The majority of early experiments were concerned with measuring. Pennsylvania Medical School

Transcription

1 182 J. Physiol. (I94I) 99, I I2.74I.6I VASOCONSTRICTOR NERVES AND OXYGEN CONSUMPTION IN THE ISOLATED PERFUSED HINDLIMB MUSCLES OF THE DOG BY J. R. PAPPENHEIMER From the Department of Pharmacology, University College, London, and the Department of Physiology, University of Pennsylvania Medical School (Received 22 July 1940) REIN & SCHNEIDER [1937] have described experiments which indicate that reflex activity of the vasoconstrictor nerves to the hindlimb of the dog is accompanied by a closely associated reduction in the oxygen consumption of the limb. They have observed that during vasoconstriction elicited reflexly from the carotid sinus the arterio-venous difference in oxygen content (A.V. 02-di.ff) may be diminished despite an unchanged or even a diminished blood flow through the limb. They found further that the arterio-venous temperature difference (A.V. T.-diff.) was diminished during the constriction, and that the magnitude of this diminution was of the order which might be expected from the calculated decrease in oxygen consumption. They have concluded that the sympathetic nerves exert a direct action in lowering the heat production and oxygen consumption of muscle. The experiments of Rein & Schneider are in contrast to earlier investigations of a possible action of the sympathetic nervous system on metabolism. Previous investigations have been made with a view to obtaining evidence for or against a sympathetic control of tonus in skeletal muscle, and the presumption has been that if any change in metabolism should result from sympathetic activity it would be in the direction of an increase. The majority of early experiments were concerned with measuring the oxygen consumption or heat production of the whole animal or of muscle preparations before and after sympathetic denervation [Mansfeld & Lukacs, 1915; Nakamura, 1921; Freund & Janssen, 1923; Newton, 1924; Cannon, Newton, Bright, Menkin & Moore, 1929]. The results of

2 VASOCONSTRICTOR NERVES 183 these experiments have given no decisive support to the view that sympathetic nerves may influence the oxygen consumption of muscle. Thus Cannon et al. [1929] conclude that in the cat "....removal of the sympathetic chain does not reduce the metabolic rate more than 10 %". It is noteworthy, however, that Nakamura [1921] tried the effects of electrical stimulation of the sympathetic chain on the oxygen consumption of the hindlimb muscles of the cat: under these conditions he observed a profound reduction in the oxygen consumption calculated as the product of the blood flow and A.V. 02-diff., a reduction for which he was unable to account. An explanation of the results of Rein & Schneider and of Nakamura is, however, possible which would make unnecessary the conclusion that the sympathetic nerves exert a direct action in lowering the metabolism. If the action of the nerves were to divert blood into regions in which the oxygen consumption and surface area available for heat loss were low, then both the A.V. 02-diff. and the A.V. T.-diff. would be reduced. The product of blood flow and A.V. 02-diff. would be in this case only an apparent measure of the oxygen consumption which might in reality continue unchanged so long as adequate store of oxygen were available to the tissue deprived of a circulation. In this paper it will be shown that electrical stimulation of the vasoconstrictor nerves to the isolated, perfused hindlimb or gastrocnemius muscle of the dog results in changes of blood flow and oxygen saturation similar to those which Rein & Schneider have described as occurring in the intact hindlimb during reflex vasoconstriction. Evidence will be presented that these changes are brought about by vascular rather than by metabolic means, and for this reason the product of A.V. 02-diff. and blood flow will be termed the "apparent oxygen consumption". The isolated hindlimb provides a convenient preparation in which to investigate the phenomenon. The blood flow and oxygen saturation may be reliably measured and recorded over periods of several hours by the methods described by Kramer & Winton [1939] and the effects of changes in blood flow due to non-nervous sources such as variations in arterial pressure or in the composition of the blood may be studied and controlled. METHODS (1) Perfusion. The hindlimb or the gastrocnemius muscle alone was perfused at constant pressure with defibrinated blood from a pump-lung circulation. The apparatus and technique of perfusion were similar to

3 184 J. R. PAPPENHEIMER those described by Kramer & Winton [1939] for the isolated kidney. In the hindlimb experiments the perfusion was made through the femoral artery and the venous blood collected from the femoral vein. Exclusion of the skin from the circulation did not affect the results to be described. In the single muscle preparations the hindlimb was skinned and the circulation confined as far as possible to the gastrocnemius muscle as described by Kramer & Quensel [1938]. The femur was then sawn through, and the muscle together with its nerve transferred to the perfusion circuit. (2) The A.V. 02-diff. The oxygen saturation of arterial and venous blood was measured photoelectrically and recorded by the methods of Kramer & Winton [1939] with the modifications described by Eggleton, Pappenbeimer & Winton [1940]. Kramer & Winton have indicated that the relative error in the measurement of A.V. 02-diff. by these methods may approach vol. %. A more accurate appraisal of the absolute error involved in the measurement of oxygen combined with haemoglobin in blood of any given oxygen capacity is now available from a series of calibrations made with the manometric blood gas method (Van Slyke- Neill). The standard deviation of 97 gas analyses from the best straight lines in 31 calibration curves of the type shown by Kramer & Winton was vol. %. The oxygen content of the blood as determined by gas analysis was corrected for dissolved oxygen on the assumption that the blood was in equilibrium with the 5 % C02 used to ventilate the lungs in the perfusion circuit. The curves covered the range % saturation. (3) Blood flow. The venous outflow was measured with a Gaddum Outflow Recorder calibrated at intervals during the experiment with a stopwatch and measuring cylinder. The excursion of the recorder was proportional to the blood flow within an error of about 3 %. (4) Arterio-venous temperature difference. Single or multiple constantan-manganin junctions were inserted into the arterial and venous cannulae and the E.M.F. led to the same galvanometer-amplifier-recording system as that used for recording the oxygen saturation of the blood. The sensitivity of the system was generally adjusted by means of a shunt across the recording galvanometer so that a change of 0.10 C. produced an excursion of the recording milliammeter of 2 mm. (5) Stimulation. The motor endings were paralysed completely by the addition of curarine or purified curare to the perfusing blood, and the mixed nerve supplying limb or muscle stimulated with induction shocks or with alternating current of controllable frequency. I am indebted to Dr H. King for giving me the curarine and advice concerning the purification of curare. Neither preparation had a detectable effect on the

4 VASOCONSTRICTOR NERVES 185 oxygen consumption: the purified curare sometimes caused a slight vasodilatation, and when this occurred the oxygen content of the venous blood always increased also. The alternating current was generated by interrupting a narrow beam of light focused on a photoelectric cell, with a rotating brass disk whose edges formed a sine wave in polar co-ordinates. The disk was mounted on ball bearings and driven through a gear box by a motor whose speed was recorded with an automobile speedometer. The amplified output of the photocell was approximately sinusoidal, and the apparatus was so arranged that the frequency could be conveniently varied from 2 to 300 c./sec. EXPERIMENTAL Fig. 1 shows the effects of stimulating the tibial nerve on the blood flow and oxygen content of venous blood in the isolated, curarized gastrocnemius muscle. The following description applies equally well, however, to the effects of stimulating the sciatic nerve in the whole hindlimb preparation. As the flow is diminished byvasoconstriction the venous blood becomes more saturated with oxygen and the A.V. 02-diff. is diminished. The apparent oxygen consumption is therefore reduced and more than in proportion to the blood flow. The magnitudes of these changes vary from one preparation to another, and in the same preparation with the frequency of the stimulus as will be shown below. With maximal stimulation a reduction in the apparent oxygen consumption to one-half the resting rate is usual, although a reduction to a quarter of the resting rate has been observed. When the reduction of blood flow is slight the reduction of apparent oxygen consumption may occur largely as a result of a diminished A.V. 02-diff. When the reduction of blood flow is great the A.V. 02-diff. may be unchanged or even slightly increased. In no case, however, has the apparent oxygen consumption failed to be substantially reduced. The recovery of blood flow following release of the stimulus is accompanied by a decrease in the oxygen content of venous blood, and the magnitude of this decrease is such as to increase the apparent oxygen consumption above the initial value as seen in the analysis of the record. The apparent increase in oxygen use as measured by the area under the curve above the resting rate of 0-62 c.c. 02/min. was 0-27 c.c. 02 as compared with the apparent decreased use of 0-52 c.c. 02 which occurred during the stimulation. In the example shown, therefore, there occurred a net decrease in oxygen use of 0-25 c.c. 02 as a result of stimulation.

5 -- :40 : APPARENT OXYGEU CONSUMIPTION Fig. 1. The effects of stimulating the tibial nerve on the oxygen content of venous blood and on the blood flow in the isolated perfused gastrocnemius muscle. The arterial oxygen content was measured before and after the stimulation as indicated by the discontinuous sections of the venous oxygen record. Despite the reduction of blood flow the A.V. 02-diff. was diminished during the stimulation as indicated by the increase in the oxygen content of venous blood. In calculating the apparent oxygen consumption (product of blood flow and A.V. 02-diff.) corrections have been made for the curvatures of the recording levers and for the dead space between the photocell unit and the vein.

6 VASOCONSTRICTOR NERVES 187 Evidence concerning the mechanism of the phenomenon has been obtained from the following lines of enquiry: I. Comparison of the effects of changes in blood flow produced by nerve stimulation with those produced by (a) change of perfusion pressure, (b) the action of adrenaline. II. The effects of varying the frequency of stimulation. III. Quantitative comparison of the apparent decrease in oxygen use with the apparent increase occurring during the recovery period. IV. The action of ergotoxine. V. The arterio-venous temperature difference. I. (a) Perfusion pressure The effects of changes in blood flow caused by changing the perfusion pressure have been observed in thirty-two hindlimbs and four gastrocnemius muscles. An increase in A.V. 02-diff. has never failed to follow a decrease in blood flow caused by a lowering of the pressure, and conversely a decreased A.V. 02-diff. has followed an increase in blood flow caused by a rise in pressure. A typical experiment is seen in Fig. 2. It is seen that the oxygen consumption of the gastrocnemius muscle is relatively unafected by large changes in flow. In fifty-two experiments on eleven hindlimbs the mean diminution in oxygen consumption following a lowering of perfusion pressure has been 1-4 % per 10 c.c./min. reduction in blood flow over the range c.c./min. Two points concerning these observations should be made which will be discussed in greater detail elsewhere: (i) The values of oxygen consumption given in Fig. 2 are equilibrium values taken not less than 2 min. after a change of pressure. Following a sudden increase of pressure there may be a transient increase of A.V. 02-diff. accompanied by a vasodilatation lasting some 20 sec. (ii) As the blood flow is reduced below about 50 c.c./min. in the hindlimb the oxygen consumption may be considerably reduced although the A.V. 02-diff. continues to increase. It is clear, however, that a reduction of blood flow is not in itself capable of producing the changes in apparent oxygen consumption which occur during stimulation of the vasoconstrictor nerves. (b) Adrenaline If the fall in apparent oxygen consumption during stimulation were due to a direct action of the sympathetic fibres on metabolism, it might be expected that a sympathomimetic substance such as adrenaline would

7 188 J. R. PAPPENHEIMER exert a similar action. If, on the other hand, the action of the nerves were to divert blood from localized parts of tissue then adrenaline might -0*60- c.c./min. Consumption j 0* Vols. % - 40 _ omm. Hg I Arterial pressure 1*4 I'82 10 n_ l ll_ - 60.c.c./min Time (min.) Fig. 2. The effects of changes in blood flow caused by changes of arterial pressure on the oxygen consumption of the isolated gastrocnemius muscle. The data are depicted diagrammatically in that only equilibrium values of blood flow and A.V. 0,-diff. are shown as explained in the text. The oxygen consumption at the lowest flow was 10% lower than that at the highest flow. Weight of muscle 46 g. act differently, for in this case a more uniform vasoconstriction might be expected.

8 VASOCONSTRICTOR NERVES Adrenaline in doses sufficient to cause a diminution of blood flow has without exception caused an increased A.V. 02-diff. With a moderate reduction of blood flow there may even be an increase in apparent oxygen consumption which returns to its initial value before the blood flow has recovered as shown in Fig. 3. Von Euler [1931] has reported a similar increase in oxygen consumption of the perfused hindlimb following 1'80 c.c./min. Apparent 02-ICOnsumption 1*60X 1*40 -mi Vols. % A.V. 02-diff. 1'50- I o c.c./min Blood flow 90 -_\ 80 I ~~~~~~Time(Lmin.) Fig. 3. The action of adrenaline on the blood flow, A.V. 0,-diff., and apparent oxygen consumption of the isolated hindlimb. At the arrow 2-5,ug. adrenaline were'addedto the perfusing blood (ca. 11.). The transient increase in apparent oxygen consumption has not been observed in all cases although the A.V. 02-diff. has never failed to increase. small doses of adrenaline. We have not observed the increase in every case and do not feel that the data at present warrant a more detailed description. It is clear, however, that although adrenaline and stimulation of the vasoconstrictor nerves have similar effects on the overall blood flow, they have widely different actions on the oxygen content of venous blood. II. The effects of varying the frequency of stimulation Valdecasas [1935] has shown that stimulation of the vasoconstrictor nerves to the hindlimb of the dog with alternating current of low frequency (18 c./sec.) results in a greater diminution of blood flow than PH. XCIX. 13

9 190 J. R. PAPPENHEIMER does stimulation with higher frequencies ( c./sec.). It seemed possible that if the effect of stimlulating the sciatic nerve on the apparent oxygen consumption of the curarized limb were due to a direct nervous action, then such an action might be distinguished from vascular changes by varying the frequency of stimulation. The observations of Valdecasas that the vasoconstrictor nerves are more sensitive to stimulation with low than with high frequencies are.m -1- Z.-. Fig. 4. The effects of stimulating the vasoconstrictor nerves with alternating current of different frequencies on the blood flow and oxygen content of venous blood in the isolated hindlimb. The sudden partial recovery of blood flow following stimulation with 4-6 c./sec. is accompanied by a sudden release of unsaturated venous blood. The gradual recovery of blood flow following stimulation with 18 c./sec. is accompanied by an equally slow change in the oxygen content of venous blood. Stimulation with 290 c./sec. produced a just perceptible effect on both blood flow and venous oxygen content. confirmed on the isolated preparation. We have also investigated the effects of stimulating with frequencies below 18 c./sec.; this range has not previously been investigated. Fig. 4 shows the effects of four successive stimulations of varying frequencies. Although the absolute values of the frequencies involved vary greatly from one preparation to another, their effects may, for descriptive purposes, be divided into three main groups: (i) Low frequencies, 2-15 c./sec. It is characteristic of this range that when the stimulus is stopped the blood flow recovers partially within a few seconds. The remainder of the recovery occurs over a period of minutes.

10 VASOCONSTRICTOR NERVES 191 (ii) Intermediate frequencies, c./sec. It is characteristic of this range that although the reduction of blood flow is of the same magnitude as that occurring during low-frequency stimulation, the recovery takes place gradually and may require several minutes to reach completion. (iii) High frequencies, c./sec. As the frequency becomes higher the magnitude of the reduction of blood flow becomes less: at frequencies of 300 c./sec. or above little or no response may be obtained, as found by Valdecasas. If the effects of stimulation on the apparent oxygen consumption are due to vascular changes then the characteristic effects on the blood flow might be expected to be associated with characteristic effects on the oxygen saturation of venous blood. This is found to be the case. It is seen that the sudden partial recovery of blood flow following stimulation with 4f6 c./sec. was accompanied by a sudden release of unsaturated venous blood. At the intermediate frequency, on the other hand, the poststimulation increase of apparent oxygen consumption was delayed along with the delayed recovery of blood flow. At 290 c./sec. the effects on both blood flow and apparent oxygen consumption were just perceptible. The complex nature of the recovery at the lower frequencies could be explained by the existence of two or more sets of vasoconstrictor nerves having different excitabilities and different loci of action. Evidence in support of this hypothesis is shown in Fig. 5. In this experiment the frequency was changed during the stimulation from 150 to 4-4 c./sec. and then back to 150. The initial stimulation of 150 c./sec. caused a small diminution in both flow and A.V. 02-diff. When the frequency changed to 4-4 c./sec. there occurred a further diminution of apparent oxygen consumption. When the frequency returned to 150 c./sec., however, the blood flow recovered partially almost immediately, and the oxygen content of the venous blood fell rapidly despite the fact that the stimulus of 150 c./sec. continued. The interpretation of these events in terms of an indirect vascular mechanism would be as follows: During the initial period of stimulation at 150 c./sec., blood was diverted from certain localized parts of the limb through regions of lowered oxygen consumption. When the frequency changed to 4-4 c./sec. nerve fibres supplying new tissues were excited, and this resulted in a further diversion of blood and a further reduction in apparent oxygen consumption. But when the frequency returned to 150 c./sec. venous blood from regions which had been deprived of blood by the action of 13-2

11 192 J. R. PAPPENHEIMER fibres responding to low frequencies was released, so that the oxygen content of the venous blood diminished despite continued stimulation at high frequency. m~~~~ T Fig. 5. The effects of sudden changes in frequency of stimulation of the vasoconstrictor nerves on the blood flow and A.V. 02-diff. of the isolated hindlimb. Following stimulation with 4-4 c./sec. the oxygen content of venous blood diminished rapidly despite continued stimulation with 150 c./sec. The interpretation of these events in terms of a direct nervous action on the oxygen consumption would be difficult. III. Quantitative relations If the action of the vasoconstrictor nerves were to divert blood from parts of the muscle, it might be expected that the tissues deprived of a blood supply would continue to consume oxygen at the expense of oxygen

12 VASOCONSTRICTOR NERVES contained in the stagnated blood and combined with muscle haemoglobin. Provided that the store of oxygen were sufficient and that the rate of oxygen usage remained unchanged, the apparent increase in oxygen use Apparent increase in oxygen use X 100 Apparent decrease in oxygen use I Period of stimulation or of clamping I I I'l Fig. 6. The effects of clamping the circulation entirely for varying periods (black circles) or of stimulating the vasoconstrictor nerves for varying periods (open circles) on the ratio of the increase in apparent oxygen use following release of the clamp or of the stimulus to the apparent decrease in oxygen use which occurred during the period in which the stimulus or the clamp was applied. The broken line is the curve expected if the muscles continued to consume oxygen at an unchanged rate during the period of clamping or of stimulation until a 2 min. supply of oxygen were exhausted. Points to the right of this curve therefore indicate an increase in oxygen use greater than that obtainable from a 2 min. supply of oxygen stored in muscle. Compiled from observations on eight hindlimbs and two muscles. In no one experiment, however, were the effects of clamping compared with those of stimulation. following release of the stimulation should exactly equal the apparent decrease which occurred during the stimulation. Millikan [1937] has shown that when the blood supply to the resting soleus muscle of the cat is clamped off, the muscle haemoglobin is reduced at the rate of approximately 1 %/sec. This would indicate that the supply of oxygen available

13 194 J. R. PAPPENHEIMER to resting muscles deprived of a circulation is sufficient to last less than 2 min. In the example in Fig. 1 the stimulation lasted 92 sec., and the increase in apparent oxygen use as measured by the area under the curve above the resting rate of 0-62 c.c./min. was only 52 % of the apparent decrease which occurred during the stimulation. In order to investigate this question more fully we have compared the effects of clamping the circulation entirely for varying periods with those of stimulating the vasoconstrictor nerves for varying periods. The results are shown in Fig. 6. It is seen that the ratio of the increase in apparent oxygen use following release of the clamp or of the stimulus to the apparent decrease which occurred during the period of clamping or of stimulation bears no obvious relation to the period during which the stimulus or the clamp was applied. There is, moreover, little to distinguish the effects of stimulation from those of clamping in this respect. The broken line indicates the curve expected if the muscles continued consuming oxygen at an unchanged rate until a 2 min. supply of oxygen were exhausted. It may be noted that four of the points lie to the right of this curve, indicating that the increase in apparent oxygen use following the release of the clamp or stimulus was greater than that sufficient to supply the muscle for 2 min. The possibility exists, however, that the muscles may continue to metabolize anaerobically after the supply of oxygen has been exhausted, and that the debt so created may be repaid when the circulation is restored. Fenn [1930] and Rotta & Stannard [1939] have shown that this may be true of resting isolated frog's muscle which has been deprived of an oxygen supply. Rein & Schneider [1937] reported that a considerable net decrease in oxygen use occurred as a result of reflex vasoconstriction, and considered the decrease as evidence for a direct sympathetic control of the metabolism. The results shown above indicate that a net decrease in oxygen use cannot be taken as evidence of direct nervous action, for clamping the circulation entirely for short periods may produce a similar net decrease. IV. The action of ergotoxine The results of stimulating at different frequencies have revealed a close association between vascular changes and changes in apparent oxygen consumption. If the effects on the apparent oxygen consumption are due to vasoconstriction then they should be abolished by the action of ergotoxine. Fig. 7 shows the effects of stimulating the nerve i hr. after the addition of 5 mg. ergotoxine ethansulphonate (B.D.H.) to the per-

14 VASOCONSTRICTOR NERVES 195 fusing blood (ca. 1 1.). Stimulation caused a vasodilatation accompanied by an initial fall in the oxygen content of venous blood followed by an increase which was of such a magnitude that the apparent oxygen consumption remained unchanged. Fig. 7. The effects of stimulation following the addition of 5 mg./l. of ergotoxine ethansulphonate to the perfusing blood. The initial fall in venous oxygen content is of interest, for it may be evoked by various agents causing vasodilatation and is presumably due to the release of small quantities of reduced blood from capillary fields which have been opened by the agent causing vasodilatation. V. Arterio-venous temperature difference At any given environmental temperature, the A.V. T.-diff. of the hindlimb is dependent on three factors: (a) the metabolism of the limb, (b) the area of cooling surface, (c) the velocity of flow. In the perfused

15 196 J. R. PAPPENHEIMER limb, blood in the venous cannula may be as much as 1.50 C. lower than that of blood'in the arterial cannula. Under these conditions the heat liberated by metabolism is small compared to the heat loss from the Fig. 8. A comparison of the effects of approximately equal changes of blood flow caused by adrenaline and by electrical stimulation of the vasoconstrictor nerves on the A.V. T.-diff. of the isolated hindlimb. At D the record was stopped until recovery from the effects of adrenaline had taken place. The recording levers were not correctly aligned as may be seen from the record at D. The temperature changes actually occur within a few seconds of the changes in flow. surface. Thus if the utilization of 1 c.c. of oxygen results in the liberation of 5 cal., then for a typical limb consuming 2-0 c.c. oxygen/min. at a blood flow of 100 c.c./min. the warming of venous blood due to metabolism should be only 0-1 C. If the blood flow were changed by mechanical or

16 VASOCONSTRICTOR NERVES 197 pharmacological means the main factor determining the A.V. T.-diff. should be the velocity of flow since the area of cooling surface might be expected to remain constant. If, on the other hand, the reduction of blood flow involves the diversion of blood from large areas of tissue through regions of "lowered oxygen consumption" then alterations in the area of cooling surface might occur. Fig. 8 shows a comparison of the effects of similar changes in blood flow caused by adrenaline and by stimulation of the vasoconstrictor nerves on the A.V. T.-diff. A reduction of blood flow caused by the action of adrenaline or by a lowering of the perfusion pressure has been found to result in an increased A.V. T.-diff., conversely an increase in blood flow caused by the action of histamine or by raising the perfusion pressure has been found to result in a decreased A.V. T.-diff. In all these cases the limb may be compared to a crude form of thermostromuhr, the A.V. T.-diff. varying inversely with the flow. When the flow is reduced by stimulation of the vasoconstrictor nerves, however, the A.V. T.-diff. decreases instead of increasing. In the example shown in Fig. 8 the A.V. T.-diff. during constriction caused by nerve stimulation was 0.60 C. lower than during a similar constriction caused by the action of adrenaline. Tlis difference is at least five times as great as could be accounted for by a change of metabolism. It seems reasonable, therefore, to conclude that the effect of stimulating the nerve is to divert blood through regions having a small surface available for the loss of heat. DISCUSSION It has been shown that the oxygen consumption of the perfused hindlimb or gastrocnemius muscle is apparently greatly reduced when the vasoconstrictor nerves are stimulated electrically. Two possible explanations of the effect have been considered, a direct action of the nerves on the metabolism and an indirect vascular mechanism. While none of the evidence which has been obtained excludes the possibility that the action is a direct one, it renders such an hypothesis untenable unless further assumptions are made. It would thus be necessary to assume: (1) An explanation of the otherwise anomalous differences between the action of adrenaline and the action of the vasoconstrictor nerves. (2) That when stimulation is stopped another mechanism is involved which increases the oxygen consumption.

17 198 J. R. PAPPENHEIMER (3) That the mechanism assumed in (2) is affected by the frequency with which the nerves had been stimulated. (4) That the nerves causing the changes in metabolism have the same electrical excitability as the vasoconstrictor nerves. (5) That they are affected by ergotoxine in the same way as are vasoconstrictor nerves. On the other hand, all the facts which have been observed admit of an explanation in terms of a vascular shunting mechanism without further assumption. The question arises on the vascular hypothesis as to where the blood rnay be diverted and what tissues are deprived of a circulation during vasoconstriction. The regions through which blood may be diverted must, on the vascular hypothesis, consume oxygen at a rate lower than that of the muscle as a whole, for the apparent oxygen consumption is reduced more than in proportion to the reduction in blood flow. It has been shown that such regions have a reduced surface available for the loss of heat. These conditions would be fulfilled if the blood were diverted from capillary fields in the muscle tissue through arterio-venous anastomoses. Perhaps the most widely used method of measuring the oxygen consumption of mammalian organs has involoved the withdrawal of samples of venous and arterial blood and the calculation of the oxygen consumption as the product of the A.V. 02-diff. and the blood flow measured at the time of sampling. The inconstancy of the results obtained by the application of this method to resting mammalian muscle is well known. The variations have been emphasized by Barcroft [1934] and more recently by Looney & Freeman [1938] and by Holling [1939]. In this paper it has been shown that the oxygen consumption of the gastrocnemius muscle as measured by this method may vary fourfold within a minute as a result of stimulating the vasoconstrictor nerves (Fig. 1). In experiments on the innervated preparation in which the perfused limb has remained connected to the anaesthetized dog only through the sciatic nerve, we have found that large changes in the oxygen saturation of blood in the femoral vein may occur as a result of vasomotor activity in the dog. It would seem possible that such changes may be in part responsible for the variations in the apparent oxygen consumption which have been observed in previous investigations. The evidence presented in this paper suggests that the oxygen saturation of venous blood from resting muscle may be an indicator of the state of the intimate circulation within the muscle, rather than a measure of the metabolic rate.

18 VASOCONSTRICTOR NERVES 199 SUMMARY 1. The oxygen consumption of the isolated perfused hindlimb or gastrocnemius muscle of the dog is relatively unaffected by changes in blood flow caused by changes of perfusion pressure. In eleven hindlimbs the mean diminution in oxygen consumption following a lowering of perfusion pressure was 1-4 % per 10 c.c./min. reduction of blood flow over the range c.c./min. 2. When the blood flow through the hindlimb or muscle is reduced by stimulation of the vasoconstrictor nerves the oxygen consumption calculated as the product of blood flow and arterio-venous oxygen difference (apparent oxygen consumption) is greatly reduced; the A.V. 02-diff. is usually diminished. The changes are similar to those which Rein & Schneider [1937] have described as occurring in the intact limb during reflex vasoconstriction. 3. When the blood flow is similarly reduced by the action of adrenaline the A.V. 02-diff. is increased and the apparent oxygen consumption may be increased also. 4. When stimulation of the vasoconstrictor nerves is stopped the apparent oxygen consumption is increased. This increase may be equal to or less than the diminution in apparent oxygen use occurring during the stimulation; no simple relation between the two quantities has been found. 5. The observations of Valdecasas [1935] that stimulation of the vasoconstrictor nerves with alternating current of medium frequencies ( c./sec.) produces a greater reduction of blood flow than stimulation with high frequencies ( c./sec.) are confirmed on the isolated preparation. 6. The effects of stimulation with frequencies lower than those which have been investigated previously (2-15 c./sec.) are described. It is shown that the blood flow recovers partially within a few seconds after such stimulation. 7. The characteristic effects on the blood flow of stimulating with different frequencies are accompanied by closely associated changes in apparent oxygen consumption. 8. The effects of stimulation on the apparent oxygen consumption are abolished by ergotoxine. 9. The arterio-venous temperature difference is increased when the blood flow is reduced by lowering the perfusion pressure or by the action of adrenaline. When a similar change in blood flow is caused by nerve

19 200 J. R. PAPPENHEIMER stimulation the A.V. T.-diff. is decreased. The changes observed are greater than can be accounted for by changes in metabolism. 10. The evidence suggests that the action of vasoconstrictor nerves is to divert blood from parts of the muscle through regions in which the oxygen consumption and surface available for heat loss are small. These regions may be arterio-venous anastomoses. It is, therefore, unnecessary to conclude that sympathetic nerves have a direct action in lowering the oxygen consumption of muscle. I have pleasure in thanking Prof. Winton for his advice and help throughout the course of this work. This research was aided by a grant from the Faculty Research Fund, University of Pennsylvania. REFERENCES Barcroft, J. [1934]. The Architecture of Physiological Function, Chap. xm. Camb. Univ. Press. Cannon, W. B., Newton, H. F., Bright, S. M., Menkin, V. & Moore, R. M. [1929]. Amer. J. Physiol. 89, 84. Eggleton, M. G., Pappenheimer, J. R. & Winton, F. R. [1940]. J. Physiol. 97, 363. Fenn, W. 0. [1930]. Amer. J. Physiol. 93, 124. Freund, H. & Janssen, S. [1923]. Pflug. Arch. gem. Physiol. 200, 96. Holling, H. [1939]. Clin. Sci. 4, 103. Kramer, K. & Quensel, W. [1938]. Pflug. Arch. gee. Physiol. 239, 620. Kramer, K. & Winton, F. R. [1939]. J. Physiol. 96, 87. Looney, J. M. & Freeman, H. [1938]. Arch. Neurol. Psychiat., Lond., 39, 276. Mansfeld, G. & Lukacs, A. [1915]. Pftug. Arch. ge8. Physiol. 161, 467. Millikan, G. [1937]. Proc. Roy. Soc. B, 123, 218. Nakamura, H. [1921]. J. Phy8iol. 55, 100. Newton, F. C. [1924]. Amer. J. Physiol. 71, 1. Rein, H. & Schneider, M. [1937]. Pflug. Arch. gee. Physiol. 239, 464. Rotta, A. & Stannard, J. N. [1939]. Amer. J. Physiol. 127, 281. Valdecasas, F. G. [1935]. Z. Biol. 96, 28. Von Euler [1931]. C.R. Soc. Biol., Paris, 108, 246.