that tyramine has no dilator action on the denervated pupil of

Transcription

1 459 J. Physiol. (1938) 91, :6 I THE ACTION OF TYRAMINE AND ADRENALINE ON THE DENERVATED NICTITATING MEMBRANE BY EDITH BtTLBRING AND J. H. BURN From the Pharmacological Laboratory, College of the Pharmaceutical Society, London (Received 8 November 1937) IN the course of examining the action of cocaine on physiological responses to adrenaline and tyramine, Burn & Tainter [1931] made the observation that tyramine has no dilator action on the denervated pupil of the cat's eye. Their experiments were performed 9 and 16 days after removal of one superior cervical ganglion, using urethane as an anaesthetic; doses of 0.5 mg. tyramine, injected intravenously, caused the normal pupil to dilate, while doses as large as 8-0 mg. had no action on the denervated pupil. Later Burn [1932] showed that this was also true of the iris when isolated in a bath of Ringer, though the isolated iris is a relatively insensitive organ; he further investigated the action of tyramine on the blood vessels of the cat's foreleg and found that after removal of the stellate ganglion and subsequent degeneration of the sympathetic fibres, tyramine failed to cause constriction of the foreleg, although constriction occurred in the opposite normal foreleg. Similar differences between the action on the denervated and on the innervated structures were found for ephedrine, though large doses of ephedrine (10 mg.) had a slight dilator action on the denervated pupil. Since the effect of adrenaline on the pupil is increased by denervation and that on the blood vessels is at least not appreciably diminished, Burn argued that a qualitative difference must exist between the point of action of tyramine and that of adrenaline; he drew the conclusion that tyramine in general must act on the sympathetic nerve ending itself, so that after denervation there was no longer a site of action for it. Recently, however, Bacq [1936] has described the results of examining tyramine and other substances on the denervated nictitating membrane; he found that, as with adrenaline, the effect of tyramine was greater on the

2 460 4E. B(JLBRING AND J. H. BURN denervated than on the normal membrane. So far as can be judged, the number of observations made with tyramine was not great, since Bacq tested very many compounds; his experiments were performed with dial as anaesthetic. Because his result was so completely at variance with those on the pupil and the vessels of the forelimb, we have examined the action of tyramine ourselves, and also that of dioxyphenylethylamine, sympatol and adrenaline. Sympatol has the same structure as adrenaline except that there is only one -OH group in the ring; dioxyphenylethylamine has the same structure as tyramine except that there are two -OH groups in the ring. EXPERIMENTAL METHODS The removal of the superior cervical ganglion of one side was performed at different periods varying from 1 to 75 days before the final experiment. In the final experiment the cat was anaesthetized with ether, the abdomen was opened and the suprarenal glands were excluded from the circulation by ligatures; the abdomen was closed. Artery clips were then placed temporarily on both carotid arteries, the spinal cord was divided at the second cervical vertebra and the brain was destroyed. The foramen magnum was plugged with plasticine and a cork. Ether was then discontinued, and the cat artificially respired. The clips on the carotid arteries were removed. To record the contractions of the nictitating membranes the head of the cat was fixed rigidly in a clamp, and the outer corners of the lids were incised. A silk thread through the middle of each membrane passed by way of a pulley wheel to an isotonic lever with a frontal writing point. The levers were of equal length and weight, and the threads were attached at the same distance from the fulcrum. The blood pressure was recorded from the femoral artery of one side, and a cannula for injection was inserted into the femoral vein of the other. The cervical sympathetic chain of the unoperated side was divided to exclude impulses reaching the ganglion from the spinal cord. I. Results with tyramine The characteristic response. At the outset of the experiments it did not seem likely that the results could be other than simple; to our surprise, however, this was not so; they varied from one experiment to another, and it is not easy to select one as typical of all the rest. We begin, however, by describing the experiment shown in Fig. 1 which is characteristic of tyramine in that similar records have never been

3 ACTION OF DRUGS ON NICTITATING MEMBRANE 461 obtained with adrenaline, dihydroxyphenylethylamine or sympatol. The experiment was carried out 24 days after extirpation of the right superior cervical ganglion. When 04 mg. tyramine (i.e. 0x66 mg. tyramine acid phosphate) was injected intravenously, there was no effect on the normally innervated membrane, but a small contraction of the denervated membrane took place which was delayed in onset and prolonged. This is seen in Fig. 1 (a). At this point the denervated membrane was more sensitive than the normal. In Fig. 1 (b) is shown the action of C 8 mg. tyramine. A contraction occurred in the denervated membrane, slightly earlier than a contraction of the same height, though of shorter duration in the normal membrane. In Fig. 1 (c) and (d) are shown the Fig. 1. Contractions of nictitating membranes of spinal cat. The superior cervical ganglion was excised on the right side 24 days previously. In (a) a dose of Q.4 mg. tyramine causes contraction of the denervated membrane, but not of the normal. In (c) and (d) doses of 1-6 and 4-0 mag. tyramine cause a much greater contraction of the normal membrane than of the denervated. effects of higher doses of tyramine, namely 1-6 and 4-0 mg.; these effects are strikingly different, for in each the contraction of the normal membrane is greater than that of the denervated membrane. The contractions of the denervated membrane in response to the different doses are all very much alike, showing little increase in size in proportion to the dose; they are, moreover, prolonged contractions. The cont-ra'ctions of the normal membrane increase rapidly in size according to the dose, and they are contractions of short duration. Variations according to the interval after ganglionedtomy. In one experi- ment only did tyramine fail to stimulate the denervated membrane in w~~~~~~ _ any dose, and in all others the lower doses made the denervated membrane contract without causing a contraction of the normal membrane; this sensitization of the denervated membrane is illustrated in Fig. 2, which in (a) shows the response in a cat 2 days after ganglionectomy, PH. XCI. 29

4 462 E. B(JLBRING AND J. H. BURN and in (b) shows it in a cat 75 days after ganglionectomy. These results resemble the observation of B a cq already mentioned. Fig. 2. Upper record, denervated nictitating membrane; lower record, normal membrane. (a) is a record of the effect of 04 mg. tyramine 2 days after removal of the ganglion, (b) is a record of the effect of 05 mg. tyramine 75 days after removal of the ganglion. Fig. 3. Records of the denervated (upper) and normal (lower) nictitating membranes from different experiments. In (a), (b), (c), (d) and (e) the effect of injecting 4*0 mg. tyramine is shown. In (f ), which is taken from the same experiment as (e), the effect of injecting 8.0 mg. tyramine is shown. (a) was 2 days after extirpation of the ganglion, (b) was 4 days, (c) 14 days, (d) 21 days, (e) and (f) 75 days. In (a), (b), (c), (d) and (f) the contraction of the normal membrane was greater than that of the denervated membrane. In most of the experiments the larger doses of tyramine caused a greater contraction of the normal membrane than of the denervated. w

5 ACTION OF DRUGS ON NICTITATING MEMBRANE 463 In Fig. 3 are results obtained by injecting 4 mg. tyramine in cats at different intervals after ganglionectomy. In Fig. 3 (a), 2 days after ganglionectomy, the contraction of the denervated membrane is still large, though not so large as that of the normal; in Fig. 3 (b), at 4 days' interval, the contraction of the denervated membrane is already small; similarly at (c) after 14 days and at (d) after 21 days. But in Fig. 3 (e) after 75 days the contraction of the denervated membrane is relatively larger; in this cat a contraction in the normal membrane exceeding that in the denervated was obtained only with a dose of 8x0 mg. (Fig. 3 (f)). Thus we were unable to discover any progressive change in the response of the denervated membrane to tyramine as time after ganglionectomy lengthened, or to foretell the size of contraction in any experiment. Contraction of the denervated membrane. The form of the contraction of the denervated membrane in response to tyramine varied greatly as can be seen in Figs. 1, 2 and 3. In some cats the contraction rose slowly to a maximum which was reached later than the maximum in the normal membrane; it also relaxed more slowly (see Fig. 1). In others the contraction began steeply; it then reached a plateau which was maintained longer than the contraction in the normal membrane (Fig. 3 (d)). The plateau gave the impression that there was some change in the muscle which made it unable to contract further; when other substances were tested on the same muscle, however, it contracted further, and the form of contraction was very similar to that of the normal membrane. Summary of the results with tyramine. We performed fifteen experiments in which the response of the two membranes to different doses of tyramine was recorded. In eleven of these the results were as already described, and we have expressed the mean of these results graphically in Fig. 4. The ordinates are the mean heights of contraction of the two membranes, while the abscissse are the doses of tyramine. For doses below 2-0 mg. the continuous line, representing the response of the denervated membrane, is above the dotted line, representing the response of the normal membrane. For doses above 2-0 mg. the dotted line rises steeply above the continuous line. It should be mentioned that the size of the contraction in the denervated membrane did not depend on the order in which the doses were given. When the large doses were given first the same differences in the contractions of the two membranes were observed. Unusual results with tyramine. Not less important than the results of these eleven experiments, however, were the results of four others. In two of these the responses of the denervated membrane were very small 29-2

6 464 E. BULBRING AND J. H. BURN to all doses, and the responses of the normal membrane were always greater. In the remaining two experiments the results were quite different, for no matter what dose was used the contraction of the denervated membrane was greater than that of the normal membrane. One experiment was carried out on the day following the removal of the 30 - //' +~~~~. ~~~// 20 j + X, ~+1 /, /, 7+ y/ / O mg. tyramine Fig. 4. The curves were drawn by plotting the mean points of eleven experiments in which doses of tyramine were injected. Abscis#ae are doses of tyramine (mg.), ordinates are heights of contraction of the nictitating membranes (mm.). The continuous line represents the contractions of the denervated membrane and the dotted line the contractions of the normal membrane. ganglion; in this the greatest difference between the denervated membrane and the normal was seen with the highest dose; there was no suggestion that with still higher doses the normal membrane would contract as much or more than the denervated. The other exrperiment was carried out 68 days after removal of the ganglion, and although the denervated membrane contracted more than the normal in response to the highest dose injected, it loboked as though with a still higher dose the responses might have been reversed. Apart from this surmise, however, the main

7 ACTION OF DRUGS ON NICTITATING MEMBRANE 465 point of these two experiments is that the response of the denervated membrane to tyramine was always greater than that of the normal membrane, and the action of tyramine was similar to that which till now we have always supposed to be the action of adrenaline. II. Results with other substances Dihydroxyphenylethylamine and sympatol. In examining the action of dihydroxyphenylethylamine we have observed results of two kinds. There were results in which the denervated membrane contracted more than the normal in responses to all doses, and there were also results like Fig. 5. Contractions of the nictitating membranes to doses of dihydroxyphenylethylamine after excision of the right superior cervical ganglion 33 days previously. the majority of the tyramine results in which the denervated membrane contracted more than the normal in response to low doses, but contracted less than the normal in response to high doses. An example of this is given in Fig. 5. Out of a total of thirteen experiments, there were five in which the denervated membrane contracted more than the normal for all doses, and six in which the relationship was reversed according to the size of the dose. In the two remaining experiments the e-ffect of large doses was the same in both membranes. The form of the contraction of the denervated membrane was always' similar to that of the normal membrane, though the contraction of the denervated membrane was more prolonged. The number of experiments in which different doses of sympatol were examined was smaller because the effect of this substance on the

8 466 E. BLJLBRING AND J. H. BURN nictitating membrane was very prolonged. In six out of nine experiments the denervated membrane contracted more than the normal in response to all doses; in three out of nine the relationship was reversed according to the size of the dose as shown in Fig. 6. In all experiments with these substances the increase of the contraction with increase of dose was steeper in the normal membrane. Fig. 6. Contractions of the nictitating membranes to doses of sympatol after excision of the right superior cervical ganglion 21 days previously. Results with adrenaline. The majority of the results with adrenaline showed, as would be expected, that the denervated membrane contracted more than the normal in response to all doses. Nevertheless, when the mean results of eight experiments were calculated, it was found here also that as the dose increased the contraction of the normal membrane increased more in proportion than did that of the denervated. We would have concluded that this was because the contraction of the denervated membrane was reaching a maximum, but for the surprising observation that in two other experiments, the relative size of the contractions was reversed. One of these experiments was made 1 day after ganglionectomy and the second was made 33 days after ganglionectomy. This second experiment is shown in Fig. 7. Adrenaline and tyramine infusions. Some experiments were also made in which adrenaline and tyramine were administered by slow intravenous infusion. The results were similar to those given by large single injections, but they brought out more clearly the changed response

9 ACTION OF DRUGS ON NICTITATING MEMBRANE 467 in the denervated membrane. In Fig. 8 (a) is shown the effect of an adrenaline infusion; the contraction began much earlier in the denervated membrane, but when the normal membrane began to contract it did so more rapidly and the contraction was twice as great. The resemblance of this record to that of the infusion of tyramine in Fig. 8 (b) Fig. 7. Contractions of the nictitating membranes to doses of adrenaline after excision of the right superior cervical ganglion 33 days previously. Note that the normal membrane contracts more than the denervated when the doses are 40 and looy. (a) Fig. 8. The effect of slow infusion of adrenaline and of tyramine in a cat 8 days after excision of the right superior ganglion. Both adrenaline and tyramine cause contraction earlier in the denervated membrane, but the contraction is greater in the normal membrane. indicates the similarity of the effects of adrenaline and tyramine, although in other animals an infusion of adrenaline produced a contraction of the denervated membrane which was throughout greater than that of the normal membrane. (b)

10 468 E. B(JLBRING AND J. H. BURN Comparison of the four substances. The actions of the four substances examined can be grouped in three classes; (1) an increased response of the denervated membrane to all doses; (2) an increased response of the denervated membrane to low doses and a decreased response to high doses; (3) a decreased response of the denervated membrane to all doses. In Table I are given the number of experiments with each substance TABLE I. The figures show the number of experiments in which the indicated response of the denervated membrane was observed Increased response Increased to low doses; Decreased response to decreased response response to all doses to high doses all doses Adrenaline Sympatol Dioxyphenylethylamine Tyramine which fall into each of these classes. The table indicates that in passing from adrenaline to tyramine we pass from a substance most of the responses to which fall in the first class, to a substance most of the responses to which fall in the second class with occasional responses falling in the third class. This transition is supported by a consideration of individual experiments; thus the two experiments in which the response of the denervated membrane was decreased to high doses of adrenaline were the same experiments as those in which the response to all doses of tyramine was decreased. Similarly, the two experiments in which the response of the denervated membrane was increased to all doses of tyramine were experiments in which the response was increased to all doses of other substances. It can be said that in an experiment the results with tyramine are most likely to fall in the second class; the chances are about equal that the results with dihydroxyphenylethylamine will fall in either the first or second class; the chances are in favour of the results with sympatol falling in the first class, and heavily in favour of the results with adrenaline falling in this class. The division of the results into these three classes is certainly justified by the observations themselves, but it is likely to be an artificial classification, which has no real existence. The impression left by the different experiments is that for every substance examined there is a stage after extirpation of the ganglion in which the results would always fall in the first class. As time goes on there is a second stage in which a proportion of the results would fall in the second class; this proportion is low for adrenaline, but becomes greater in passing to sympatol, dioxyphenylethylamine and

11 ACTION OF DRUGS ON NICTITATING MEMBRANE 469 tyramine, for which it is high. Finally, there is a third stage in which a proportion of the tyramine results would fall in the third class. Evidence that this transition occurs is entirely circumstantial, for it is impossible to follow it in one animal, and there is so much variation among different animals that even an approximate idea of the duration of each stage cannot be formed. Ephedrine. An attempt has been made to investigate the behaviour of ephedrine because it is one of the substances which Blaschko et al. [1937b] have found not to be affected by the enzyme they have described. Ephedrine, however, exerts a very prolonged action on the nictitating membrane, and it is impossible to observe the effect of a series of doses in one experiment unless they are small. There is no doubt that in response to small doses, 03-0O5 mg., the denervated membrane contracts more than the normal membrane. In one experiment this relation was reversed when 4 0 mg. was injected. At the end of another experiment in which a large number of injections had been made, 1.0 mg. ephedrine caused a contraction of the normal membrane but not of the denervated membrane. In a few experiments we have given an initial dose of 4-0 mg. ephedrine, and in these the heights of contraction of the two membranes were very similar. The evidence on the whole indicates that the action of ephedrine is like that of the other substances examined. The one peculiarity of the action of ephedrine, which distinguishes it from the other substances, is that relaxation occurs earlier in the denervated than in the normal membrane. Extirpation of both ganglia. The possibility occurred to us that some of the phenomena seen in the denervated membrane might be due to incomplete denervation because of postganglionic fibres crossing from the ganglion of the other side. We excluded this by making observations in cats in which both ganglia were removed. The results obtained agreed with those in the other experiments. Use ofancestheties. Various workers have used dial as an ansesthetic in making observations on the nictitating membrane. We performed one experiment under urethane, and in this there was no contraction of the denervated nictitating membrane whatever in response to tyramine, though it contracted well to adrenaline. Since we believe that the anaesthetic may influence the results, we have been careful to use the spinal preparation throughout.

12 470 E. BULBRING AND J. H. BURN DiSCUSSION The evidence indicates that the view previously put forward by Burn is incorrect; there is no qualitative difference between the site of action of tyramine and that of adrenaline; the difference between the action of these substances is quantitative and not qualitative. So far as the nictitating membrane is concerned, a simple conclusion that tyramine acts on the nerve ending while adrenaline acts beyond it is in no way justified. Nevertheless, the reactions of the denervated nictitating membrane to large doses of tyramine fall completely in line with the reactions described by Burn for the pupil and for the vessels of the forelimb; in the majority of animals the contraction of the denervated membrane is much less than that of the normal membrane. But the observation has now been made that in a minority of animals this is also true of adrenaline. Hitherto there has been little possibility of speculating about the changes which follow denervation; most workers have been content to accept it as a physiological law that denervated tissues are more sensitive to chemical stimuli than normal tissue. The results described indicate, however, that there are at least two changes which are independent of one another. The one change leads to the increased response of the denervated membrane to low doses; if this change occurs alone it leads to an increased response to all doses. The other change is that which is responsible for the slower rate of increase of contraction of the denervated membrane with increase of dose, illustrated in Figs. 1, 4. Prof. Gaddum has suggested to us that this may be explained by the vascular conditions; that if the vessels are more sensitive after denervation they will be more constricted by bigger doses of tyramine, and the tyramine will not reach the nictitating membrane; therefore, there would be less increase of effect with increase of dose. One of us found, however [Burn, 1932], that tyramine causes less vaso-constriction in a forelimb which is denervated by removal of the stellate ganglion; for this reason we do not think that the suggestion is likely to be correct. It is possible to explain the increased response of the denervated membrane to low doses in terms of Straub's hypothesis of potential action. Straub considers that for many drugs the intensity of action is proportional to the steepness of the concentration gradient of the stimulating substance across the cell membrane. In a normally innervated tissue it may be that there is always a small amount of the humoral transmitter inside the responsive cell, the presence of which is due to the

13 ACTION OF DRUGS ON NICTITATING MEMBRANE 471 continuous liberation of the transmitter by tonic impulses. If the tissue is denervated, this small amount disappears, and if now a drug is injected into the circulation which can act in the same way as the humoral transmitter, it follows that the concentration gradient necessary to cause a visible response will be reached in the denervated tissue before it is reached in the normal tissue; thus denervated tissues are sensitized to the action of drugs in low doses. We doubt, however, whether this explanation is sufficient to explain the increased response of the denervated membrane to high doses which is usually seen when the substance injected is adrenaline. When the dose injected is large, the concentration gradient across the cell membrane must be almost the same on the normal and denervated sides. The diminished response of the denervated membrane to large doses of tyramine (and occasionally to large doses of adrenaline) is still more puzzling. We have tried to approach this also from the point of view of Straub's theory, since the physiological importance of concentration gradient was impressed upon one of us by the influence of cocaine on the action of adrenaline in the perfused cat's heart. (Burn & Tainter [1931] found that when cocaine was diffusing into or out of the heart, the effect of adrenaline was increased; but when cocaine was present in unchanging concentration, then the action of adrenaline was diminished.) Now in a recent communication [1937] Straub has emphasized that a drug can act efficiently only if there is a mechanism inside the cell for destroying it, for if the drug accumulates the concentration gradient between the outside and the inside of the cell becomes less steep and therefore the cell fails to respond to the drug. The slow removal of the drug would explain, for example, the failure of a second dose of acetylcholine applied to the motor end-plate of the lizard to cause a contraction of the muscle as described recently by Buchthal & Lindhard [1937]. Now Blaschko et al. [1937 a, b] have shown that there is an enzyme system in the tissues which destroys adrenaline and other similar substances. If it is assumed that this system often becomes less efficient after denervation, then when a large dose of a substance such as tyramine is injected there will soon be no concentration gradient between the outside and inside of the denervated cell, and, therefore, on Straub's hypothesis the contraction of the denervated cell will be very small. Furthermore, this small contraction will last longer than the larger contraction in the normal membrane; it will persist until the chemical stimulant has diffused from the cell back into the blood stream since there is no other way for its removal. This is what is observed.

14 472 E. BULBRING AND J. H. BURN The results obtained with ephedrine do not altogether conform to this explanation. Blaschko et al. found that ephedrine is not destroyed by the enzyme system in the tissues, and we have observed that the effect of ephedrine is actually very prolonged. On the other hand, its action is on the whole similar to that of adrenaline and tyramine; this would not be expected if the rate of removal of the drug inside the cell played an important part in deciding its effectiveness. As already mentioned, ephedrine is peculiar in that relaxation occurs earlier in the denervated membrane. If ephedrine is not destroyed by any enzyme and can be removed only by diffusion from the tissue cells back into the blood, relaxation will occur earlier in the side with the better blood supply. Now when the forelimb is denervated by extirpation of the stellate ganglion, the blood supply of the soft pads of the foot becomes better than on the normal side. If the same vascular change occurs in the nictitating membrane the denervated membrane will have a better blood supply, and therefore the relaxation after ephedrine will occur earlier on that side. Knowledge of cell structure and of the mode of action of drugs is still too fragmentary to make it likely that any explanation of the phenomena of denervation which agrees with all the facts can be put forward. To be able to discuss these phenomena in terms of any hypothesis, however, seems to us an advance on the previous view that they were the manifestation of some mysterious law. SUMMARY When the effect of tyramine on the denervated nictitating membrane is compared with the effect on the normal membrane in the spinal cat, it is usually observed that when the dose is small the denervated membrane contracts more than the normal membrane, but when the dose is large, the normal contracts much more than the denervated. In a few cats the denervated membrane does not respond to any dose of tyramine, and on the other hand in a few cats the denervated membrane always responds more than the normal. The interesting observation has been made that in some cats the response of the denervated membrane to large doses of adrenaline also is less than that of the normal membrane. Hence it is not an invariable rule that denervation increases the response to adrenaline; this rule is true for lower doses only.

15 ACTION OF DRUGS ON NICTITATING MEMBRANE 473 In passing from adrenaline to sympatol, then to dioxyphenylethylamine and finally to tyramine, there is a gradual change in the effect of denervation on the response to large doses; to adrenaline denervation usually increases the response, to tyramine denervation usually decreases the response. REFERENCES Bacq, Z. M. (1936). Mem. Acad.-M&. Belg. 25, 1. Blaschko, H., Richter, D. & Schlossmann, H. (1937a). J. Physiol. 90, 1. Blaschko, H., Richter, D. & Schlossmann, H. (1937b). Ibid. 89,39P. Buchthal, F. & Lindhard, J. (1937). Ibid. 90, 82P. Burn, J. H. & Tainter, M. L. (1931). Ibid. 71, 169. Burn, J. H. (1932). J. Pharmacol., Baltimore, 46, 75. Straub, W. (1937). Proc. Roy. Soc. B, 121, 584.