs' solution (Fur- ness, 1969) at 36 ~ C and vigorously oxygenated.

The drugs used and their conditions of application are listed below. Reserpine (Serpasil, in ampoules from Ciba). This was injected intraperitoneally or

subcutaneously. Guanethidine sulphate (Ismelin, in ampoules from Ciba). Intraperitoneal injection. 1-Noradrenaline bitartrate (Levophed, in ampoules from Winthrop). Applied in vitro

to isolated tissue. 1-~-Methyl-noradrenaline hydrochloride (neocobefrin, as the salt from Sterling Pharma-

ceuticals). Intraperitoneal injection or applied in vitro. Pargyline hydrochloride (Eutonyl, as the salt from Abbott Laboratories). Intraperi-

toneal injection or applied in vitro. ~qialamide hydrochloride (as the salt from Pfizer). Dissolved in distilled water made

slightly acid with HC1. Intraperitoneal injection or applied in vitro. Desmethylimipramine hydrochloride (D.M.I. as the salt from Geigy). Dissolved in dilute

HC1. Applied in vitro. Phenoxybenzamine hydrochloride (Dibenzyline, as the salt from Smith, Kline & French).

Dissolved in distilled water at 40 ~ C. Applied in vitro. a-Methylparatyrosine hydrochloride (H44/68 as the salt from Kistner, Sweden). Intra-

peritoneal injection. dl-3,4-Dihydroxyphenylalanine (dopa, as the salt from Calbiochem). Dissolved in dilute

HC1. Intraperitoneal injection or applied in vitro. 1-u-Methyl-dopa (as the salt from Merke, Sharp & Dohme). Dissolved in dilute HCh

Intraperitoneal injection or applied in vitro. Nl-dl-Seryl-N2-(2,3,4 trihydroxybenzyl) hydrazine hydrochloride (RO 4-4602, as the salt

from Hoffman-La Roche). Intraperitoneal injection. Bis (4-methyl-l-homopiperazinyl thiocarbonyl) disulphide (FLA 63, as salt from Hassle).

Dissolved in ethyl alcohol. Intraperitoneal injection. 2,4,5-Trihydroxyphenylethylamine hydrochloridc (6-OH-DA, as the salt from Kistner,

Sweden). Injected into the exposed jugular vein under light ether anaesthesia. Dissolved in distilled water containing 2 mg/ml ascorbic acid.

Results

Methodological Considerations

In this work, an a t t e m p t has been made to es t imate the degree of deplet ion

of the fluorescence of adrenergic nerves in s t re tch prepara t ions of the myenter ic and submucous networks of the guinea-pig intest ine. Several factors mus t be considered in eva lua t ing such an es t imat ion. I n the normal preparat ion, there is a spec t rum of fibre intensit ies, f rom ve ry br ight to just detectable. I n addi t ion,

the fibres do not appear to be uni formly affected by drugs. This means t h a t a general decrease in ca techolamine stores results in a decrease in the number of fibres. I n the ear ly stages of deplet ion, a decrease in the to ta l number of fibres w i thou t a decrease in the appa ren t brightness of the most in tensely f luorescent

fibres has been observed. In the case of the br ightes t fibres, i t is probable t h a t a small decrease in the a m o u n t of ca techolamine does not result in a decrease

366 M. Costa and J. B. Furness:

i 0 0 ~ c~ D

50 ~ ~

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Fig. 1. A graphical representation of the time course of depletion of the fluorescence reaction of the adrenergic fibres in the myenteric plexus following the injection of reserpine (5 mg/kg, intraperitoneal). Individual observations in the proximal colon are indicated by the squares and those in the distal colon are indicated by the circles. Lines have been drawn by eye to show the progress of depletion suggested by the results. In the distal colon (and in the duodenum and ileum) there is a rapid depletion of the fluorescence between the first and fourth hours and no fibres are observed after 6 hours. In the proximal colon, depletion is in two stages and the nerves do not completely disappear until 15 or 16 hours. Abscissa,

hours after injection. Ordinate, percentage of the control number of fibres

in the apparen t intensi ty . This m a y arise from sa tura t ion in the format ion of

fluorophores, the exci ta t ion produced by the fluorescent lamp or the discrimina- t ion of the detector (Ritz6n, 1967). The progressive disappearance of the fainter

fibres is possibly a more sensit ive measure of deplet ion than is a visual es t imate of change in fibre intensi ty . The quan t i t a t ive est imations of deplet ion are there-

fore g iven in terms of the number of surviving fibres. Wi th this method, the authors ' independent es t imates of the percentage of the control number of fibres

present in an exper imenta l prepara t ion have near ly always agreed to within 10, and have usual ly agreed to wi thin 5, percentage points. In evaluat ing the results, i t must be kept in mind tha t the relat ion between apparent fibre intensi ty,

proport ions of surviving fibres and catecholamine content are not known.

Figs. 2-7. The appearance of the adrenergic fibres of the myenteric plexus under control conditions and a comparison of the depleting action of reserpine in different areas. Pigs. 2 and 3 are control and Figs. 4-7 show different areas of the myenteric plexus of the one animal sacrificed 3 hours after the intraperitoneal injection of reserpine (5 mg/kg). Fig. 2. The normal appearance of the myenteric plexus of the guinea-pig ileum. • 130. Fig. 3. Part of the myenteric plexus of the proximal colon from an untreated animal. • 120. Fig. 4. The myenteric plexus of the ileum, almost complete disappearance of the fluorescence reaction after 3 hours. • 130. Fig. 5. The duodenum. The plexus is markedly depleted, but not quite as much as in the ileum. • 130. Fig. 6. Proximal colon. The reserpine treatment has caused only a slight reduction of the plexus. • 130. Fig. 7. An area of the distal colon following the injection of reserpine, showing that, although the plexus is substantially depleted, a single fibre with

a normal fluorescent appearance is still observed. • 130


Figs. 2-7


Figs. 8-11

Histoehemistry of Drug Actions on CAs of Intrinsic Intestinal Neurones 369

The Action o/Reserpine Depletion. A graphica l r ep resen ta t ion of the progressive decrease in the

f luorescence of the myen te r i c p lexus of the guinea-pig in tes t ine following the i n t r ape r i t onea l in jec t ion of reserpine (5 mg/kg) is g iven in Fig. 1. I t can be seen f rom this g raph tha t , af ter a shor t pe r iod of s t ab i l i ty , the f luorescence in the myen te r i c plexus in the d is ta l colon fell r ap id ly to a ve ry low value, so t h a t ve ry few nerves could be de tec ted af te r 4 hours. The d i sappea rance of f luorescence was s imilar in the i leum and d u o d e n u m (see below). I n the p rox ima l colon, the f luorescence of the t e rmina l s decreased r ap id ly f rom abou t the second hour to reach 40-50% of normal a t 4 hours. The number of f luorescent t e rmina l s seen in the p rox ima l colon be tween the four th and n in th hour af ter in jec t ion r ema ined a p p r o x i m a t e l y cons tant . The number of f ibres r ap id ly again decreased af te r the t en th hour. Very few fibres could be de t ec t ed a t 13 hours and af ter 16 hours there was a lmost a lways comple te d i sappea rance of fluorescence. I n a few cases, however, shor t segments of f luorescent axons were seen up to 24 hours a f te r the inject ion.

I n the i leum, there was a sl ight decrease in the number of f ibres a n d in f ibre in t ens i ty a f te r 0.5 hours. A t the end of the f i rs t hour, dep le t ion was subs t an t i a l a n d a t 2-3 hours, less t h a n 10% of the f ibres r ema ined (Fig. 4). The fibres of the in t e rnoda l s t rands , which in control p repa ra t ions are the mos t fa int , were the first to d i sappear , b u t a t the same t ime the nodes had become subs t an t i a l l y less f luorescent . I n some of the p repa ra t ions t a k e n be tween 1.5 and 3 hours, long varicose fibres which r an th rough more t h a n one node were found. Such fibres were mos t common in the d is ta l colon and are descr ibed below.

Al though i t began somewhat la ter , the dep le t ion of the d u o d e n u m was s imi lar to t h a t of the i leum. I n p repa ra t ions wi th less t h a n abou t 40% of the no rma l n u m b e r of f ibres de tec table , a lmost all surv iv ing t e rmina l s were in the nodes. Clear va r i a t ions be tween ind iv idua l f ibres were a p p a r e n t a t 3-4 hours ; a l though less t h a n 20% of the fibres could stil l be observed, some of those r ema in ing had an in t ens i ty of f luorescence s imilar to the b r igh tes t f ibres of control p repara - t ions (Fig. 5).

The t ime course of the d i sappea rance of the fluorescence reac t ion of f ibres in the d is ta l colon was s imilar to t h a t in the duodenum. However , e x a m i n a t i o n when less t h a n 20% of the te rmina ls r ema ined (about 3-5 hours a f te r the in jec t ion of reserpine) revea led m a n y fibres which were var icose for long dis tances, pass ing th rough several nodes and following the p r i m a r y s t r ands be tween the nodes (Fig. 7). These axons were of ten followed for 5 or 6 m m and somet imes t h e y could be t r aced for a t least 1 cm. They were followed th rough from 2 to 6 nodes. I n control p repara t ions , the number of fibres in the nodes is too g rea t to be able to t race ind iv idua l axons (Gabel la and Costa, 1967; Furness , 1970; Costa and Gabel la , 1971).

The in i t ia l decrease in f luorescence in the p rox ima l colon p r inc ipa l ly invo lved the fibres of the in t e rnoda l s t r ands and the fa in te r f ibres of the nodes. A t 3 hours,

Figs. 8-11. The time course of depletion of the fluorescence of the myenteric plexus in the proximal colon. In 8, the control appearance. Figs. 9-11 are from animals sacrificed 4, 8 and t2 hours, respectively, after the intraperitoneal injection of reserpine (5 mg/kg). All • 130


Figs. 12-15


when most of the fibres in other parts of the intestine had disappeared (Figs. 4, 5, 7), the majority of fibres in the proximal colon were still observable (Fig. 6). Many of those fibres which persisted seemed to be identical with the very bright clumps of fluorescent fibres which are characteristic of the nodes of this part of the myenteric plexus (Costa and Gabella, 1971; Furness and Costa 1971). Although their disposition was similar to the bright clumps of fluorescent fibres seen in the control, their intensity gradually decreased with time (Figs. 8-11). In partially depleted preparations of up to 10 hours, occasional fibres which passed through the nodes, similar to those of the distal colon, were seen. Fluorescent cells were observed in the myenteric plexus of the proximal colon, although in decreasing numbers, up to 9 hours after the injection.

After the injection of reserpine, a dull green fluorescence developed in the ganglion cells of the myenteric plexus, particularly in the duodenum and ileum. The background fluorescence in the longitudinal muscle also increased.

Varicose fibres with a swollen appearance, similar to degenerating terminals, were seen sometimes in normal preparations. These were more resistant to the action of reserpine than were other fibres in the duodenum, ileum and distal colon, being amongst the last fibres to lose their fluorescence.

The fluorescent fibres of the submucosa, both around the blood vessels and associated with submucous ganglia, disappeared at a similar rate in the duodenum, ileum and proximal colon. The fluorescence decreased rapidly between 1.5 and 3 hours and no fibres could be seen in the duodenum and ileum after about 5 hours. However, in the submucosa of the proximal colon, a few fibres, less than 5 % of the normal number, were observed in some preparations between 5 and 9 hours. Other preparations were completely depleted after 5 hours. The fluorescence of perivascular nerves of the mesentery and of the vessels of the inferior mesenterie ganglia had completely disappeared by 5 hours. The adrenergic nerves of the atrium were very sensitive, complete abolition of their fluorescence being achieved within 3 hours of the injection of reserpine.

Subcutaneous injection of reserpine was more effective in causing depletion of adrenergic nerves than was intraperitoneal injection. The threshold dose for the depletion by reserpine at 18-24 hours was 0.1 mg/kg, subcutaneous, in the ileum. However, the normal complement of fibres was still observed in the myenteric plexus of the proximal colon after such treatment. A higher dose, 0.5 mg/kg, while completely depleting the plexus in the ileum (Fig. 12), caused only a partial disappearance of the fluorescence reaction of axons in the myenteric plexus of the proximal colon (Fig. 13). The subcutaneous injection of 0.75 mg/kg was required to deplete the proximal colon in 18-24 hours. On the other hand,

Figs. 12 and 13. The fluorescence reaction of the adrenergic nerves in the myenteric plexus of the ileum (12) and the proximal colon (13) of a guinea-pig sacrificed 18 hours after a single

sub-cutaneous injection of reserpine, 0.5 mg/kg. Both • 125

:Figs. 14 and 15. The recovery of the fluorescence reaction of adrenergic nerves following an injection of reserpine (2.5 mg/kg, subcutaneous). Both preparations were taken 9 days after the injection. There is no significant difference in recovery between the proximal colon (14)

and the ileum (15). Both • 130


Figs. 16 19


a dose of 0.1 mg/kg on each of the 3 days prior to sacrifice caused a complete disappearance of the fluorescence reaction in the myenteric plexus o3 the proximal colon.

Recovery. In contrast to the depletion by reserpine, the repletion of the adrenergic fibres (after an injection of 2.5 mg/kg reserpine, subcutaneously) was similar in the proximal colon to that in the myenteric plexus of other parts of the gut. There was no indication of a recovery of fluorescence in the cells before the terminals reappeared; neither cells nor fibres were observed at one day or up to 7 days after the injection of reserpine. A few faint fibres and cells were observed at 8 days. On the ninth day, the recovery varied between about 20% and almost complete restitution of the plexus (Figs. 14, 15). At 10 days, the plexus was indistinguishable from control; the ceils were as numerous and as intensely fluorescent as normal.

Depletion by Guanethidine The depletion of the adrenergic nerve terminals of the myenteric plexuses

of the ileum and proximal colon were compared (Figs. 16-18). The fluorescence reaction of the nerves was examined 20 hours after intraperitoneal doses of 0.5 to 500 mg/kg of guancthidine. In the ileum, 0.5 or 1 mg/kg had no observable effect. However, a dose of 5 mg/kg reduced the fluorescence to 20% of normal. With 10 mg/kg no terminals were seen in one animal (Fig. 16) and less than 2% in a second. Doses of 50, 100, 200 and 500 mg/kg completely depleted the terminals. In the proximal colon, the effectiveness of depletion with guanethidine increased as the dose increased up to 50 mg/kg, but higher doses were slightly less effective. No reduction was observed at 0.5 and 1 mg/kg. After 5 mg/kg, 50 % of the fluorescence remained and 10 mg/kg caused depletion to about 30 % (Fig. 17). The greatest depletion achieved, with 50 mg/kg, was a survival of 5-10 %0 of fibres (Fig. 18). Injections of 100, 200 and 500 mg/kg caused depletion to between 20 and 50% of normal, the different doses being about equally effective. The difference in the sensitivity of the proximal colon and ileum is illustrated in Figs. 16 and 17, both preparations being from the same animal. The sensitivity of the adrenergic terminals of the myenteric plexus of the duodenum and distal colon to the action of guanethidine was similar to that in the ileum.

The appearance of the plexus during depletion by guanethidine resembled that seen after reserpine treatment. In the proximal colon, the groups of densely crowded and intensely fluorescent terminals were most persistent. In cases of partial depletion, the paths of individual axons could be traced. Varicose axons

Figs. 16-19. The depletion of the myenteric plexus by guanethidine and its restoration by ~-mothyl-noradrenaline. Guanethidine was administered intraperitoneally, 20 hours prior to sacrifice. Fig. 16. The plexus in the ileum following 10 mg/kg of guanethidine. • 130. :Fig. 17. The proximal colon, showing the depletion caused by guanethidine 10 mg/kg, x 130. Fig. 18. Depletion of the myenteric plexus of the proximal colon by 50 mg/kg of guanethidine. x 200. Fig. 19. The adrenergic nerves of the myenteric plexus in the proximal colon loaded with a-methyl-noradrenaline after depletion by guanethidine. Guanethidine (500 mg/kg) was injected 20 hours before and u-methyl-noradrenaline (10 mg/kg) was injected 1 hour before

sacrifice. • 130

25 Z. Zellforsch., Bd. 120


Figs. 20 and 21. The uptake and retention of ~-methyl-noradrenaline in the myenteric plexus of the proximal colon. The guinea-pig was injected intraperitoneally with reserpine (10 mg/kg), 22 hours before sacrifice. Two pieces of proximal colon were taken and incubated for 30 min with ~-methyl-noradrenaline (10 ~g/ml). In one case, no other drug was present and the fluorescence of the plexus was restored (Fig. 20). In the other, desmethylimipramine (10 -6 g/ml) was included in the incubation medium and the restoration of the plexus was prevented.

Both • 130

were followed through several nodes of the myenterie plexus in the proximal colon and also in other parts of the gut. The fluorescence of the adrenergic nerves of the in tes t ine could be restored by the in t raper i toneal inject ion of e-methyl noradrenal ine (Fig. 19).

The Uptake and Retention o/ Cateeholamines

The res t i tu t ion of the fluorescence of adrenergic axons of reserpinised animals was examined. Reserpine (10 mg/kg) was injected intraperi toneal ly, 18-22 hours before t r ea tmen t with catecholamines. The animals were injected intraperitoneal ly or excised tissue was incubated in Krebs ' solution conta in ing the catecholamine.

e-Methyl-Noradrenaline. In jec t ion of e-methyl-noradrenal ine (2 mg/kg) 15 or 30 min before sacrifice, or incuba t ion of tissue in 10 .7 g/ml e-methyl-noradrenal ine restored the fluorescence in the myenter ie plexus of the proximal colon (Fig. 20) and of other parts of the intestine. The in tens i ty of the fluorescence of varicose terminals was similar to tha t in the control, bu t the non-varicose fibres of the


internodal strands were considerably brighter than those of untreated preparations. The cell bodies also reappeared, some of them brighter than in control, but others were still quite faint. The restitution of fluorescence was similar if noradrenaline (10 -7 g/ml) in the presence of pargyline or nialamide (10 -6 g/ml) was used (see below).

Desmethylimipramine (DMI) or phenoxybenzamine completely inhibited the reappearance of the fluorescence in the terminals and in the cell bodies of the myenteric plexus of the proximal colon (Fig. 21). The tissue was pre-incubated for 15 min in DMI (10 -6 g/ml) or phenoxybenzamine (10-~g/ml) and then was incubated for a further 30 rain with r162 (10 -7 g/ml) in addition to the uptake inhibitor. Whether or not DMI or phenoxybenzamine was present, there was a slight increase in the fluorescence of the background muscle.

The uptake of ~-methyl-noradrenaline by the fibres of the myenteric plexus, in vitro, was compared in the proximal colon and ileum using different concentrations of the amine. In these experiments, ileum and proximal colon segments were incubated in the same container. The threshold concentration for the appearance of fibres in the myenteric plexus of the proximal colon was 10 -s g/ml. Only a few faint fibres were seen at this concentration, but none were observed in the ileum. After incubation at 3 x 10 -s g/ml there was an homogeneous fluorescence of the fibres in the ileum, but their intensity was only 3 0 4 0 % of normal. Restoration in the proximal colon was similar, but it was notable that the bright clumps of terminals normally observed were not prominent. Incubation in 10 -7 g/ml restored the fluorescence to normal in both parts, except that non- varicose fibres were slightly brighter and the closely packed clumps of the proximal colon did not stand out. At 3 x 10-L there was further increase in fluorescence intensity of non-varicose fibres in both the ileum and proximal colon. In the latter, there was also an increase in the intensity of fluorescence of the closely packed clumps of terminals. With increasing concentration (10 -6, 3 x 10 -6 and 10 -5 g/ml) the relative intensity of fluorescence of the background muscle increased, but there was not such a pronounced increase in fibre intensity. The clumps of fibres in the proximal colon increased in brightness as the concentration of ~-methyl-noradrenaline was increased up to 10 -5 g/ml. Cell bodies in the proximal colon were observed after incubation at 10 -7 g/ml and became brighter with greater concentrations. At 10 -6 g/ml many of the cell bodies were brighter than in control.

Loading experiments were also performed using the proximal colon from guinea-pigs reserpinised 4 days after interruption of the extrinsic nerves. In this case, 60-70% of the number of fibres in the control appeared in the myenteric plexus after exposure to ~-methyl-noradrenaline, but there was no reappearance of fluorescence in the submucous plexus.

Noradrenaline. The reappearance of fluorescence was examined in tissue from guinea-pigs reserpinised in the manner described above. Noradrenaline was used alone or in combination with an inhibitor of the action of monoamine oxidase. In vivo application was by intraperitoneal injection of 1-2 mg/kg, 30 min before sacrifice. Otherwise, incubation with 10 -7 g/ml noradrenaline in Krebs' solution was used. Some tissue was incubated with nialamide or pargyline (10 -6 g/ml)

25*


Figs. 22-25


for 15 min before the noradrena l ine was a d d e d and then wi th the monoamine oxidase inh ib i to r and noradrena l ine toge ther for a fur ther 30 rain.

W i t h o u t monoamine oxidase inhib i t ion , there was no res to ra t ion of the myen te r i c plexus of the i leum (Fig. 22) or of the submucous plexus of the p rox ima l colon. On the o ther hand, monoamine oxidase inh ib i t ion a l lowed comple te res to ra t ion of these plexuses b y noradrena l ine (Fig. 23).

I n the myen te r i c p lexus of the p r o x i m a l colon there was usua l ly some res tora- t ion of the fluorescence even wi thou t the inac t iva t ion of monoamine oxidase. The in jec ted noradrena l ine gave 50-90% res to ra t ion in 4 exper iments , bu t in 3 o ther exper iments less t han 5 % recovery of the plexus was observed. I n v i t ro , the res to ra t ion was 30-70% (Fig. 24). The plexus had a s imi lar appea rance to t h a t af ter loading wi th ~-methyl -noradrena l ine . Br igh t var icose f ibres were seen in the nodes and the non-var icose f ibres were more in tense ly f luorescent t h a n in no rma l t issue. The ada'energic cells of the p rox ima l colon were b r igh te r t h a n in control , b u t t hey were also more evenly f luorescent so t h a t the nuclei were obscured.

The same exper iments were done using ex t r ins ica l ly dene rva t ed sections of p rox ima l colon and i leum. No fluorescence appea red in the i leum or the submucosa of the p r o x i m a l colon. The myen te r i c p lexus of the p r o x i m a l colon was res tored to abou t 60% of t h a t of the denerva ted , b u t no t otherwise t r ea ted , plexus. The appea rance of the fibres was s imi lar to t h a t of non -dene rva t ed p repa ra t ions f rom reserpinised an imals which were loaded wi th noradrenal ine .

The res to ra t ion of the plexus in the p r o x i m a l colon was more comple te if monoamine oxidase was inh ib i t ed (Fig. 25). I n combina t ion wi th n ia lamide or pargyl ine , noradrena l ine caused the r eappea rance of all the plexus. The non- var icose fibres appea red s l igh t ly br igh te r t han in control p repara t ions , b u t the fluorescence of the dense c lumps of t e rmina l s was no t fu l ly res tored. Adrenerg ic eel1 bodies a p p e a r e d s imilar to normal . I n add i t ion , in jec t ion of n ia lamide alone (100 mg/kg, in t raper i tonea l , 3 hours before sacrifice) caused a def ini te increase in the fluorescence in t ens i ty of var icose axons, of the non-var icose axons and of the cell bodies in the myen te r i c plexus of the p rox ima l colon (Figs. 26, 27). Similar resul ts were ob ta ined if pa rgy l ine was used in s t ead of n ia lamide .

The Synthesis o] Catecholamines. The neurona l a p p a r a t u s for the synthes is of ea techolamines was tes ted b y a t t e m p t i n g to res tore the fluorescence in dep le ted nerves b y prov id ing the amine precursors dopa and ~ -methy l -dopa and also b y

Figs. 22-25. Comparison of the uptake and retention of noradrenaline and the effect of inhibition of monoamine oxidase in the myenteric plexuses of the ileum and proximal colon. The tissue was taken from a reserpinised animal (10 mg/kg, intraperitoneal, 20 hours prior to sacrifice). I t was then incubated with noradrenaline (10 -6 g/ml) either alone or with pargyline (10 -~ g/ml). Fig. 22. Ileum from a reserpinised guinea-pig incubated with noradrenaline alone. There is no restoration of the plexus. • 130. Fig. 23. The restoration of the fluorescent fibres in the ileum from a reserpinised guinea-pig incubated with both noradrenaline and pargyline. • 130. Fig. 24. The myenteric plexus of the proximal colon of a reserpinised guinea- pig after incubation with noradrenaline. Note that there is partial restoration of the fluorescence of adrenergic nerves, x 130. Fig. 25. The restoration of the fluorescence in the proximal

colon by noradrenaline plus pargyline. • 210


Figs. 26-29


t ry ing to prevent this restoration, or to deplete control preparations, with inhibitors of catecholamine synthesis, ~-methyl-p-tyrosine ; RO 4-4602 and F L A 63.

The tyrosine hydroxylase inhibitor, u-mcthyl-p-tyrosine, was ineffective in causing depletion of the adrenergic fibres of any par t of the guinea-pig intestine. Preparat ions taken from animals sacrificed 21 hours after 500 mg/kg or 3 hours after the last of a series of 4 injections of 100 mg/kg at 3 hour intervals were not significantly different f rom normal. To test if a conversion of :c-methyl- p-tyrosine to g-methyl-noradrenal ine (Maitre, 1965; Orden et al., 1970) might explain this result, guinea-pigs were injected with reserpine (5 mg/kg, intraperitoneal) 16 hours after ~-methyl-p-tyrosine (500 mg/kg, intraperitoneal) and the animals were sacrificed after a fur ther 6 hours. No protect ion of the catceholamines f rom depletion with reserpine could be a t t r ibu ted to ~-methyl-p-tyrosine. An appreciable format ion of ~-methyl-noradrenaline in the present experiments therefore seems unlikely.

The fluorescence reaction of nerve terminals and of the adrenergic cells which had been depleted by reserpine (10 mg/kg, intraperi toneal 16-20 hours before) could be restored by the injection of sr (100 mg/kg), 1-4 hours before sacrifice. A similar restorat ion was achieved by the injection of pargyline, 100 mg/kg, 3 hours before and dl-dopa, 100 mg/kg, 1 hour before sacrifice (Fig. 28). I n some experiments an inhibitor of dopa-decarboxylase (1%O 4-4602; Burkard, Gey and Pletscher, 1964; Bartholini and Pletscher, 1 9 6 8 ) o r the inhibitor of dopaminc-fl-hydroxylase, F L A 63 (Corrodi, Fuxe, Hamberger and Ljungdahl , 1970) was used. The inhibitors (100 mg/kg) were injected one hour after the pargyline. RO 4-4602 almost completely prevented the restorat ion of fluorescence in the plexus in the proximal colon (Fig. 29) as well as in the other areas of the gut. Under the same conditions F L A 63 did not affect the restorat ion of the fluorescence reaction of the plexus. Trea tment with F L A 63 alone (50-200 mg/kg, injected intraperitoneally, 4-8 hours prior to sacrifice) had no effect on the fluorescent reaction of adrenergic nerves in any par t of the intestine.

I n some experiments, a fluorescence reaction appeared in a sma]l propor t ion of the cells of the myenter ic plexus of the ileum and duodenum after the injection of ~-methyl-dopa or of dopa plus pargyline into reserpinised animals.

Fig. 26. The appearance of the myenteric plexus of the proximal colon in a control preparation. • 130

Fig. 27. The increase in the fluorescence intensity of the fibres of the myenteric plexus by the inhibition of monoamine oxidase. Pargyline (100 mg/kg) was administered by intraperitoneal injection, 3 hours before sacrifice of the animal. Compare this with Fig. 26. • 130

Figs. 28 and 29. The restoration of the fluorescence in the myenteric plexus of the proximal colon by dopa. The animals were both injected with reserpine (10 mg/kg, intraperitoneal, 20 hours before sacrifice), pargyline (100 mg/kg, intraperitoneal, 3 hours before sacrifice) and dl-dopa (100 mg/kg, intraperitoneal, 1 hour before sacrifice). In addition, the animal from which Fig. 29 is taken was injected with the dopa-decarboxylase inhibitor, RO 4-4602 (100 mg/kg, intraperitoneal), 2 hours before sacrifice. In the presence of an inhibitor of monoamine oxidase, dopa restores the fluorescence reaction of the adrenergic nerves (Fig. 28) but if the synthesis step from dopa to dopamine is blocked, there is no restoration (Fig. 29).

Both • 130


Figs. 30 33


The Action o/6-OH-DA. I t has been shown previously that 6-OH-DA causes a long-lasting depletion of noradrenaline from peripheral tissues, but that adrenergic ganglia are significantly less sensitive to its action (Laverty, Sharman, and Vogt, 1965; Mamlfors and Sachs, 1968; Thoenen and Tranzer, 1968; Tranzer and Thoenen, 1968). In the present work, guinea-pigs were sacrificed 18-24 hours after the injection of 100-350 mg/kg of 6-OH-DA into a jugular vein.

The myenteric plexus of the stomach, ileum, caecum and distal colon and the submucous plexus of all parts of the gut were similarly affected by 6-Ott-DA ; the number of varicose, terminal fibres was reduced to less than 5% of normal. Many of the smooth, non-terminal fibres became more brightly fluorescent after 6-OH-DA treatment. Some of the remaining axons were greatly swollen for short distances. This gave the appearance of segments of varicose axons with the varicosities as large as 30 ~ across. As with reserpine or guanethidine depletion, the sensitivities of individual fibres were different and single surviving fibres could sometimes be traced for several hundred microns.

The adrenergic neurones of the myenteric plexus of the proximal colon were less susceptible to depletion by 6-Ott-DA than were fibres in other parts of the gut; between 20% and 60% of the terminal fibres were still observed 24 hours after the injection (Figs. 30, 31). The degree of depletion of the fluorescence showed considerable variation between fibres (Figs. 32, 33). This variation was eliminated if the animal was also injected with :r (10 mg/kg ; intraperitoneal), 1 hour before sacrifice (Figs. 30, 31). I t appeared that only those fibres whose fluorescence survived until 24 hours after 6-OH-DA were capable of taking up ~-methyl-noradrenaline; loading with this drug did not increase the number of fibres observed. The ceil bodies appeared brighter after the injection of 6-Ott-DA (Figs. 30, 31 ; see also Furness and Costa, 1971). As in other parts of the gut, the course of single fibres could easily be followed after 6-Ott-DA (Figs. 32, 33). The increase in fluorescence intensity of non-terminal fibres and the exaggerated swelling of some fibres were also observed in the proximal colon.

Discussion

The effects of drugs on the uptake and retention of catecholamines was qualitatively similar for the intrinsic adrenergic neurones of the proximal colon and for the extrinsic adrenergic neurones supplying other parts of the intestine or other organs. The intrinsic adrenergic nerves can be depleted of catecholamines by reserpine or guanethidine, in common with other neurones containing noradrenaline (Muscholl and Vogt, 1958; Sheppard and Zimmermann, 1959; Cass,

Figs. 30-33. The appearance of the myenteric plexus of the proximal colon of the guinea-pig, 24 hours after the intraperitoneal injection of 6-OH-DA. Fig. 30. The plexus after an injection of 250 mg/kg 6-OH-DA. The guinea-pig was injected with u-methyl-noradrcnaline (10 mg/kg, intraperitoneal) 1 hour before sacrifice. • 200. Fig. 31. The plexus after 6-OH-DA (200 mg/kg) and ~-methyl-noradrenaline (10 mg/kg, intraperitoneal, 1 hour). • Fig. 32. The differ- ential effects of 6-OH-DA (200 mg/kg) on adrenergic fibres of the plexus. Some single fibres are as brightly fluorescent as normal and can be readily traced through the plexus. Other fibres are only faintly fluorescent. • 130. Fig. 33. Single axons remaining after the depletion

of most of the plexus by 6-OH-DA (200 mg/kg). • 130


Kuntzman and Brodie, 1960). These neurones are also capable of concentrating and retaining catecholamines, and this uptake mechanism is blocked by desmethylimipramine or phenoxybenzamine, potent inhibitors of the transport of catecholamines into adrenergic neurones (Axelrod, Whitby and Hertting, 1961; Axelrod, Hertting and Potter, 1962). The fluorescence of the intrinsic adrenergic neurones of reserpinised animals is restored by the monoamine precursor, dopa, and this restoration is prevented by inhibition of the conversion of dopa to dopamine. All this evidence strongly suggests that the intrinsic adrenergic neurones are very similar to other adrenergic neurones in the mechanisms of concentration, synthesis and storage of catecholamines.

However, there are some distinct quantitative differences in the effects of" drugs on the intrinsic adrenergic neurones compared with extrinsic adrenergic neurones which supply the gut. The intrinsic neurones are more resistant to depletion by reserpine, guanethidine or 6-OH-DA. On the other hand, they are more able to store noradrenaline following depletion by reserpine. The depletion of the terminals in the myenteric plexus of the proximal colon by reserpine was in two stages. The initial depletion was similar to that in other intestinal plexuses but this was followed by a period of stability, whereas depletion con- tinued in other areas. I t was previously shown that the myenteric plexus of the proximal colon contains fibres of both extrinsic and intrinsic origin; the latter form the dense clumps of brightly fluorescent fibres in the proximal colon that survive extrinsic denervation (Furness and Costa, 1971). These clumps are still observed at a stage after reserpine injection when fibres in other parts of the intestine have disappeared. Thus it is likely that the initial depletion of nerve terminals seen after reserpine is from extrinsic fibres and that the intrinsic fibres are more resistant to depletion. The extrinsic adrenergic terminals in the perivascular and submucous plexuses of the proximal colon are depleted by reserpine with a similar time course to those of the plexuses of other parts of the gut. A different exposure to reserpine is therefore an unlikely explanation for the reserpine resistance of the fibres of the proximal colon.

Several other instances of a variation of reserpine sensitivity between groups of adrenergic terminals are known. Terminals of the adrcnergic neurones of the pelvic plexus which innervate the genital organs are more slowly depleted by reserpine than are those of organs which are not so close to the ganglia which supply them (Nilsson, 1964; Owman and Sj6berg, 1967; Sj6strand and Swedin, 1968). Norberg (1965) observed that reserpine was more effective in depleting terminals of the submaxillary blood vessels than of the acini or of the muscula- ture of the vas deferens. I t has been suggested that short neurones may represent a "specific entity of adrenergic tissue" different from the more usual long adrenergic neurones (Sj6strand and Swedin, 1968). The terminals of the adrenergic neurones of the proximal colon are certainly close to the cell bodies and they are therefore, morphologically, "short neurones". The recovery after reserpine in both the intrinsic and extrinsic terminals of the guinea-pig intestine took about the same time. This is in contrast to the comparison of short and long adrenergic neurones made in other studies; it has usually been found that short neurones are faster to recover than are long neurones (Norberg, 1965; Owman and Sj6berg, 1967).


The differences observed in the present work could involve a difference in the activity of monoamine oxidase in the terminals of intrinsic and extrinsic adrenergic neurones of the gut, the activity in the terminals of intrinsic neurones being lower. Reserpine prevents the retention of cateeholamines in the granular storage particles but does not act on the uptake of catceholamines into the cytoplasm or on their cytoplasmic storage (Euler and Lishajko, 1963; Stj~rne, 1964 ; Hamberger, 1967). The presence of monoamine oxidase inhibits the increase in cateeholamine content in the cytoplasm. However, if the intraneuronal oxida- tion of eatccholamines is inhibited, depletion by reserpine is antagonised (Shore, Alpers and Busfield, 1965). A lower monoamine oxidase activity could also allow a selective retention of noradrenaline in the terminals of intrinsic neurones of reserpinised animals. This selective retention was observed following in vitro application of noradrenaline, so the difference does not arise from a difference in exposure. I t is also possible that a reserpine resistant uptake into amine storage granules (Lundborg, 1967) occurs in the intrinsic but not in the extrinsic adrenergic axons of the intestine. Lesser monoamine oxidase activity could also contribute to the relative resistance of intrinsic adrenergic neurones to depletion by guanethidine. Chang, Costa and Brodie (1965) found that peripheral adren- ergically innervated tissues contained two binding sites for guanethidine, which they described as specific and non-specific. Guanethidine competed with noradrenaline for binding at the specific site and this binding was antagonised by reserpine. I t thus seems that guanethidine displaces noradrenaline from granule storage sites. Lundborg and Stitzel (1968) have shown that guancthidine causes a proportionately greater displacement of eateeholamine from granular storage sites than it does from other sites. The retention of catecholamines in the cytoplasm may be greater in the intrinsic neurones than in the terminals of extrinsic neurones supplying the gut, if monoamine oxidase activity is lower. I t was also found, in the present work, that 6-OH-DA was less effective in depleting the intrinsic neurones of the proximal colon than it was in depleting adrenergic terminals of extrinsic origin. The reported delaying effect of monoamine oxidase inhibition on the depletion of adrenergic terminals by 6-OH-DA (Malmfors and Sachs, 1968) supports an hypothesis of low monoamine oxidase activity in the intrinsic neurones. I t is not suggested that there is no monoamine oxidase activity in the intrinsic neurones. In fact, it was found that these neurones are more able to retain noradrenaline after monoamine oxidase inhibition and that the inhibition of this enzyme increased the fluorescence intensity of the terminals of otherwise untreated animals. Although an hypothesis that monoamine oxidase activity differs between neurones in consistent with the observations, it is recog- nised that differences in other properties, such as binding within granules, could be contributory factors.

Some unusual neurones which do not normally contain sufficient cateeholamine to be observed can be revealed in the ciliary ganglion after the application of 1-dopa plus nialamide (Ehinger and Falck, 1970). These authors found similar neurones in the intramural ganglia of the rat intestine. The type of neurone observed by Ehinger and Falek (1970) is clearly different from the intrinsic adrcnergic neurones of the guinea-pig proximal colon. In the proximal colon, the cell bodies of the intrinsic neurones are readily observed without any pre-


t r e a t m e n t (Costa, Furness and Gabella, 1971; Furness and Costa, 1971) and

at least some of thei r processes appear to be ext remely br ight ly fluorescent (Furness and Costa, 1971). Nei ther the cell bodies nor their terminals in the ciliary

ganglion are normal ly observed. In the present work, some fluorescent cells were observed in the myenter ic plexus of the small intest ine after the inject ion

of dopa and the inhibi t ion of monoamine oxidase. Fur ther exper iments are required to test if these neurones in the intest ine are comparable to those reported

by Ehinger and Fa lck (1970). I t is l ikely tha t there is a range of types of adrenergic neurone which includes those described by Ehinger and Falck, those of the myenter ic plexus of the proximal colon and the several types found in other gangl~ .

Despite quan t i t a t ive differences, i t is considered that , in their mechanisms

of uptake, storage and synthesis of catecholamines, the intrinsic adrenergic neurones of the myenter ic plexus of the guinea-pig proximal colon are quali-

t a t ive ly similar to the adrenergic neurones of pre- and para- ver tebral ganglia.

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J . B. Furuess Department of Physiology The Medical School Birmingham University Birmingham, 15 U.K.

M. Costa Department of Zoology University of Melbourne Parkville, 3052 Victoria, Australia

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