J. Physiol. (1977), 267, pp. 767-789 With 11 text-figure Prine in Grea Britain

767

THE SITE AND RECEPTORS RESPONSIBLE FOR THE INHIBITION BY SYMPATHETIC NERVES OF INTESTINAL SMOOTH MUSCLE AND ITS PARASYMPATHETIC MOTOR NERVES

BY J. S. GILLESPIE AND M. A. KHOYI* From the Department of Pharmacology, Univer8ity of (iiagow, Glasgow (12 8QQ

(Received 19 October 1976) SUMMARY

1. The effects of three inhibitory stimuli, sympathetic nerve stimulation, noradrenaline (NA) and isoprenaline have been examined on three forms of motor activity in the rabbit colon, the response to pelvic (parasympathetic) nerve stimulation, acetylcholine (ACh) and spontaneous tone. 2. The response to pelvic nerve stimulation is most effectively inhibited by sympathetic nerve stimulation, much less effectively by NA and hardly at all by isoprenaline. The sympathetic nerves can inhibit the pelvic response at frequencies of stimulation which do not affect spontaneous tone. The inhibitory effect of sympathetic stimulation, and of NA, on the pelvic response is reduced by phentolamine 5 x 10-6 M and unaffected by propranolol 5 x 10-6 M suggesting the effect is mediated via a receptors. 3. The response to ACh is inhibited by all three stimuli equally. The inhibitory effect of sympathetic nerve stimulation and of isoprenaline is reduced by propranolol 5 x 10-6 M. The inhibitory effect of NA is also reduced by propranolol but to a lesser extent. Phentolamine 5 x 10-6 M has a small effect in reducing the inhibitory effect of sympathetic nerve stimulation or of NA. This effect of phentolamine is lost if the participation of motor nerves in the response to ACh is excluded by either tetrodotoxin 10-7 g/ml. or cold storage for 10- 14 days. These results suggest that inhibition of the ACh response takes place mainly at the muscle by activation of /8 receptors but that ACh may have a small indirect stimulant action through motor nerves and this is susceptible to inhibition through a receptors. 4. All three stimuli are equally effective in lowering smooth muscle tone. This inhibitory effect of sympathetic nerve stimulation and of isoprenaline * Present address: Department of Pharmacology, University of Tehran, Tehran 14, Iran.

J. S. GILLESPIE AND M. A. KHOYI 768 is reduced by propranolol 5 x 106M and unaffected by phentolamine 5 x 106 M. The inhibitory effect of NA is reduced by propranolol but again is less sensitive to block than the other two inhibitory stimuli. Phentolamine is without effect on the inhibitory action of NA and the combination of phentolamine with propranolol is no more effective than propranolol alone. These results suggest that NA liberated by sympathetic nerves and isoprenalins inhibit myogenic tone in the smooth muscle by an action on fi receptors but the action of NA added to the bath cannot be fully explained in this way. INTRODUCTION

Sympathetic nerve stimulation has long been known to inhibit the smooth muscle of the gut. Until the development of fluorescent microscopical techniques capable of visualizing adrenergic nerves it was assumed this was due to a direct sympathetic innervation of the smooth muscle cells. Fluorescence microscopy, however, showed that the great majority of adrenergic nerve terminals ended on the ganglion cells of Auerbach's plexus (Norberg, 1964). This observation raises the possibility that part or all of the observed smooth muscle inhibition is due to inhibition of motor nerves involved in the maintenance of tone. This possibility has previously been investigated by studying the effects of exogenous catecholamines on the response of the isolated intestine to transmural stimulation or acetylcholine (Kosterlitz, Lydon & Watt, 1970). There are at least three difficulties in applying such results to the physiological inhibition by the extrinsic sympathetic nerves. Firstly, exogenous catecholamines may have access to sites not available to the NA liberated by the sympathetic nerves. Secondly, transmural stimulation will affect all neuronal structures and in addition will give rise to both orthodromic and antidromic nerve action potentials. Thirdly, since it is known that ACh acting through muscarinic receptors on adrenergic nerve terminals can inhibit NA release, sympathetic nerves might be relatively ineffective at a cholinergic synapse. The intention of the present experiments was to obtain direct information on the effectiveness of sympathetic nerve stimulation on a known motor cholinergic pathway to the gut by using the doubly innervated rabbit colon preparation (Garry & Gillespie, 1955). In this preparation the' inhibitory effect of extrinsic sympathetic nerve stimulation on the motor response to orthodromic excitation of the extrinsic pelvic (parasympathetic) nerve can be examined and compared with the effects of exogenous catecholamines and the receptors involved in any inhibition analysed by studying the effects of a and , adrenoceptor and muscarinic blocking agents.

769 INHIBITION OF INTESTINAL MOTOR NERVES A preliminary account of these experiments has previously been published (Gillespie & Khoyi, 1974). METHODS Rabbits weighing between 1-5 and 2 kg were killed by a blow on the head, bled and lengths of approximately 4 cm of terminal colon retaining the extrinsic lumbar colonic (sympathetic) and both pelvic (parasympathetic) nerves were isolated as described by Garry & Gillespie (1955). These preparations were mounted in a 100 ml. bath of Krebs saline under a resting tension of 3-4 g. Contractions were recorded isotonically and displayed on a Servoscribe recorder. The sympathetic and pelvic

nerves were drawn through separate Ag/AgCl rubber-diaphragm electrodes. Both pelvic nerves were included in a single electrode. The Krebs saline was maintained at 37°C and gassed with a mixture of 95 % 02 + 5 % CO2. The nerves were stimulated with 0 5 msec pulses of supramaximal voltages. Submaximal motor responses to stimulation of the pelvic (motor) nerve at frequencies between 2 and 10 Hz or approximately matching submaximal responses to ACh were used and frequencyresponse curves for the inhibition of these motor responses were constructed by stimulating the sympathetic nerves at frequencies between 0-5 and 50 Hz. This produced inhibition increasing from about 20% at 5 Hz to over 90 % at 50 Hz. Dose-response curves over a similar range of inhibition were constructed with appropriate concentrations of NA or isoprenaline The contact time for ACh was 40 sec and the duration of motor nerve stimulation 5 sec. The timing of the inhibitory stimuli in relation to the test motor stimulus was such as to produce maximum inhibition. For sympathetic nerve stimulation this meant beginning stimulation 15 sec before and continuing 5 see after the end of the test stimulus, a total duration of sympathetic nerve stimulation of 25 sec when the test stimulus was the pelvic nerve and 60 see when the test was ACh. When inhibition was produced by the catecholamines these were added 60 see before the test motor stimulation. The receptor blocking agents phentolarnine, propranolol or atropine were added at least 20 min before the agonist. The various inhibitory stimuli not only reduced the test response to pelvic nerve stimulation or ACh but also lowered the tone of the preparation. In calculating the percentage inhibition of the test motor response, the change in base line was ignored and the amplitude of the response measured from the new base line expressed as a percentage of the control response previous to inhibition. There are difficulties in this method but since all three inhibitory stimuli were almost equally effective in lowering the base line, comparisons between them are probably valid. The following drugs were used: acetylcholine chloride (Koch-Light), ascorbic acid (B.D.H.), atropine sulphate (B.D.H.), cocaine hydrochloride (Macarthys), 17/) oestradiol (Sigma), ethylene diamine tetra-acetate (EDTA) (Sigma), isoprenaline sulphate (Burroughs Wellcome), mecamylamine hydrochloride (Sigma), noradrenaline bitartrate (Koch-Light), phentolaminemesylate (Ciba), propranolol hydrochloride (J.C.I ), tetrodotoxin (Sankyo). Doses refer to the base. Stock solutions of catecholamines were made up in 0-01 N-HCl containing 10 #ag ascorbic acid/ml. and the Krebs saline when these drugs were used contained EDTA 10 #sg/ml. and ascorbic acid 20 ,sg/ml.

770

J. S. GILLESPIE AND M. A. KHOYI RESULTS

Inhibition of the parasympathetic motor response The ability of three stimuli, extrinsic sympathetic nerve stimulation, NA and isoprenaline, to reduce the height of the motor response to pelvic nerve stimulation was examined. Each inhibitory stimulus was started before the pelvic nerves were stimulated and continued for an interval which resulted in maximum inhibition (see Methods). The greatest inhibitory effect was by sympathetic nerve stimulation. Frequencies between 1 and 50 Hz produced a graded inhibition of the pelvic motor response varying from 20 % at 5 Hz up to almost complete suppression of contraction at 50 Hz (Figs. 1, 2). Sympathetic nerve stimulation was more effective in suppressing the parasympathetic contraction than in lowering spontaneous tone so that, as Fig. 1 illustrates, low frequencies within the physiological range of 1-5 Hz were able to inhibit the pelvic response with no effect on either tone or rhythmic activity. Even lower frequencies of sympathetic nerve stimulation, 0 5 and 1 Hz, were also examined to see whether low release rates of NA could potentiate the parasympathetic response by facilitating ganglion transmission. For these experiments test stimulation of the pelvic nerve at 2 and 5 Hz which produce submaximal motor responses were used in addition to the standard 10 Hz which produces a near maximal motor response. No evidence of potentiation was found in any combination. The inhibition of the parasympathetic motor response by sympathetic nerve stimulation was reduced by phentolamine 5 ,M without interfering with the inhibition of tone and rhythmic activity (Figs. 2, 3). Propranolol 5 and 10/EM had no effect (Fig. 3). Noradrenaline in concentrations between 0 04 and 10-2 ,ug/ml. also inhibited the parasympathetic motor response though less effectively than sympathetic nerve stimulation and never in concentrations which did not themselves lower tone (Figs. 1, 3). The doses of NA used roughly matched the inhibitory effect of sympathetic nerve stimulation on spontaneous tone yet were less than half as effective as the sympathetic nerves in inhibiting the parasympathetic motor response. Nevertheless, this inhibitory effect on the parasympathetic response was reduced by phentolamine 5 /zM and unaffected by propranolol 5 or 10 /M (Figs. 2, 3). Isoprenaline in concentrations up to 10-2 /zg/ml. had no significant inhibitory effect on the response to pelvic nerve stimulated at 2, 5 or 10 Hz in spite of producing powerful relaxation and a fall in tone of the preparation (Figs. 1, 3).

INHIBITION OF INTESTINAL MOTOR NERVES r P

-_ 1

Hz

2 Hz

c

-

or 5 Hz

10 Hz

771

20 Hz

P

j P~it1 h, p ra jl jl *

*

*

@

*

*

S

iI a

0

S

5

6

0

.

Fig. 1. The effect of lumbar sympathetic nerve stimulation (upper row), noradrenaline (middle row) and isoprenaline (lower row) on the response of the rabbit colon to pelvic (parasympathetic) nerve stimulation. The records are from three separate experiments. In each row the control response to pelvic stimulation (P) is illustrated first, in the upper record the frequency of stimulation of the lumbar sympathetic nerves in Hz is given below each response; in the remaining two rows the comparable figures give the concentration of noradrenaline and isoprenaline in pg/ml. For details see text.

J. S. GILLESPIE AND M. A. KHOYI

772 rj-y-

r

5 Hz 10 Hz

0

IT--- r 5 Hz

20 Hz 50 Hz

10 Hz

20Hz

r-50Hz

I1

L4 --. -L-ir n-4-IP -L-i0. 64 0'16 2*56

10.2

"iv

N

T

0 LrLy -L2Ir0-16 0'64 2-56

Al

10 2

10'2

I Ig

I.1 %,

Fig. 2. The effect of phentolamine on the inhibition of the pelvic response (P) by sympathetic nerve stimulation (top row) or noradrenaline (bottom row). Between the first and second panel in each row phentolamine was added to the bath to produce a concentration of 5 x 10-6 M. The figures below the stimulus markers indicate the frequency of sympathetic nerve stimulation in Hz or the bath concentration of noradrenaline in /,g/ml. Phentolamine reduces the inhibitory effect of both sympathetic nerve stimulation and noradrenaline on the pelvic motor response but has no effect on the ability of these two types of stimulation.to lower tone and reduce rhythmic activity.

INHIBITION OF INTESTINAL MOTOR NERVES

773

Inhibition of the response to ACh The test response to ACh in these experiments was matched to that of the response to pelvic nerve stimulation; this usually required a bath concentration of between 2 x 10-8 and 3 x 10-7 g/ml. Sympathetic nerve stimulation inhibited the response to ACh in a frequency related fashion E 100 E

Ex

80

0

60

I:

0 C

40

-

,

4

,/|

20

C

-

10 -4 10 5

20 10 Frequency (Hz)

50

0-04 0-16 0-64 2-56 10-2 0-04 0-16 0-64 2-56 10-2 Noradrenaline (pg/ml.) Isoprenaline (pg/ml.)

Fig. 3. The inhibitory effect of sympathetic nerve stimulation, of noradrenaline and of isoprenaline on the response of the rabbit colon to pelvic nerve stimulation and the effect on this of phentolamine or propranolol. Each graph shows the control response (@-0) and the response in the presence of either phentolamine 5 x 10-6 M (0---0) or propranolol 5 x 10-6 M ( x - x ). Sympathetic nerve stimulation is much mole effective than noradrenaline and isoprenaline in causing inhibition. The inhibitory effect of both sympathetic nerve stimulation or noradrenaline is reduced by phentolamine and unaffected by propranolol suggesting an action mediated by a receptors. Each point is the mean of either 12 (control sympathetic nerves and noradrenaline) or six experiments and the vertical bars are + one s.E. The effect of phentolamine is statistically significant at the P < 0-05 level or better at all points for sympathetic nerve stimulation and at all points other than 0-04 and 0-64 for noradrenaline.

varying from 25 % inhibition at 5 Hz to 79 % inhibition at 50 Hz (Figs. 4, 6). This inhibition was reduced by propranolol 5 /%M (Figs. 5, 6). Phentolamine 5/tM was less effective; at low frequencies of sympathetic nerve stimulation it had no effect but at 20 and 50 Hz it did significantly reduce the response (Fig. 6). Noradrenaline in concentrations between 0-02 and 2-56 ,ug/ml. also inhibited the response to ACh and in contrast to inhibition of the parasympathetic response NA was more effective than sympathetic nerve

774

J. S. GILLESPIE AND M. A. KHOYI --r

LJL

-

5Hz

0

y

-

10Hz

0

002

0*04

0

Lf

20Hz

0*16

50Hz

0'"el#

004

0.16

0-64

TF

0 0*32

2*56

10-2

Fig. 4. The effect of sympathetic nerve stimulation (upper row), noradrenaline (middle row) and isoprenaline (lowe rrow) on the motor response

to ACh. The addition of ACh is marked by a black dot. In each row the first response is to ACh alone, in the experiment illustrated in the upper records in a bath concentration of 1O-7 g1ml and in the other two experiments in a concentration of 5 x 10-8 g/ml. The remaining responses show the effect of sympathetic stimulation at the frequencies shown or the catecholamines in the concentrations shown on this response to ACh. All three stimuli can almost completely abolish the response.

INHIBITION OF INTESTINAL MOTOR NERVES

mLj~~5Hz

10Hz

0

0

20Hz

5 Hz

*

0.16

5Hz

r-

10Hz

0

o-t-F-L 0-16

0

-'r ,L -20Hz

50Hz

0 64

2-56

*

0-64

2X56

0@04

0*16

0 -64256

004 016

-I-r--

775

56@

0

0064

0

-1r r 2-56

10-2

0

0

*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

*

0*

Fig. 5. Three separate experiments illustrating the effect of propranolol 5 X 10-6 M on the inhibitory effect of sympathetic nerve stimulation (upper row), noradrenaline (middle row) and isoprenaline (lower row) on the response of the rabbit colon to ACh and on its tone and rhythmic activity. The frequency of sympathetic nerve stimulation or the dose of catecholamine isshown undertheappropriate event marker. The black dot marks the addition of ACh. Propranolol 5 x 10 6M was added between the two panels in each row and reduces the ability of the three stimuli to inhibit the response to ACh and also their ability to lower tone and inhibit rhythmic activity.

776 J. S. GILLESPIE AND M. A. KHOYI stimulation (Fig. 6). The inhibitory effect of all concentrations of NA was reduced by propranolol 5,UM, though the effects were small compared with the action of propranolol on inhibition by sympathetic nerve stimulation. Phentolamine 5/UM reduced the inhibitory effect only at high concentrations of NA (Fig. 6). 100 0

.Va..)

U -r

< 60

-

v

/

I

:~40 2080 0 0~~~~~~~~~~I

04

20

X

I

5

10

20

Frequency (Hz)

50

J

0404 0-16 0-64 2-56 10-2 0-04 0-16 0-64 2-56 10-2 Noradrenaline (#cg/ml.) Isoprenaline (,#g/ml.)

Fig. 6. The inhibitory effect of sympathetic nerve stimulation, noradrenaline or isoprenaline on the response of the rabbit colon to ACh (e-e) and the effect on that inhibition of phentolamine 5 x 10-6 M (0- - -) or pro pranolol 5 X 10-6 M ( X -X ). All three stimuli are effective in inhibiting the response, propranolol can reduce the inhibitory effect of sympathetic nerve stimulation or isoprenaline but is less effective on noradrenaline. In each of the first two graphs the means of the controls for the experiments with both phentolamine or propranolol are combined and represent fifteen experiments. The responses in the presence of phentolamine or propranolol are means from between five and nine experiments. The isoprenaline responses are the means of four experiments. The reduction of inhibition by propranolol is statistically significant at P < 0 01 or better at all points in the &iAt graph and at P < 0- 02 or better at 0- 16 and 0- 064 ,ug/ml in the second and at 2-56 and 10-2 in the third. Reduction of inhibition by phentolamine is significant at P < 0-02 or better at 20 and 50 Hz in the first graph and at P < 0 01 or better at 0-64 and 2-56 in the second.

Isoprenaline produced a dose-related inhibition of the response to ACh ranging from 18 %/ inhibition at 0-04 ,ug/g up to 93 %/ inhibition at 10-2 ,ug/ ml. (Figs. 4, 6). This inhibitory effect was in sharp contrast to the ineffectiveness of isoprenaline on the parasympathetic motor response. The inhibitory effect of is'oprenaline on the motor response to ACh was reduced by propranolol 5 ,UM (Fig. 6). The effectiveness of isoprenaline in inhibiting the motor response to ACh suggested that 8b receptors were mainly involved.

777 INHIBITION OF INTESTINAL MOTOR NERVES The greater sensitivity of inhibition produced by sympathetic nerve stimulation to propranolol than to phentolamine agreed with this interpretation and contrasted with the results reported in the previous paragraph on sympathetic inhibition of the parasympathetic pathway which appeared to be mediated purely by ax receptors. There remained, however, the inconsistency that phentolamine did reduce the inhibitory effect of high frequencies of sympathetic nerve stimulation or high concentrations of NA, an effect which suggested the involvement of az receptors at these high concentrations. One possible explanation of these discrepancies was that in addition to its direct motor effect ACh also stimulated the smooth muscle cells indirectly through motor, presumably cholinergic, intramural nerves. The direct effect would be antagonized by catecholamines acting through f receptors on the smooth muscle and the indirect by acting on a receptors on neurones. This hypothesis was tested in two groups of experiments, each intended to exclude the participation of motor nerves in the response to ACh. In the first the inhibitory effect of NA was examined in the presence of tetrodotoxin 1o-7 g/ml. In the second the participation of nerves was excluded by storing the tissue for 10-14 days at 40 C. The results are summarized in Fig. 7. Tissue treated with tetrodotoxin or stored in the cold no longer responded to stimulation of either sympathetic or parasympathetic extrinsic nerves though ACh still produced contraction. In the tetrodotoxin treated preparations this ACh response remained as sensitive to inhibition by NA as in untreated tissue, but NA was less effective in the cold stored tissue with a maximum inhibition of 60 %. In neither preparation was the inhibitory effect of NA, even at high concentrations, sensitive to phentolamine, though in the -cold stored tissue propranolol remained as effective as ever.

Inhibition of smooth muscle spontaneous activity and tone All three stimuli, sympathetic nerve stimulation, NA and isoprenaline powerfully inhibited the spontaneous activity and tone of the preparations. In comparing their effectiveness and the action of a and ,f blocking agents we measured the fall in tone and expressed this as a percentage of the maximum fall in tone produced by the most effective stimulus. The results for all three stimuli are summarized graphically in Fig. 8 and the effect in individual experiments can be seen and compared in Figs. 1, 2, 4 and 5. Figs. 2 and 5 illustrate the effects of phentolamine and propranolol respectively. Isoprenaline had the most easily analysed action. This drug produced a dose-related inhibition varying from 25 % at 0-04 jug/ml. up to 100 % at 10-2 /zg/ml. This response was reduced by propranolol 5 gm (Figs. 5, 8). Sympathetic nerve stimulation produced a frequency-related inhibition

J. S. GILLESPIE AND M. A. KHOYI 778 varying from 50 % at 5 Hz to 98 % at 50 Hz. This effect also was reduced by propranolol but unaffected by phentolamine. NA had similar effects to nerve stimulation, a drug-related inhibition varying from 30 % at 0-04 ,sg/g up to 91 % at 10-2 jug/ml. This inhibition was reduced by propranolol 100 r

C

0

TTX

Cold-stored

80 F

-c

U 0

60

F

C

._2

-c

.C

401-

i -C

C

20

F

1t'~

I1

0

a-0 -20 004

016 0-64

2-56

0 04 0-16 0-64 2 56 10-2

Noradrenaline (ug/ml.)

Fig. 7. The inhibitory effect of noradrenaline on the response to ACh in the rabbit colon either treated with tetrodotoxin (TTX, 10-7 g/ml.) or stored at 40C for 10-14 days and the effect of phentolamine 5 x 10-6 M (0-0) or propranolol 5 x 10-6 M ( ) on this inhibition. In both TTX-treated and cold stored tissue, phentolamine is no longer able to reduce the inhibitory effect. In the experiments with TTX each point is the mean + s.E. of six experiments. In the cold stored experiments, the control points are the means of ten experiments and the phentolamine or propranolol treated the means of five experiments. x

x

5 /UM, though only at the lower concentrations of NA, and unaffected by phentolamine 5 #M (Fig. 8). These results suggested that the direct inhibition of smooth muscle by sympathetic nerve stimulation or either catecholamine was mediated by f, receptors only. Once again, however, the actions of NA were inconsistent, in this instance in its resistance to block by propranolol. In the gut NA

INHIBITION OF INTESTINAL MOTOR NERVES

779 is known to be a powerful fi receptor agonist (Furchgott, 1960). None the less, it is not as powerful as isoprenaline and, therefore, it was to be expected that propranolol would block the ft actions of NA even more effectively than those of isoprenaline, the opposite of our findings. One possibility was that the nerves in the gut wall contributed to the background motor * 100 E

o

Ie

I

80

I,I

E..

X40 80

I

5 |

I /

0 0~~~~~~~~~~

20

.2 0 ~ 0 1

5

20 10 Frequency (Hz)

50

0-04 0-16 0-64 2-56 10-2 0-04 0-16 0-64 2-56 10-2 Noradrenaline (pg/mi) Isoprenaline (pg/ml.)

Fig. 8. The inhibitory effect of sympathetic nerve stimulation, of noradrenaline and of isoprenaline on tone in the rabbit colon. Control responses (@-@); responses in the presence of phentolamine 5 x 10-6 M (0--0) and in the presence of propranolol ( x -x ). All three stimuli produce comparable inhibition, propranolol reduces the inhibitory effect of sympathetic nerve stimulation and of isoprenaline but has relatively little effect on the response to noradrenaline. Phentolamine was tested only on sympathetic nerve stimulation and noradrenaline and was without effect. Each point is the mean + s.E. of between five and eight values when a blocking agent is present and between nine and fifteen for the controls. The difference between control and propranolol in the first graph is not statistically significant at any point by the paired t test. In the second the difference at 0-04 and 0-16 is significant at the P < 0-005 level and in the final graph there is a statistically significant difference at all points at P < 0-02 or better.

tone and that part of the NA inhibition was neurogenic and part myogenic. Three kinds of experiment were carried out to clarify this point, all aimed at the elimination of a neural component. Firstly, the experiments were repeated in the presence of atropine 10-7 g/ml. and mecamylamine 10-5 or 2 x 10-5 g/ml. Secondly, the experiments with tetrodotoxin and cold storage previously mentioned gave information on the effect of denervation on the inhibitory action of NA. Finally, the additive effect of

J. S. GILLESPIE AND M. A. KHOYI 780 phentolamine and propranolol was examined since together they should inhibit both the a (neurogenic) and /, (myogenic) effects. In the presence of atropine and mecamylamine both sympathetic nerve stimulation and NA inhibited tone. Propranolol 5 /m was now more effective in reducing the inhibition caused by sympathetic nerve stimulation but there was little difference with NA. Phentolamine 5 /M had no effect on the inhibition produced by either (Fig. 9). 100 E E

~80

E

0

Z

40

40-

2060

X

5

20

5

401,06

-61

C

0

10

10

QO

20

5

10 20 Frequency (Hz)

50

I 0.04 0-16 0-64 2-56 10-2 Noradrenaline (,ug/ml.)

Fig. 9. The inhibitory effect of sympathetic nerve stimulation and of noradrenaline on tone in the rabbit colon in the presence of mecamylamine 2 x 10-5 g/ml and atropine 2 x 10-v g/ml. Control inhibition (I-e), inhibition in the presence of phentolamine 5 X l0-6 M (O-.-0), inhibition in the presence of propranolol 5 X 10-6 M ( x - x ). Each point represents the mean ± S.E. of seven experiments in the presence of propranolol or phentolamine in the first graph and six experiments in the second graph, with the exception of 2-56 and 102 #sg NA/ml. where only four values were available. The controls are common and represent the mean of fourteen experiments in the first graph or between six and thirteen experiments in the second. In the presence of mecamylamine and atropine propranolol is more effective in reducing the inhibitory response to lumbar nerve stimulation but not more effective in reducing that to noradrenaline (compare with Fig. 8). The differences between control and propranolol are all statistically significant at the level P < 0-02 or better by Student's paired t test in graph 1 but statistical difference is reached at only two points, 0 04 and 0-16 lzg/g in the corresponding curves in the second graph for NA.

INHIBITION OF INTESTINAL MOTOR NERVES 781 Preparations treated with tetrodotoxin maintained normal tone and were inhibited by NA but this inhibition was little affected by phentolamine. In cold-stored tissue tone and spontaneous rhythmic activity was reduced in comparison with fresh tissue but this residual tone was inhibited 100

100

80

r

80 F

4)

0

0.

60

I

-C

U 0

F

/~~~~

-o ._

/I/IISl Il

._

-_

60

I

/~~1_~~~~~1

- -

40F 0

20

IX 016 0-64 2-56 10*2 04 Noradrenaline (fig/ml.)

2

5

10

20

50

Frequency (Hz)

Fig. 10. The additive effect of propranolol and phentolamine in reducing the inhibitory effect of noradrenaline on the response to ACh (left hand graph) and the inhibitory effect of sympathetic nerve stimulation on tone (right hand graph). Control responses to noradrenaline or sympathetic nerve stimulation were measured in the absence of all other drugs (a-0), propranolol 5 x 10-i M was then added and responses elicited in its presence ( x -x ) and, finally, phentolamine 5 x 10-" M added and responses again observed in the presence of both drugs (0-. -0). Propranolol inhibits the response and phentolamine adds little to this inhibition. Each point is the mean of eight experiments in the left hand graph and of seven experiments in the right. The controls have been combined in each.

by NA and again the inhibitory effect was no more sensitive to block by propranolol than in fresh tissue. In cold-stored tissue phentolamine actually increased the inhibitory action of NA but the effect was not statistically significant and can probably be attributed to the ability of phentolamine itself to cause a rise in tone in the tissue, an effect more noticeable in cold stored tissue.

J. S. GILLESPIE AND M. A. KHOYI 782 The results of experiments to measure the additive effects of a and fi blocking agents in reducing the inhibitory action of sympathetic nerve stimulation or NA are illustrated in Fig. 10 for NA on the response to ACh and sympathetic nerve stimulation on spontaneous tone. In these experiments propranolol 5 /tM was added first and its effect on inhibition noted, phentolamine 5/M was then added and the effects re-examined. Propranolol reduced the inhibitory effect on tone and on the response to ACh of sympathetic nerve stimulation and to a lesser extent of NA. The addition of phentolamine had little additional effect. Finally, since phentolamine may interfere with the uptake of NA and in this way potentiate the inhibitory effect we re-examined the inhibitory effect of NA on ACh contractions in the presence of cocaine 10-5 g/ml and 17/J oestradiol 5 x 10-6 g/ml. No evidence of an increased sensitivity to NA or of a greater ease of block by phentolamine was found in the presence of these agents. DISCUSSION

We have compared the inhibitory effect of sympathetic nerve stimulation with that of noradrenaline and isoprenaline on three forms of motor activity in the rabbit colon; spontaneous activity responsible for tone and rhythmic activity, the response to ACh and, finally, the response to pelvic parasympathetic nerve stimulation. The results with the first and last of these are clear. On spontaneous activity sympathetic stimulation, NA and isoprenaline are all effective inhibitory stimuli each capable of abolishing myogenic tone almost completely. Phentolamine is unable to antagonize any of these inhibitory stimuli and all are antagonized, though to a variable degree, by propranolol. These results suggest that inhibition of spontaneous activity is mediated almost exclusively through ,? receptors. In contrast, the ability of these stimuli to inhibit the pelvic response varies; sympathetic nerve stimulation is highly effective. At low physiological frequencies it can reduce the response without affecting spontaneous myogenic activity and at high frequencies it will completely suppress contraction; NA is considerably less effective and isoprenaline is almost ineffective in the doses tested. The inhibitory effect of sympathetic nerve stimulation and of NA are antagonized by phentolamine but unaffected by propranolol, suggesting that on this pathway inhibition is mediated entirely through a receptors. Inhibition of the motor response to ACh was less straightforward. All three inhibitory stimuli were effective, but both phentolamine and propranolol produced some antagonism of the response. The most effective antagonist was propranolol, suggesting a main inhibitory influence through f8 receptors. Phentolamine reduced inhibition only when this was produced by high concentrations of NA or high frequencies

783 INHIBITION OF INTESTINAL MOTOR NERVES of sympathetic nerve stimulation. The experiments with tetrodotoxin or cold storage, which cause the loss of the antagonist action of phentolamine, suggest that ACh may stimulate neural structures as well as the smooth muscle directly and that it is this indirect component of the response which is inhibited through ax receptors sensitive to phentolamine. The simplest interpretation of these results is that inhibition can be produced at two sites, one on neural structures through a receptors and the other on the smooth muscle directly through ft receptors. The hypothesis that intestinal smooth muscle possesses only ft receptors is in opposition to the bulk of a quite extensive literature suggesting the presence of both a and ft inhibitory receptors on gut smooth muscle. The initial, and still the basic, observation supporting the presence of both receptors was that inhibition of tone in the canine ileum by adrenaline or NA could be blocked only if both at and ft blocking agents were simultaneously present (Ahlquist & Levy, 1959). This requirement has been confirmed in other species and in other sites including the colon (Furchgott, 1960; Bucknell & Whitney, 1964; Brody & Diamond, 1967; Dzoljic, 1967; Antonio, 1968; Day & Warren, 1968). In some of these experiments inhibition of the response to transmural stimulation rather than myogenic tone was studied bringing with it the possibility of an additional a site of inhibition on nerves. Some experiments, however, were so designed as to make such a contribution unlikely. For example, where the tissue was exposed to high potassium so as to depolarize both muscle and nerve, effects attributable to both a, and ft receptors have been reported (Jenkinson & Morton, 1967). Ahlquist & Levy (1959) and other authors have used more selective agonists, usually phenylephrine and isoprenaline, and demonstrated that inhibition by these agents can be blocked by either an a or ft blocking agent alone but that sympathetic nerve stimulation, NA or adrenaline require both blocking agents again suggesting the presence of both receptors (Bowman & Hall, 1970). Other reports do suggest that only ft receptors are involved in inhibition of the smooth muscle cells. Lawson & Mackenna (1969) found that inhibition of the rabbit ileum or colon by sympathetic nerve stimulation was antagonized by ft but unaffected by a blocking drugs. These same authors, however, confirmed that exogenous NA or adrenaline required both cx and ft blocking agents. Lawson & Mackenna suggested, therefore, that the transmitter liberated by sympathetic nerve stimulation acted only on ft receptors. Species variation may also be important. In the chick rectum, inhibition even by phenylephrine appears to be mediated by f8 receptors (Bartlet & Hassan, 1970). Kosterlitz et al. (1970) compared the inhibitory effect of adrenaline, NA and isoprenaline on the response of guinea-pig

J. S. GILLESPIE AND M. A. KHOYI 784 ileum to ACh and to field stimulation. The ACh response was optimally inhibited by isoprenaline and the effect of all three catecholamines was abolished by propranolol and unaffected by phenoxybenzamine. In contrast, inhibition of field stimulation was optimal with adrenaline; isoprenaline was less effective and the inhibition was antagonized by phenoxybenzamine, unaffected by propranolol and was accompanied by a reduction of ACh output. The authors' interpretation of these results was that inhibition is mediated by /J receptors on the muscle and a receptors on neural tissue. Our own results are in agreement with this. Nevertheless, we are left with the same difficulty as previous authors; in the rabbit colon NA is highly effective in inhibiting both spontaneous activity and the response to ACh yet this effect is relatively resistant to block by either propranolol or phentolamine. The presence of both a and f8 receptors on the smooth muscle cell is not a convincing explanation since the addition of phentolamine to the bath already containing propranolol does not in this preparation greatly reinforce the blocking effect of the propranolol on NA inhibition, particularly of spontaneous activity (Fig. 10). We have no convincing hypothesis to overcome this difficulty. It may be that the receptors in smooth muscle are not uniform and that those closest to the myenteric plexus have been modified to the conventional , form under the trophic influence of NA diffusing to them from adjacent sympathetic nerves. A more radical explanation would be that NA can stimulate intramural inhibitory neurones whose final transmitter is not NA. Ohkawa & Prosser (1972) reported ganglion stimulation in the intestine by NA in experiments with extracellular recording and Gershon (1967) found synaptic transmission between the vagus nerve and the intrinsic inhibitory neurones in the guinea-pig stomach facilitated by sympathetic nerve stimulation. With intracellular recording recent reports, however, have all confirmed the apparently universal cholinergic nature of transmission in the myenteric neurones in the intestine (Hirst, Holman & Spence, 1974) and neither NA nor sympathetic nerve stimulation caused excitation (Nishi & North, 1973; Hirst & McKirdy, 1974). Fig. 11 summarizes our interpretation of the present results. The sympathetic nerves end largely on the ganglion synapses in the myenteric plexus where they inhibit the presynaptic release of ACh through an action on a receptors. The reasons for preferring the ganglion synapse rather than the post-ganglionic terminals, and within the ganglion a presynaptic rather than post-synaptic site, are as follows. Fluorescence histochemistry of this region of the gut shows that the great majority of fluorescent adrenergic terminals surround the ganglia and few are found in the smooth muscle (Gillespie, 1968). Catecholamines and sympathetic nerve stimulation inhibit the release of ACh from both small and large intestine

INHIBITION OF INTESTINAL MOTOR NERVES 785 (Schaumann, 1958; Gershon, 1967; Paton & Vizi, 1969; Beani, Bianchi & Crema, 1969; Kosterlitz et al. 1970) suggesting the effect is on nerve terminals releasing ACh rather than post-synaptic receptors. Preganglionic rather than post-ganglionic cholinergic nerve endings seem more likely because of the greater inhibitory effectiveness of NA from sympathetic nerves compared with NA added to the bath. If the site of action of the endogenous NA was on post-ganglionic cholinergic nerves then it would need to reach these by diffusion from its site of release in the myenteric plexus and consequently it could not be expected to be more effective than exogenous NA. Paton & Thompson (1953) have already shown that adrenaline in the perfumed superior cervical ganglion of the cat does inhibit the release of ACh from' preganglionic cholinergic nerves. Furthermore, direct intracellular recording from myenteric neurones in the guinea-pig have shown that both NA (Nishi & North, 1973) and sympathetic nerve stimulation (Hirst & McKirdy, 1974) reduce or suppress excitatory post-synaptic potentials without affecting the electrical properties of the post-ganglionic neurones. Intracellular recording cannot identify the function of the single neurones sampled; the experiments reported in this paper show that this form of presynaptic inhibition applies to at least one known synaptic pathway, that of the extrinsic parasympathetic nerves. De Groat & Krier (1976) have reported ganglia on the pelvic pathway on the surface of the cat colon. Sympathetic nerve stimulation at frequencies which inhibit the gut smooth muscle do not impair transmission through these ganglia. The authors have suggested that the parasympathetic motor fibres to the colon may synapse twice, once in these external ganglia and again in the myenteric plexus. Our results are consistent with such an arrangement but an alternative, and one which retains the accepted pattern of the autonomic nervous system, is that the fibres relaying in the external ganglia are not motor to the gut but have some other function such as vasodilatation, and this is their only synapse. Some of the NA liberated in Auerbach's plexus, especially at high frequencies of stimulation, diffuses to the adjacent longitudinal smooth muscle and from an action on I8 receptors is able to inhibit both spontaneous tone and directly acting agonist drugs. The inhibitory action of NA added to the bath is, for reasons already given, less clear. A minor effect is to inhibit synaptic transmission at the ganglion but its effectiveness here is small compared to its ability to inhibit the smooth muscle (compare Figs. 3, 6 and 8). This latter inhibition is most likely exercised on some kind of f8 receptor on the smooth muscle but, if so, its properties, particularly its resistance to block by propranolol, are not identical with the receptors acted on by the NA diffusing from the sympathetic nerves. An alternative and less likely explanation is that NA stimulates an

J. S. GILLESPIE AND M. A. KHOYI

786 SYMP

NA

Ach

Isop.

PARA

1

-I

Fig. 11. This represents diagrammatically our interpretation of the experimental results. The parasympathetic nerves enter from the serosa, synapse in the myenteric plexus, and the post-ganglionic fibres end on the longitudinal smooth muscle which they excite through muscarinic receptors. The sympathetic nerve fibres end mainly around the ganglion cells, the noradrenaline they liberate acts on a receptors on the endings of the preganglionic fibres to inhibit the release of ACh and block transmission. Some NA diffuses to receptors on adjacent smooth muscle to cause inhibition of tone and of the response to ACh. ACh added to the bath causes contraction mainly by a direct action on muscarinic receptors on the smooth muscle but has a minor indirect stimulant action either through ganglion Continued on opposite page so

787 INHIBITION OF INTESTINAL MOTOR NERVES intramural, non-adrenergic inhibitory neurone. Isoprenalinein contrast has only one site of action, on inhibitory fi receptors on the muscle. This agrees both with our findings that isoprenaline does not inhibit the motor response to pelvic nerve stimulation and with the observation that isoprenaline does not reduce the release of ACh from the transmurally stimulated guineapig ileum (Paton & Vizi, 1969; Kosterlitz et al. 1970). Our results and the anatomical disposition of the fibres suggest that sympathetic nerves are mainly involved and much more effective in controlling synaptic transmission rather than directly inhibiting smooth muscle. Our experimental evidence concerns the synapses on the direct parasympathetic pathway but reports in the literature suggest that block also occurs at the synapses mediating the peristaltic reflex (McDougal & West, 1954; Lee, 1960; Crema, Frigo & Lecchini, 1970). Taken together these results suggest that the major function of the sympathetic nerves in the gut is to isolate the effector cells from motor nervous control. One final point deserves comment. Isoprenaline can very effectively inhibit tone and the response to exogenous ACh (Figs. 4, 6 and 8). This effect, mediated through fi receptors, is presumably an example of physiological antagonism rather than any interference with the action of ACh at the muscarinic receptor. It is, therefore, paradoxical that isoprenaline is so ineffective against ACh liberated by the post-ganglionic parasympathetic nerves (Fig. 1). The situation is reminiscent of the similar difficulty in inhibiting the parasympathetic response with atropine. Atropine resistance has been attributed to impeded access of the atropine to the site of action of the ACh released by the nerves (Dale & Gaddum, 1930). Such an explanation is not applicable to the failure of isoprenaline since there is no reason to believe the , receptor to be confined to the site of cholinergic innervation. We are grateful for a grant from the Medical Research Funds (Wood Boyd) of Glasgow University.

cells or cholinergic nerve terminals. The response to ACh is inhibited mainly by activation of /? receptors but the small indirect component is inhibited by an action on az receptors. Isoprenaline inhibits only through an action on fi receptors and has no significant inhibitory effect on the parasympathetic pathway. Noradrenaline added to the bath is a powerful inhibitory agent acting directly on the smooth muscle and to a lesser extent on the parasympathetic nerve terminals on the ganglia. The action at the

ganglia is on az receptors but the direct action on smooth muscle, unlike the action of noradrenaline diffusing to the muscle from sympathetic nerves, is resistant to block by propranolol, suggesting that at least some of the receptors on which it acts do not have the properties of typical fi receptors.

788

J. S. GILLESPIE AND M. A. KHOYI

AHLQUIST, R. & LEvy, B. (1959). Adrenergic receptive mechanism of canine ileum. J. Pharmac exp. Ther. 127, 146-149. ANTONIO, A. (1968). The relaxing effect of bradykinin on intestinal smooth muscle. Br. J. Pharmnc. Chernother. 32, 78-86. BARTLET, A. L. & HASSAN, T. (1970). Adrenoceptors of the chick rectum. Br. J. Pharmac. 39, 817-821. BEANI, L., BIANCHI, CLEMENTINA & CREMA, A. (1969) The effect of catecholamines and sympathetic stimulation on the release of acetylcholine from the guinea-pig colon. Br. J. Pharmac. 36, 1-17. BOWMAN, W. C. & HALL, MOIRA T. (1970). Inhibition of rabbit intestine mediated by a- and ft- adrenoceptors. Br. J. Pharmac. 38, 399-415. BRODY, T. M. & DIAMOND, J. (1967). Blockade of the biochemical correlates of contraction and relaxation in uterine and intestinal smooth muscle. Ann. N.Y. Acad. Sci. 139, 772-780. BUCKNELL, A. & WHITNEY, B. (1964). A preliminary investigation of the pharmacology of the human isolated taenia coli preparation. Br. J. Pharmac. Chemother. 23, 164-175. CREMA, A. FRIGO, G. M. & LECCHINI, S. (1970). A pharmacological analysis of the peristaltic reflex in the isolated colon of the guinea-pig or cat. Br. J. Pharmac. 39, 334-345. DALE, H. H. & GADDUM, J. H. (1930). Reactions of denervated voluntary muscle and their bearing on the mode of action of parasympathetic and related nerves. J. Physiol. 70, 109-144. DAY, M. D. & WARREN, P. R. (1968). A pharmacological analysis of the responses to transmural stimulation in isolated intestinal preparations. Br. J. Pharmac. Chemother. 32, 227-240. DE GROAT, W. C. & KRIER, J. (1976). An electrophysiological study of the sacral parasympathetic pathway to the colon of the cat. J. Phy8iol. 260, 425-445. DzoLjic, M. (1967). Stimulatory effect of tolazoline on smooth muscle. Br. J. Pharmacy. 30, 203-212. FURCHGOTT, R. F. (1960). Receptors for sympathomimetic amines. In Adrenergic Mechani8rne, Ciba Foundation Symposium, ed. VANE, J. R., pp. 246-252. London: Churchill. GARRY, R. C. & GILLESPIE, J. S. (1955). The responses of the musculature of the colon of the rabbit to stimulation, in vitro, of the parasympathetic and of the sympathetic outflows. J. Physiol. 128, 557-575. GERSHON, M. D. (1967). Inhibition of gastro-intestinal movement by sympathetic nerve stimulation: the site of action. J. Phy8iol. 189, 317-327. GILLESPIE, J. S. (1968). Electrical activity in the colon. In Handbook of Phy8iology, vol. IV, section 6, ed. CODE, C. F., pp. 2093-2120. Washington D.C.: American Physiological Society. GILLESPIE, J. S. & KHoYI, M. A. (1974). A pharmacological analysis of the inhibitory effects of the sympathetic nerves on the rabbit colon. J. Phy8iol. 244, 42-43P. HIRST, G. D. S., HOLMAN, MOLLIE E. & SPENCE, I. (1974). Two types of neurones in the myenteric plexus of duodenum in the guinea-pig. J. Physiol. 236, 303-326. HIRST, G. D. S. & McKIRDY, H. C. (1974). Presynaptic inhibition at mammalian peripheral synapse? Nature, Lond. 250, 430-431. JENINSON, D. H. & MORTON, I. K. M. (1967). The effect of noradrenaline on the permeability of depolarized intestinal smooth muscle to inorganic ions. J. Phy8iol. 188, 373-386. KOSTERLITZ, H. W., LYDON, R. J. & WATT, A. J. (1970). The effects of adrenaline, noradrenaline and isoprenaline on inhibitory a- and /?-adrenoceptors in the longitudinal muscle of the guinea-pig ileum. Br. J. Pharmac. 39, 398-413.

INHIBITION OF INTESTINAL MOTOR NERVES

789

LAWSON, W. R. & MACKENNA, B. R. (1969). The inhibitory adrenergic receptors of the rabbit intestine. J. Physiol. 202, 35-36P. LEE, C. Y. (1960). The effect of stimulation of extrinsic nerves on peristalsis and on the release of 5-hydroxytryptamine in the large intestine of the guinea-pig and of the rabbit. J. Physiol. 152, 405-418. McDOuGAL, MARY & WEST, G. B. (1954). The inhibition of the peristaltic reflex by sympathomimetic amines. Br. J. Pharmac. Chemother. 9, 131-137. Nism, S. & NORTH, R. A. (1973). Presynaptic action of noradrenaline in the myenteric plexus. J. Phygiol 231, 29-30P. NORBERG, K. A. (1964). Adrenergic innervation of the intestinal wall studied by fluorescence microscopy. Int. J. Neuropharmac. 3, 379-382. OHKAWA, H. & PROSsER, C. L. (1972). Functions of neurons in enteric plexuses of cat intestine. Am. J. Phyfiol. 222, 1420-1426. PATON, W. D. M. & THOMPSON, J. W. (1953). The mechanism of action of adrenaline on the superior cervical ganglion of the cat. Ab8t. XIXth Int. Congre88, pp. 664-665. PATON, W. D. M. & Vizi, E. S. (1969). The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip. Br. J. Pharmac. 35, 10-28. SCHAUMANN, W. (1958). Zusammenhange zwischen der Wirkung der Analgetica und Sympathomimetica auf den Meerschweinen-Dunndarm. Arch. exp. Path. Pharmak. 233, 112-124.