[ ] showed that in distilled water aeration increased the destruction

Transcription

1 : DURATIONS OF RESPONSES TO ADRENALINE, TYRAMINE, AND EPHEDRINE. By A. J. CLARK and J. RAVENT6S. From the Department of Pharmacology, University of Edinburgh. (Received for publication 218t December 1938.) THE experiments described in this paper were made with the object of analysing the factors determining the durations of action of adrenaline, tyramine, and ephedrine. The isolated frog's auricle and the intact cat were used. In the latter case the responses of the nictitating membrane, blood-pressure, and gut were studied. The breakdown of adrenaline in vitro is known to be dependent on a number of factors. Oliver and Schiafer in 1895 noted that adrenaline was destroyed more rapidly in Ringer's fluid than in blood. Sugawara [ ] showed that in distilled water aeration increased the destruction of adrenaline about threefold, that the destruction was more rapid in 0-85 per cent. saline than in distilled water, and still greater in Locke's solution or in Tyrode's solution. The times he found for reduction of a solution of adrenaline (1 in 106) to half-strength in saline, Locke's fluid, Tyrode's fluid, and in blood were respectively 4 hours, 45 min., 11 min., and 60 min. Analysis of his figures shows an approximately linear relation between time and the logarithm of the amount of adrenaline remaining except in the case of adrenaline in blood, where this relation is not linear. The former cases therefore resemble a simple form of enzyme action. Various authors have shown that the destruction of adrenaline in saline solutions can be augmented by many agencies, e.g. alkaline reaction [Sugawara, , and Blix, 1929], presence of minute traces of metals such as copper or iron [Barker, Eastland, and Evers, 1932; Schild, 1933], or even exposure to glass surfaces [Welch, 1934]. There is an extensive literature regarding the inactivation of adrenaline by living tissues, which has been reviewed by Gautrelet, Halpern, and Corteggiani [1935]. Several authors at the commencement of this century proved that adrenaline was inactivated by several tissues. Tatum [1912] showed that the introduction of strips of mammalian arteries or veins into test-tubes containing adrenaline solution greatly accelerated the adrenaline inactivation. Blaschko, Richter, and Schlossmann [1937] showed that tissues contained a cyanide insensitive oxidase that inactivated adrenaline. The extract 185

2 186 Clark and Raventos from 1 g. rat's liver could inactivate 1 mg. adrenaline in 15 min. On the other hand, it has been shown that a wide variety of substance act as stabilisers to adrenaline, e.g. amino acids (Abderhalden and Gellhorn, 1993; Wiltshire, 1931], glutathione and ascorbic acids [Welch, 1934]. Bain, Gaunt, and Suffolk [1937] have shown that the inactivation of adrenaline in whole blood is a different process from its inactivation in Ringer's fluid or in serum, since in the former case most of the adrenaline which disappears is fixed by the corpuscles and can be recovered by laking. In blood serum and in plasma, on the other hand, adrenaline is slowly inaetivated, presumably by an enzyme. The inactivation of adrenaline in vivo is probably a complex process, since it is exposed to the specific tissue oxidase but is protected by the presenee of a wide variety of stabilisers, and may also be fixed by erythrocytes and possibly by other cells. Bacq [1936 c] pointed out that the action of a moderate dose of adrenaline in a cat only lasted a few minutes, and hence its duration could not be affected by some of the mnechanisms described above, which took hours to remove the drug. Cramer [1911] showed that formaldehyde inactivated adrenaline and tyramine in a few minutes, and Toscano Rico and Malafaya Baptista [1935] suggested that adrenaline was inactivated in vivo by aldehydes formed as intermediate products of carbohydrate metabolism. Trendelenburg [1910, 1916] found that 75a per cent. of an adrenaline injection disappeared from the blood-stream of cats in 15 seconds. This can be accounted for by diffusion into the extracellular fluids. He also quoted several authors who showed that intra-arterial adrenaline injections were inactive, and that circulation through the liver and intestine destroyed adrenaline, but that the lungs did not do so. Hess [1921] found intra-arterial injection to be inactive in man. Markowitz and Mann [1929] showed that the rate of destruction of adrenaline was not markedly affected in dogs either by hepatectomy or evisceration. It would appear that adrenaline, when injected intravenously, diffuses into the tissues very rapidly, and is subsequently inactivated in the tissues, and that all tissues possess this power. The relation between the duration of response to adrenaline and the persistence of adrenaline in the blood has been studied by many authors. Trendelenburg [1916] found in rabbits that the pressor effect and adrenalinaemia were of equal duration with doses of less than 041 mg. adrenaline, but that with massive doses (1 mg.) the bloodpressure returned to normal at a time when adrenaline was still present in the blood. Bacq [1936 a] found in the dog that the adrenalinaemia and the pressor effect were of equal duration, but that after pyrogallol the duration of adrenalina,mia was unaltered, whilst the pressor effect was prolonged. Joseph [1912] and Githens and Meltzer [1916] described a dilatation

3 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 187 of the pupil lasting for some hours which was produced by small doses of adrenaline in pupils of cats and rabbits which had been denervated some hours previously. Githens and Meltzer [1916] concluded that this response was different in nature from the response of the normal pupil to adrenaline. The authors found in the cat that the duration of response of the recently denervated pupil was similar to the durations of responses of the b.p. and n.m. Therefore the results mentioned above do not provide any certain evidence that adrenaline can persist in some tissues much longer than in others. A consideration of the literature regarding the destruction of adrenaline in the intact mammal indicates that several mechanisms are concerned. There is, firstly, a rapid diffusion into the interstitial fluids, which reduces the plasma concentration to about one-quarter. The adrenaline, localised in the tissues on which it acts, usually is destroyed at about the same rate as the adrenaline in the blood-stream, but may persist longer. In view of the complex problem presented by the mammal it is obviously desirable to obtain as much information as possible from the simpler system represented by the isolated frog's auricle. DURATION OF ACTION OF ADRENALINE ON THE FROG'S AURICLE. Burridge and Seth [ ] showed that the disappearance of adrenaline from Ringer's fluid could be measured by means of the augmentor effect produced by adrenaline on a frog's heart which had been depressed by calcium deficiency. We used strips of frog's auricle suspended in Ringer's fluid according to the method described by Dale [1937]. In some experiments the sinus was included, and in other experiments the sinus was cut out and artificial stimulation (12 per min.) was maintained. Ringer's fluid with phosphate buffer was used (ph 7 4), but the calcium chloride content was reduced to 1/2 or 1/3 of the usual concentration (0012 per cent. CaCl2) because this facilitated measurement of the augmentor response to adrenaline. Our object was to determine the rate of destruction of adrenaline by the auricle, and hence it was necessary to estimate the rate of destruction of adrenaline in the absence of the auricle. Oxygenation of adrenaline solution (1 in 1 million in Ringer's fluid) in a test-tube showed a slow rate of destruction (half-destruction in 60 min.), a result which agrees with those obtained by Sugawara [ ]. The rate of adrenaline destruction in the bath containing the auricle was considerably greater than in the test-tube, but it varied widely in different experiments. One source of error was the presence of stabilisers in commercial preparations of adrenaline hydrochloride solution. This was avoided by preparing solutions from the pure base, but even so there was considerable variation in different experiments.

4 188 Clark and Ravent6s Fig. 1 shows examples of the duration of adrenaline action with the auricle in 10 c.c. of fluid (A and D), and with the moist auricle strip (B, C, E, and F). The recovery of the auricle in the bath takes more than four times as long as does the recovery of the moist auricle. Hence it appears that, the inactivation of adrenaline in the latter case is due chiefly to the action of some esterase in the tissue. FiG. I. Duiratioin of action of adreinaline oin frog's aurlicle. A anld( D, auricle in 10. bathl; B, C, E, ain(d F, bath einptied at airow an(d wet atiricle suispendedc In air. Log. conc. adrenaline: A, - 7; 13, - 7; C, - 8; D, - 7-5; E, - 7-5; F, - 6. TimIe, 30 sees. WVe have previously described [Clark and Raventos, 1938] the calculationi of the rate of hydrolysis of acetylcholine from the duration of its action on the wetted auricle strip, and similar calculations were applied to the results obtained with adrenaline, which are summarised in Table I. These results with adrenaline Nere less consistent and less accurate than those obtained by a similar method with acetylcholine, but since they cover a 2000-fold range of concentrations they suffice to indicate the general nature of the process of inactivation. The time for half-destruction is nearly constant with concentrations ranging from S) to 1000 parts per 1000 mnillion, but with higher concentrations this time increases. A similar relation was found in the case of acetylcholine, and the simplest explanation for such a relation is to assume that the drug is destroyed by a limited quantity of enzyme. With low concentrations of drug the amount destroyed per unit of time is proportional to the concentration of the drug, but when the concentration of drug reaches a certain level partial enzyme saturation occurs, and the amount destroyed per unit of time is no longer proportional to the concentration. The hypothesis that the cessation of action of adrenaline was due

5 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 189 to inactivation was tested by measuring the influence of stabilisers. Ascorbic acid is known to be a powerful stabiliser of adrenaline [Bacq, ]. We found that addition of 1 part per million of ascorbic acid to 3 parts per 100 million of adrenaline was sufficient to reduce to 1/4 the rate of oxidation of adrenaline both in the 10 c.c. bath and in the wetted auricle. The results suggested that adrenaline and ascorbic acid compjjted for the same catalyst. TABLE I.-RATE OF INACTIVATION OF ADRENALINE BY THE FROG'S AURICLE (AVERAGES OF 6 EXPERIMENTS). Log. concentration of adrenaline I. Duration in min. until conc. deduced to 10-9 (threshold conc.): (a) Auricle in 10 c.c. fluid (b) Wetted auricle II. Time in secs. for half inactivation of adrenaline: (a) Auricle in 10 c.c. fluid (b) Wetted auricle INACTIVATION OF ADRENALINE BY THE AURICLE. The experiments described indicate that adrenaline is inactivated by some enzyme which acts on adrenaline, but which is inhibited by ascorbic acid. Hence the inactivation cannot be accounted for by fixation of adrenaline by cells in the manner described by Bain, Gaunt, and Suffolk [1937] in the case of adrenaline in contact with erythrocytes. The wide difference in the rate of inactivation of adrenaline in a 10 c.c. bath and in the fluid wetting the auricle indicates that in the latter case the drug is inactivated by the tissue. This is not absolutely certain, because there is no direct evidence regarding the rate of oxidation of adrenaline in vitro when present in a thin film of fluid. The results (Table I.) show that at concentrations of 1 part or less per million the time of half-destruction of adrenaline is constant and is about 60 secs. When the concentration is more than 1 part per million the time of half-destruction increases. Our results also indicate that each cell (3000 cu., volume) destroys per second between 1000 and 2000 molecules at a concentration of 1 part per 100 million, and about 100 times this quantity at a concentration of 1 per million. These figures are of a similar order to those calculated in the case of acetylcholine [Clark and Raventos, 1938].

6 190 Clark and Raventos DURATION OF ACTION OF ADRENALINE IN THE CAT. Frey [1914] measured the action of adrenaline on the rabbit's blood-pressure, and his figures show a duration of about 90 secs. with a dose of 3-2,ug./kg. adrenaline. Santesson [1919] made similar measurements on rabbits, and the averages of his results show a duration of 95 secs. with a dose of 20,ug., 125 secs. with 30 pg., and 170 secs. with 50 pg. These times correspond to a constant destruction at all dosages of about 0 3,ug./kg./sec. Koppanyi and Lieberson [1930] measured the duration of response of the cat's iris to intravenous doses of adrenaline (0.625,tg./kg. to 500 pg./kg.). The curves relating dosage and duration are similar in shape to those obtained by us with other responses (e.g. fig. 3), and can be interpreted as showing a constant time of half-destruction of 12 secs. with doses less than 15 jug./kg., and with doses above 15 /ug./kg. a constant and maximum rate of destruction of 0-66,ug./kg./sec. METHODS. Cats were ansesthetised with chloralose; the suprarenals and the superior cervical ganglia were removed, the vagi cut, and the responses of the nictitating membrane and blood-pressure were measured. The response of the nictitating membrane was measured with both isotonic and isometric levers, but no essential difference was found as regards the general form and duration of the responses. In other experiments the animals were anasthetised by chloralose, suprarenalectomised, the splanchnics cut, and the gut movements measured by the Cushny myocardiograph. Augmentor (n.m. and b.p.) and inhibitor (gut) responses were therefore studied. In general it was found that in any experiment the durations of different responses (e.g. gut and b.p. or n.m. and b.p.) to any particular dose of adrenaline were similar. Hence the durations measured did not appear to be dependent on accidental causes such as fatigue of the responding tissues. Bacq [1936 b] pointed out that the nictitating membrane response was exceptionally favourable for quantitative measurements of drug action, and we found it possible with this muscle to obtain graded responses over a 1000-fold range of dosage (0-25 to 330 ug./kg.). The blood-pressure response was more sensitive, but less favourable for accurate measurement. Fig. 2 shows a series of tracings taken from the isotonic responses of the nictitating membrane obtained in one experiment. Comparison of fig. 2 with fig. 1 shows that the curves obtained with the nictitating membrane resemble those obtained with the frog's auricle, since in both cases the " plateau " effect obtained with high concentrations or doses is a striking feature. Rosenblueth [1932] discussed the shape of the responses of the

7 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 191 nictitating membrane to adrenaline, and concluded that they could be interpreted by the formulae put forward by Hill [1909] to interpret the wash out of nicotine from the frog's rectus abdominis. These formulse do not, however, account for the occurrence of the " plateau." If it be assumed that the height of the response of the nictitating membrane during relaxation indicates the presence around the muscle 500 lo to100 OO secr. FIG. 2.-Action of adrenaline on nictitating membrane of cat. Isotonic records. Upper graph: tracings of records. Ordinates: left, amplitude of response; right, equivalent dosage adrenaline,ug./kg.; abscissa: time in secs. Lower graph: rate of disappearance of adrenaline calculated from upper graph. Ordinate: dosage adrenaline 4ug./kg. on logarithmic scale; abscissa: time in secs. Of the concentration of adrenaline sufficient to produce this response, then changes in the concentration of adrenaline during the recovery from a dose can be calculated. The lower graph in fig. 2 shows the results calculated from the upper graph. Fig. 3, I, shows the relation obtained in another experiment between dosage and the duration of the response (a) of the nictitating membrane and (b) of the blood-pressure. The results with the nictitating membrane fall along a curve which can he interpreted on the assumption that below 10,ug./kg. the time for half-destruction is constant and equal to 15 secs., whilst with quantities above 10,tg./kg. there is a constant destruction of 20,utg./kg. per 100 secs. The curve in fig. 3, I, showing the duration of the responses of the nictitating membrane was calculated VOL. XXIX., NO

8 192 Clark and Raventos from the constants mentioned above. The duration of responses of the blood-pressure are scattered, but they show a dosage-duration relation similar to that obtained with the n.m. The administration of large doses of adrenaline produces deleterious effects, and hence only one or two such doses can be administered in ko - A 1.o[ B1 0 Id 50 B 5 -* /- o -1. I I-,, / O +Z / I I a25 *-. t ' '- I FIG. 3. Action of adrenaline on nictitating membrane, gut, and blood-pressure of cats (suprarenalectomised and vagi cut). Ordinates: dose adrenaline log. /tg./kg.; abscissa-: time in secs. I. Durations of responses of nictitating membrane (A) and blood-pressure (B). II. Durations of responses before (A) and after (B) 40 mg./kg. pyrogallol. Dots and circles: blood-pressure response; crosses: gut response. Insets: relation between log. dose adrenaline jg.kg. (abscissae) and amplitude of response expressed as per cent. of estimated maximum. a single experiment. Table II. gives averages obtained from 10 experiments, and these show an approximately linear relation between dosage and time. The durations observed agree approximately with those calculated on the assumption that doses of adrenaline above 10,ug./kg. are inactivated at a constant rate of 1,ug./kg. per 6 secs. The relation between dosage and duration of adrenaline action on the cat's n.m. and blood-pressure shows, therefore, that with small doses the rate of inactivation is proportional to the dosage (time of half inactivation constant), whilst when the dosage is raised above 10,ug./kg. the amount inactivated per second becomes constant.

9 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 193 TABLE II.-AVERAGE DURATION OF RESPONSE (IN SECS.) TO ADRENALINE OF CAT'S N.M. AND B.P. THE FIGURES IN BRACKETS SHOW THE NUMBER OF OBSERVATIONS. THE CALCULATED FIGURES SHOW THE DURATION RESULTING FROM A DESTRUCTION OF 16-6 JuG./KG. PER 100 SECS. WITH DOSES ABOVE 10,UG./KG. Dosage. Adrenaline,ug./kg Nictitating membrane: Duration- (a) Observed (10) (12) (12) (5) (3) (b) Calculated (dose -10) Blood-pressure: Duration- (a) Observed (b) (2) (2) (2) (1) Calculated (dose -10) We compared the action of adrenaline given intravenously with that of adrenaline injected into the central end of the right renal artery. The intra-arterial injection produced the smaller response, and blood-pressure responses of equal amplitude and duration were obtained when the intra-arterial dose was equal to 6 times the intravenous dose. With large doses of adrenaline (more than 10,ug./kg.) the responses to equal doses given by the two routes were similar. Hence the tissues can only fix a limited quantity of adrenaline, but with moderate doses more than 80 per cent. of the injected dose is immediately removed from the plasma. These results agree with those described by Trendelenburg [1916]. INFLUENCE OF REDUCING AGENTS ON THE DURATION OF ACTION OF ADRENALINE. Bacq [ ] showed that a number of reducing agents greatly increased the duration of action of adrenaline. He found that pyrogallol could produce a tenfold increase in the duration and a considerable increase in the amplitude of the response of the nictitating membrane to the same dose of adrenaline. He concluded that ascorbic acid had no effect on this response, and we confirmed this fact. Bacq [1936, a and c] showed that reducing agents did not prolong the time adrenaline remained in the blood-stream, and concluded that these agents acted by delaying the oxidation of adrenaline in the tissues. The part played by oxidation in limiting the duration of the adrenaline response can therefore be estimated by a comparison of the dosageduration relations before and after administration of reducing agents such as pyrogallol.

10 194 Clark and Raventos Fig. 3, II, shows the dosage-duration relation of the response of the gut and of the blood-pressure to adrenaline before and after pyrogallol. Similar results were obtained with the n.m. Pyrogallol did not change the duration of the response to small doses of adrenaline, but markedly increased the duration of the responses to large doses. The results (fig. 3, II inset) do not show that pyrogallol produced any change in the intensity of the response to adrenaline, but the experiments involved long series of injections, and there was a long interval between the injection of comparable doses of adrenaline before and after pyrogallol. In shorter experiments a change in intensity as well as duration of response was found after pyrogallol. The effect of pyrogallol on the response to adrenaline therefore resembles very closely the effect of physostigmine on the response to acetylcholine [Clark and Ravent6s, 1938]. The results with pyrogallol indicate that the durations of the response to doses of adrenaline of 0.1 ug./kg. or less are not dependent on oxidation, whereas the durations of the responses to larger doses are dependent on oxidation. It has already been shown (Table II.) that the dosage-response relation with doses of adrenaline of 10 /g./kg. or more suggests that the amount of adrenaline oxidised in unit time is constant. Fig. 3, II, shows that in the range of dosage between 0.1 and 10 pg./kg. the duration of response is dependent on oxidation, and that there is an approximately linear relation between log. dose and duration, which indicates half-destruction of the drug in 15 secs. INFLUENCE OF COCAINE AND OF ERGOTAMINE. The response to adrenaline can be increased and decreased by cocaine and ergotamine respectively, and we made a few measurements of the influence of these drugs on the duration of the adrenaline response. Rosenblueth and Rioch [1933] found that cocaine increased both the amplitude and duration of the n.m. response to adrenaline. We confirmed this result both with the n.m. and the blood-pressure. Cocaine causes not only an increase in the amplitude, but also an increase in the duration of the response to a constant dose of adrenaline. Since, however, the threshold dosage at which the action ceases is altered by cocaine, the increase in duration does not prove any change in the rate of removal, and our results indicated that doses of equal biological activity produced responses of similar duration before and after cocaine. Ergotamine (0.15 mg./kg.) reduced the sensitivity of the bloodpressure response to about 1/3 of the normal, and also somewhat reduced the duration of the response. In this case, as with cocaine, the change in duration can be explained by the change in the threshold of sensitivity. The effects produced by potentiating and antagonising adrenaline

11 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 195 therefore suggest that the sensitivity of the tissue is altered, but that the rate of destruction of adrenaline is not changed. DURATION OF ACTION OF TYRAMINE. Tyramine has a weaker sympathomimetic action than adrenaline. Estimates of the relative pressor activity of the two substances range from 1/20 to 1/100 [Dale and Dixon, 1909; Barger and Dale, 1910; Chen and Meek, 1926]. Tyramine is broken down by isolated tissues such as the liver and heart of the rabbit [Ewins and Laidlaw, 1910]. It is oxidised by amine oxidase 1- times more rapidly than is adrenaline [Blashko, Richter, and Schlossmann, 1937]. The fact that tyramine is inactivated more rapidly than adrenaline -5- B o B6B -6 A 7- AX ~8-7- to a o t00 FiG. 4.-Duration of action of adrenaline and of tyramine. I. Duration of response of wetted frog's auricle. Ordinate: log. cone.; abscissa: time in min. Average results: A, adrenaline; B, tyramine. II. Duration of response of bloodpressure (dots and crosses) and of nictitating membrane (circles and squares). Ordinate: log. dose,ug./kg.; abscissa: time in secs. A, adrenaline; B, tyramine. Inset: relation of dosage (abscissa) and amplitude response (ordinate). appears difficult to reconcile with the well-known fact that the bloodpressure response to tyramine in the mammal is much longer than a response of equal intensity to adrenaline, but it will be shown that this apparent discrepancy can be accounted for by the gross difference in the activity of the two drugs. Fig. 4 shows the comparative duration of the actions of adrenaline (A) and tyramine (B) on the wetted auricle of the frog (I) and on the blood-pressure and nictitating membrane in the intact cat (II). Inspection of the dosage-duration curves obtained with the frog's auricle shows that the greatest difference between the two drugs is the difference in their activity. The minimum effective concentration of tyramine is more than 100 times greater than the corresponding figure for adrenaline. Hence the duration in the two cases is measured to two completely different end-points (i.e. reduction of concentration to about 2 parts per 10 million in the case of tyramine and to 2 parts per 1000 million in the case of adrenaline). Analysis of the tyramine curve

12 196 Clark and Raventos shows that with concentrations above 1 per million the destruction is at a rate of about 1 part per million in 0 5 min., whereas in the case of the same concentrations for adrenaline the destruction is at a rate of about 1 part in 2-5 min. Hence when similar concentrations are compared tyramine is found to be destroyed about 5 times as quickly as is adrenaline. If, however, equiactive concentrations be compared, e.g. 10 times the threshold dose, then owing to the great difference in threshold concentration the concentration selected falls on the steep portion of the adrenaline curve, where a tenfold reduction in concentration occurs in about 3 min., whereas in the case of tyramine the equiactive concentration falls at a point where the curve is already flattened and a tenfold reduction requires 7 min. Experiments with tyramine and adrenaline on the cat (fig. 4, II) showed a similar result. The ratio of activities in this case was 1 to 100. The duration of response to doses of tyramine above 20,ug./kg. indicated a constant destruction of about 1,ug./kg. in 3 secs. The largest dose of tyramine used in the cat was 1P6 mg./kg., and this produced a response lasting about 20 min. This time agrees with an observation by one of the authors [Clark, 1910], who found in man that doses of tyramine ranging from 0 3 to 1-2 mg./kg. produced a rise of blood-pressure lasting for min. In the experiment shown in fig. 4, II, the rate of destruction of doses of adrenaline above 3,ug./kg. was 1,ug./kg. in 14 secs. This is about half the rate shown by the general averages for adrenaline (Table II.). The results indicate that in the case of doses above 3 jug./kg. tyramine is destroyed 2-5 times more quickly than is adrenaline. In the cat, as in the frog, the greater part of the active range of dosage of tyramine shows a linear relation between dosage and duration. Hence when equiactive doses of the two drugs are compared (i.e. similar multiples of the threshold dose) the weaker drug tyramine takes longer to be reduced to the threshold than does adrenaline. DURATION OF ACTION OF EPHEDRINE. Ephedrine resembles tyramine as regards the intensity of its action on the cat's blood-pressure. Chen [1928] found adrenaline to be 140 times as active as natural ephedrine. Our results (fig. 5, inset) show a ratio of about 50 to 1, but there is a wide individual variation. Ephedrine is much more stable than either adrenaline or tyramine. It is equally active by oral or by subcutaneous administration [Miller, 1925; Ogden and Teather, 1935]. It is not oxidised by many ordinary agents which destroy adrenaline [Kendall and Witzmann, 1927]. It is not affected by the tissue oxidases which break down tyramine and adrenaline [Blaschko, Richter, and Schlossmann, 1937]. Its fate in the body appears to be unknown [Chen and Schmidt, 1930].

13 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 197 The dosage-duration relation of ephedrine action on the cat's bloodpressure, however, closely resembles that of tyramine, as is seen by comparing figs. 4 (II) and 5. The results of Chen and Meek [1926] show that similar doses of ephedrine and tyramine produce pressor effects of similar duration in the dog. The dosage-duration relation for ephedrine shown in fig. 5 agrees with results published by Chen [1928], who found the average durations of the pressor effect in pithed cats to be 540 secs. for 0 4 mg./kg. and 3000 secs. for 2 mg./kg. Our 3 0 XO FIG. 5. Action of adrenaline and ephedrine on cat's blood-pressure and nictitating membrane. Ordinate: dose of drug as log.,ug./kg.; abscissa: time in secs. A and B: duration of response of blood-pressure to ephedrine and adrenaline respectively. Inset: relation between dose of drug as log. pg./kg. (abscissa) and amplitude of response expressed as per cent. of estimated maximum (ordinate). A, ephedrine on blood-pressure; B, adrenaline on blood-pressure (circles) and nictitating membrane (crosses). figure for the former dose was 640 secs. The durations of action of ephedrine doses above 20 fig./kg. correspond to a clearance of 1,ug./kg. in 1-5 secs., whilst the corresponding figure for adrenaline in doses above 10,ug./kg. is 1,ug./kg. in 4 secs. The general average of our experiments indicated a clearance of ephedrine of about 1,ug./kg. in 1 sec. Pyrogallol was found to have no effect on the duration of action of ephedrine, a fact which agrees with much other evidence in indicating that ephedrine is not oxidised in the body. The fact that ephedrine is cleared more rapidly than adrenaline is difficult to explain and will be discussed later. When equiactive doses of ephedrine and adrenaline are compared the duration of action of the weaker drug ephedrine was the longer for the same reasons as were found in the similar case of tyramine. -6

14 198 Clark and Ravent6s DISCUSSION. Our experiments have been made on two systems, namely, the relatively simple system of the wetted frog's auricle and the much more complex system of the intact cat. The experiments with the frog's auricle are fairly easy to explain. The averages given in Table I. show that the time of half-destruction of adrenaline is constant over a considerable range of concentrations, but increases with high concentrations. This relation suggests that the adrenaline is oxidised by means of an enzyme or catalyst which is present in limited amounts. The prolongation of the duration of action by low concentrations of ascorbic acid supports this hypothesis. The chief alternative possibility is that adrenaline is removed by fixation by the cells in the manner described by Bain, Gaunt, and Suffolk [1937], but this hypothesis would not explain the prolongation of action produced by ascorbic acid. This latter effect also supports the belief that the duration of action observed measures the time until the adrenaline is inactivated, and is not simply a measure of the period for which the tissue is capable of sustaining an augmentor response. The adrenaline experiments agree with the hypothesis that the tissue enzymes inactivate adrenaline in low concentrations with a constant time of half-destruction of 60 secs., but that when the concentration exceeds 1 part in 1 million the enzyme is saturated, and the amount destroyed per unit of time is constant at 1 part per million in 150 secs. Tyramine in concentrations above 1 part per million is inactivated about 5 times more rapidly than adrenaline, a result which is in qualitative agreement with the conclusion of Blaschko et at [1937] that tyramine is inactivated 1 1 times as rapidly as adrenaline by tissue oxidases. Unfortunately ephedrine does not produce an effect on the isolated frog's auricle that is suitable for time measurements. The action of adrenaline on the intact cat's blood-pressure can be interpreted as follows. When the dose is not much greater than the minimum effective dose the duration is not affected by the dosage. It would appear that there is a certain minimum time ( secs.) required by the circulatory mechanism to re-establish equilibrium after a disturbance. Higher doses show a linear relation between log. dosage and duration, and there is a constant time of secs. for halfdestruction. When the dosage is further increased (above about 3 pg./kg.) the dosage-duration curve alters, and the amount of adrenaline destroyed becomes constant at about 1,ug./kg. per 5 secs. Pyrogallol prolongs the duration of action of adrenaline, and the results can be interpreted as showing a reduction of enzyme activity to one-fourth, so that the time of half-destruction is 64 secs., and the rate of destruction of doses above 2-5,ug./kg. is 1 pg./kg. in 20 secs.

15 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 199 Tyramine has no certain action in doses below 3,ug./kg., and its dosage-duration relation resembles that found with doses of adrenaline greater than 3,ug./kg. except that tyramine is destroyed about six times more rapidly (1,tg./kg. in 3 secs.). These results all conform to the results obtained with adrenaline and tyramine on the wetted frog's auricle. The results with ephedrine present considerable difficulty. As far as is known there is no enzyme in the body which destroys this drug, and yet the rate at which it is cleared from the body is about four times the corresponding rate for adrenaline, and at least equal to, and probably greater than, the corresponding rate for tyramine. It is necessary to assume that ephedrine is cleared by fixation by some tissue or tissues. In view of the resemblance between the shapes of the dosage-duration curves of adrenaline, tyramine, and ephedrine, the question naturally arises whether all these curves may not express the rate of fixation of the drug by the liver. Such an explanation will not, however, explain either the manner in which pyrogallol prolongs the adrenaline action or the concentration-duration curves obtained with adrenaline and tyramine on the wetted frog's auricle. It seems necessary, therefore, to assume that ephedrine is cleared by a mechanism different from that responsible for the clearance of adrenaline and tyramine, although the dosage-duration curves are so similar. The same problem was encountered in the case of the choline esters [Clark and Raventos, 1938]. The dosage-duration curves of acetylcholine in the cat before and after physostigmine showed with high doses constant clearances of 15 and 0.1 pg./kg. respectively, and this difference proved that the clearance was due to esterase activity. Carbaminoyl choline and carbaminoyl methyl choline are not acted on by the choline esterase, and produced permanent depression of the wetted frog's auricle. In the cat carbaminoyl choline showed a rate of destruction similar to that of acetylcholine after physostigmine, a result which is in accordance with expectation, but the weaker drug, carbaminoyl methyl choline, was cleared almost as rapidly as was acetylcholine in the absence of physostigmine. The examples of ephedrine and of carbaminoyl methyl choline indicate that there exist in the mammal methods for drug clearance, other than enzyme activity, which can clear drugs as quickly as do enzymes. Moreover, these two examples show that these alternative methods of clearance can produce dosage-duration curves very similar in shape to those which are produced by enzyme activity. Hence it is unsafe to deduce from dosage-duration relations the existence of tissue enzymes which can destroy drugs unless it is possible to test this hypothesis on simple systems, such as the wetted frog's auricle, in which the number of uncontrolled variables can be reduced.

16 200 Clark and Raventos CONCLUSIONS. 1. The relation between concentration and duration of action of adrenaline acting on the frog's auricle wetted with a small amount of solution indicates that the adrenaline is oxidised by a tissue catalyst. The rate of destruction varies as the concentration and the time of half oxidation is about 60 secs. 2. A comparison of the dosage-duration curves obtained in the cat with adrenaline before and after pyrogallol and with ephedrine indicates that the duration is limited by two mechanisms, namely, oxidation and, in addition, some other mechanism which does not depend on the lability of the drug. 3. The relation between dosage of adrenaline (below 3,tg./kg.) and the duration of action on the cat's blood-pressure and nictitating membrane and gut indicates that the amount of adrenaline destroyed per unit of time varies as the concentration and that half-destruction occurs in the tissues in about 20 secs. 4. When the dosage of adrenaline exceeds 3 pg./kg. the amount destroyed is between 0-1 and 0-2,Lg./kg. per sec., and this amount does not change when the dosage is increased. 5. The curves relating the concentration (or dosage) and the duration of action of tyramine on the wetted frog's auricle and on the cat's blood-pressure are similar in shape to those obtained with adrenaline. Tyramine is inactivated about five times more rapidly than is adrenaline. The expenses of this research were defrayed by a grant from the Moray Research Fund of Edinburgh University, and one of the authors (J. R.) is in receipt of a grant from Messrs. Imperial Chemical Industries, Ltd. We desire to express our thanks for this help. REFERENCES. ABDERHALDEN, E., and GELLHORN, E. (1923). Pflilgers Arch. 199, 437. BACQ, Z. M. ( ). Arch. int. Physiol. 42, 340. BACQ, Z. M. (1936 a). Ibid. 44, 15. BACQ, Z. M. (1936 b). Mem. Acad. Roy. Med. Belg. 25, 5. BACQ, Z. M. (1936 c). J. Physiol. 87, 87 P. BAIN, W. A., GAUNT, W. E., and SUFFOLK, S. F. (1937). Ibid. 91, 233. BARGER, G., and DALE, H. H. (1910). Ibid. 41, 19. BARKER, J. H., EASTLAND, C. J., and EVERS, N. (1932). Biochem. J. 26, BLASCHKO, H., RICHTER, D., and SCHLOSSMANN, H. (1937). J. Physiol. 90, 1. BLIx, G. (1929). Skand. Arch. Physiol. 56, 131. BURRIDGE, W. B., and SETH, D. N. ( ). Quart. J. exp. Physiol. 19, 201. CHEN, K. K. (1928). J. Pharmacol., Baltimore, 33, 237. CHEN, K. K., and MEEK, W. J. (1926). Ibid. 28, 59.

17 Durations of Responses to Adrenaline, Tyramine, and Ephedrine 201 CHEN, K. K., and SCHMIDT, C. F. (1930). Ephedrine. Bailliere, Tindale & Cox, London. CLARK, A. J. (1910). Biochem. J. 5, 236. CLARK, A. J., and RAVENTOS, J. (1938). Quart. J. exp. Physiol. 28, 155. CRAMER, W. (1911). J. Physiol. 42, 36 P. DALE, A. S. (1937). Ibid. 89, 316. DALE, H. H., and DIXON, W. E. (1909). Ibid. 39, 25. EWINS, A. J., and LAIDLAW, P. P. (1910). Ibid. 41, 78. FREY, E. (1914). Arch. exp. Path. Pharmak. 76, 65. GAUTRELET, J., HALPERN, N., and CORTEGGIANI, E. (1935). Medecine, 16, 726. GITHENS, T. S., and MELTZER, S. J. (1916). J. Pharmacol., Baltimore, 8, 133. HESS, F. 0. (1921). Arch. exp. Path. Pharmak. 91, 303. HILL, A. V. (1909). J. Physiol. 39, 361. JOSEPH, D. R. (1912). J. exp. Med., 15, 644. KENDALL, E. C., and WITZMANN, E. J. (1927). Proc. Soc. exp. Biol. N.Y. 24, 917. KOPPANYI, T., and LIEBERSON, A. (1930). J. Pharmacol., Baltimore, 39, 187. MARKOWITZ, J., and MANN, F. C. (1929). Amer. J. Physiol. 89, 176. MILLER, T. G. (1925). Amer. J. Med. Sci. 170, 157. OGDEN, E., and TEATHER, A. R. (1935). J. Pharmacol., Baltimore, 54, 320. ROSENBLUETH, A. (1932). Amer. J. Physiol. 101, 149. ROSENBLUETH, A., and RIocH, D. MCK. (1933). Ibid. 103, 681. SANTESSON, C. G. (1919). Skand. Arch. Physiol. 37, 185. SCHILD, H. (1933). J. Physiol. 79, 455. SUGAWARA, T. ( ). Tohoku J. exp. Med. 12, 97. TATUM, A. L. (1912). J. Pharmacol., Baltimore, 4, 151. TosCANo RIco, J., and MALAFAYA BAPTISTA, A. (1935). C.R. Soc. Biol. Paris, 120, 545. TRENDELENBURG, P. (1910). Arch. exp. Path. Pharmak. 63, 161. TRENDELENBURG, P. (1916). Ibid. 79, 154. WELCH, A. DE M. (1934). Amer. J. Physiol. 108, 360. WILTSHIRE, M. 0. P. (1931). J. Physiol. 72, 88.