Oxford, OX13PT. (Received 16 October 1969) have been studied in atropinized, isolated, ventilated lung lobes under

Transcription

1 J. Physiol. (1970), 209, pp With 8 text-figuree Printed in Great Britain THE SITE OF ACTION OF NERVES IN THE PULMONARY VASCULAR BED IN THE DOG BY I. DE BURGH DALY, D. J. RAMSAY AND B. A. WAALER* From the University Laboratory of Physiology, Parks Road, Oxford, OX13PT (Received 16 October 1969) SUMMARY 1. The effects of stimulation of the thoracic vagosympathetic nerve or upper thoracic sympathetic chain on the pulmonary vascular resistance have been studied in atropinized, isolated, ventilated lung lobes under various conditions of pulmonary circulation perfusion. Throughout the nerve-stimulation tests bronchial circulation perfusion was maintained or temporarily interrupted. 2. The pulmonary vascular resistance increase evoked by nerve stimulation (a) occurred in the absence of tidal air changes; (b) did not consistently differ during predominantly 'sluice' and 'non-sluice' conditions of pulmonary circulation perfusion; (c) was approximately one and a half times greater during constant pressure than during constant volume inflow perfusion of the pulmonary circulation; and (d) was greater during reverse than during forward perfusion. 3. In lung lobes perfused in either direction at constant volume inflow nerve stimulation produced an increase in inflow pressure and a diminution in total lung blood volume reflected by a temporary increase in blood outflow. 4. In lung lobes in which neither the pulmonary nor the bronchial circulations were perfused and the capillaries were completely blocked by high intratracheal pressures, thus isolating the pulmonary arterial system from the venous system, nerve stimulation produced a diminution in the blood volume of both systems. 5. Nerve stimulation produced a rise in bronchial arterial pressure in the absence of pulmonary circulation perfusion. 6. Further evidence is adduced that pulmonary vasomotor nerve responses do not depend upon the transfer of transmitter substances from the bronchial to the pulmonary circulation. * Present address: Institute of Physiology, The University, Oslo, Norway.

2 DE BURGH DALY AND OTHERS 7. The possible significance of these observations in relation to the site of action of pulmonary vasomotor nerves is discussed. INTRODUCTION In atropinized isolated lungs perfused through both pulmonary and bronchial circulations the predominant effect of stimulating nerve fibres derived from the upper thoracic sympathetic outflow on the pulmonary vascular bed of the dog is vasoconstriction. The fibres concerned relay in the stellate and middle cervical ganglia and for the most part proceed to the lungs in the thoracic vagosympathetic nerve trunks as post-ganglionic fibres. The pharmacological evidence supports the view that these postganglionic nerve fibres are adrenergic in character and are the only ones effective in producing vasoconstrictor responses following stimulation of the thoracic vagosympathetic nerves. Perfusion of the bronchial circulation is necessary for maintaining the functional activity of the pulmonary vasomotor nerves. The pulmonary vasoconstrictor response can be demonstrated in the absence of bronchomotor responses and also during temporary interruption of bronchial circulation perfusion, thus excluding both passive bronchomotor and passive haemodynamic bronchial vascular effects on the pulmonary vascular bed proper (for literature see Daly & Hebb, ch. ax, 1966). The precise site of the vessels in the pulmonary vascular bed which constrict in response to pulmonary nerve stimulation is unknown. It seemed possible that some information might be obtained by comparing the magnitude, direction and the latent periods of the inflow pressure and volume outflow responses to nerve stimulation under different conditions of perfusion, particularly those prevailing during forward (normal direction of flow) perfusion with those occurring during reverse perfusion, and thus a pattern might emerge which would indicate whether nerve stimulation constricted either arterial or venous vessels or both. The results of experiments performed on isolated perfused lung lobes, under these conditions, will be described. A full account will also be given of experiments, briefly reported earlier by Daly & Waaler (1961), demonstrating that stimulation of the upper thoracic sympathetic chain or thoracic vagosympathetic nerve in non-perfused atropinized lung lobes reduces the blood volume of vessels situated on both the arterial and venous side of the capillaries. METHODS Mongrel dogs varying in weight from 14-5 to 30-0 kg were premeditated with morphine hydrochloride (2 mg/kg) subcutaneously, injected intravenously with a solution of Boots's powdered heparin (3 mg/kg) and bled out under local anaesthesia. Heparin (6 mg/kg) was added to the blood-collecting vessels. Treatment and storage

3 PULMONARY VASOMOTOR NERVE RESPONSES of the blood was as described by Allison, Daly & Waaler (1961). The left apical and left cardiac lobes, or in a few experiments the left diaphragmatic lung lobe, were isolated from the body and perfused at 108 c/min by Dale & Schuster pumps through both the pulmonary and bronchial circulations by the method described by Daly (1956). The bronchial circulation was perfused at a constant volume inflow (CVI), and the pulmonary circulation at a constant head of pressure (8-24 mm Hg) by the method of Daly, Duke, Hebb & Weatherall (1948), or at a constant volume inflow. The outflow of blood was measured by a Gaddum (1929) recorder and the bronchial 319 Fig. 1. Diagram of the arrangement for changing over fromforward to reverse perfusion and vice versa of the pulmonary circulation, in isolated lung lobes (ILL). m, m', 2 mm bore vertical tube manometer and Marey tambour respectively; FR, blood flow-recorder assembly; P, pulmonary circulation pump; P', pump supplying aortic arch and descending aorta down to T 8 for bronchial circulation perfusion (BC); R, blood reservoir. PA, LA, to cannulae for pulmonary artery, left atrium (or pulmonary vein) respectively of isolated lung lobe (ILL). During forward perfusion the connexions a, c and d are open, b, e andf closed. During reverse perfusion, b, e andf are open, a, c and d closed. I I PHY 209

4 320 I. DE BURGH DALY AND OTHERS arterial pressure by a mercury manometer or on rare occasions by a membrane manometer (Hurthle type). The Gaddum recorder measured flows over successive periods of 9-7 see) and thus transient changes in flow were not easily assessable. These, however, were more readily measured by recording the changes in the blood volume of the reservoir during CVI perfusion (see later). Forward and reverse perfuwion8. The arrangement for perfusion in the forward and reverse directions is shown in Fig. 1. The lung lobes, lying horizontally within a Perspex chamber, are not shown. The inflow pressure during perfusion in either direction was recorded by a damped Marey tambour with a 2 mm bore saline manometer in parallel for direct readings from a scale placed behind the manometer. The left atrial or pulmonary lobar vein cannula opening was taken as the zero pressure line. Owing to the possibility that the greater compliance of the left atrium plus the pulmonary veins as compared with the pulmonary artery might lead to a longer latent period of response to nerve stimulation during reverse perfusion than that during forward perfusion, the vein of the perfused lobe was cannulated in later experiments. This vein was cannulated via the left auricular appendix or via the right auricular appendix and thence through a hole made septum, a method which in the interauricular gave more certain alignment of the Forward and reverse perfusion techniques have been used bycannula Daly, with the vein. Foggie & Hebb (1940), Duke (1954), Piiper (1960), Caro, Bergel & Seed (1967) and Brody, Stemmler & DuBois (1968). Left atrial and pulmonary venous pressures. These were measured by the height of blood in a vertical tube connected to the left atrial or pulmonary vein cannula. A 'Starling resistance' (Knowlton & Starling, 1912) inserted between the vertical tube and the Gaddum outflow recorder was used for varying the outflow pressure. Ventilation. The lung lobes were ventilated at 20 c/min with 6-7 % CO2 in air or in 02 by a Starling 'Ideal' pump at inspiratory and expiratory pressures of and 0-50 mm H20 respectively. Tidal air changes were recorded by the ventilation overflow method of Konzett & Rossler (1940) and the elimination of vagal bronchomotor response achieved by the administration of atropine sulphate 1-2 mg/500 ml. circulating blood. The ventilation overflow did not alter appreciably during the nerve stimulation tests. Pulmonary vascular resistance (PVR). In calculating the PVR consideration was given as to whether 'sluice' or 'non-sluice' conditions of (Banister & Torrance, perfusion 1960; Permutt, Bromberger-Barnea & predominated Dollery & Naimark, Bane, 1964; Brody 1963; West, et al. 1968). In most of our atrial pressure was set above the expiratory experiments the left pressure so that at the end of the expiratory cycle, 'non-sluice' conditions of perfusion prevailed. As soon as the lung inflation pressure rose above the left atrial pressure the perfusion conditions. Thus, unless the left atrial changed to 'sluice' pressure was set above pressure the perfusion the during each respiratory cycle peak alternated inflation sluice' and 'sluice' conditions. The relationship between the between 'non- pressure during a ventilation mean cycle and the left atrial intratracheal pressure was as determining the method for calculating the pulmonary vasculararbitrarily resistance. chosen When the left atrial pressure exceeded the mean intratracheal pressure, sluice' conditions predominated and during thus 'non- a ventilation cycle, the resistance was calculated by the formula pulmonary vascular PVR pulmonary arterial pressure (mm Hg) - left atrial pressure (mm Hg) left atrial flow(i./min) X X/m2 = ratio of total lung weight to the weight of the perfused lobes and i2 as given by = surface area in i2 using Rubner's modification of where x Rahn & Ross (1957),

5 PULMONARY VASOMOTOR NERVE RESPONSES Meek's (1879) formula, e.g. area in m2 = 0112 (wt. in kg)i. When this formula is used the PVR range for normal resting dogs as found by others is (Daly, 1961a). In those experiments in which the mean intratracheal pressure exceeded the left atrial pressure, and 'sluice' conditions predominated during a ventilation cycle, the numerator of the above PVR formula was calculated as the pulmonary arterial pressure less the calculated mean intratracheal pressure (see De Bono & Caro, 1963, and others). In experiments in which both the left apical and left cardiac lobes or the left diaphragmatic lobe was perfused, the value of x was 6-5 and 3-8 respectively (Rahn & Ross, 1957). During the experiments the pulmonary arterial pressures, left atrial pressures, peak inflation and expiratory pressures were recorded in cm saline or H20 and converted to mm Hg for calculating the PVR. Calculation of mean intratracheal pressure. The mean intratracheal pressure was calculated from the following data. The respiratory pump output at each cycle was adjusted to give a ventilation overflow of ml. during peak inflation pressure. Under these conditions records of the ventilation overflow on a fast moving drum showed that the duration of full lung inflation at the peak pressure occupied approximately two fifths of the ventilation cycle, the inflating and collapsing periods of the lung lobes occupying the remaining three fifths of the cycle. It was assumed that the mean intratracheal pressures whilst the lungs were inflating and collapsing were midway between the measured peak inflation pressure (pip) and the expiratory pressure (ep). Thus the mean intratracheal pressure during the respiratory cycle (mip) was calculated as follows: mip.(ep +J(pip-ep))+ -pip 0-3ep+0-7pip. (2) Lung blood volume change during perfu8ion. Blood volume changes following nerve stimulation were measured at constant volume inflow perfusion in both forward and reverse directions by recording the changes in blood volume of the reservoir R (Fig. 1). In these experiments the flow recorder (FR, Fig. 1) was not in circuit. The reservoir was closed and its upper portion connected to a piston recorder giving a writing point excursion of a blood volume change of 4 mm/ml. in the reservoir. At constant volume inflow perfusion a temporary increase in reservoir volume denoted an increase in blood outflow and therefore a diminution in lung blood volume (Daly, 1938). Nerve stimulation tests were carried out with the two pulmonary artery manometers clipped off in order to exclude their capacity effects during the rise in pulmonary arterial pressure. Each such test was flanked with a nerve stimulation test with the manometers in circuit in order to assess the magnitude of the vasopressor response. During each procedure perfusion of the bronchial circulation was interrupted. Nerve stimulation. Stimulation of the left vagosympathetic nerve was carried out by the application of silver electrodes to the nerve trunk at a level just cranial to the convexity of the aortic arch. In a fewexperiments the upper left thoracic sympathetic chain was separated from its connexions with the spinal cord and stimulated at levels T 3, T 4 or T 5. The nerves stimulated were either air-borne or separated from adjacent tissues by cotton wool soaked in paraffin. The stimulation parameters were 5-25 V, 10 msec duration and 47 c/s, i.e. 5-25/10/47, derived from an Attree (1950) square-wave stimulator. Stimuli were usually applied for sec at 5 ± 0 5, D0 or min intervals. Latent periods of responses to nerve stimulation. These were measured from the start of nerve stimulation to the start of the inflow pressure rise. The latent periods of the Marey tambour and piston recorder were measured by recording on a fast IT-2 321

6 322 I. DE BURGH DALY AND OTHERS drum their responses to the application of an impulse to their rubber tubing connexions, the time of application also being recorded. These latent periods were approximately 0-1 sec and less than the latent periods of the vascular responses to nerve stimulation which were always > 1-0 sec and usually sec. Owing to oscillations in the pulmonary arterial pressure and blood volume tracings produced by each ventilation and blood pump cycle it was sometimes not possible to measure with certainty the start of the responses to a greater degree of accuracy than ± 0-3sec. VR VR' St Ose Fig. 2. For legend see facing page. Experiments on non-perfused lungs Measurement of separate arterial and venous system volume changes following nerve stimulation. The two systems are defined as comprising vessels each side of the capillary bed which are not fully compressed by high intratracheal pressures. The apparatus arrangement is shown in Fig. 2, and the procedure described in detail in the legend. In brief, forward perfusion of the pulmonary circulation was stopped and the cannulae in the pulmonary artery and left atrium (or a lobar vein) were each connected by a vertical glass tube to a piston volume recorder. All other connexions with the pulmonary pump, pressure recorders and outflow recorder were clamped off. The intratracheal pressure was raised mm Hg in different experiments in

7 PULMONARY VASOMOTOR NERVE RESPONSES order to compress the capillaries sufficiently to prevent the transfer of blood from the arterial to the venous side of the pulmonary circulation when the column of blood in the arterial vertical tube was set cm higher than that in the venous vertical tube. That no such transfer took place under these conditions is shown by the column of blood in the arterial tube remaining stationary and thus it seems reasonable to assume that any volume change of the vascular system taking place on one side of the compressed capillaries would not passively affect the volume of the vascular system on the other side. In the absence of bronchial circulation perfusion upward excursions of the volume recorders VR and VR' lever denote a diminution in the volume of the arterial and venous vascular systems respectively. If during nerve stimulation the bronchial circulation is being perfused, any change taking place in the volume of the two systems is superimposed upon the slope of the VR and VR' records due to transfer of blood from the bronchial circulation. RESULTS Pulmonary vascular responses to nerve stimulation under various conditions of perfusion 'Sluice' and 'non-sluice' conditions ofperfusion. The pulmonary vascular resistance response to nerve stimulation did not consistently differ during 'sluice' conditions from that during 'non-sluice' conditions of perfusion Fig. 2. Diagram of the arrangement for measuring separately the blood volume changes on the arterial and venous side of the capillaries following pulmonary nerve stimulation in isolated atropinized lung lobes. Perfusion is initially carried out during ventilation through the pulmonary circulation of the isolated lung lobe (ILL) via a, c, e, f and h by pump P, and through the bronchial circulation (BC) by pump P'. The vertical tubes leading to the piston recorders VR, VR' are closed at b and g respectively and their side tubes st, st' are kept open. Following ventilation and perfusion of both circulations for 4 hr or so, the pulmonary nerve fibres are stimulated and the vasopressor response recorded by manometers m, m'. The clamp on b is then taken off to allow blood to rise in the vertical tube to the height of the pulmonary arterial pressure (20-25 cm blood) after which the pulmonary circulation pump (P) is immediately switched off and clamps simultaneously applied at a, c, d and e. The respiratory pump is switched off and the intratracheal pressure raised to mm Hg, in order to prevent the transfer of blood from the pulmonary arterial to the venous side of the pulmonary vascular bed following removal of clamps c and e. Bronchial circulation perfusion is then interrupted. That blood in the pulmonary arterial vessels is not being transferred to the venous side of the capillaries is demonstrated by the fact that the level of blood in the vertical tube leading to the piston recorder VR remains unchanged. Clamps are then applied to the side tubes st, st' and at h, and the clamp at g removed. When bronchial circulation perfusion is started the transferred blood flow to the arterial and venous systems can be measured for a short time by recording the rate of rise of the VR and VR' traces respectively, and also the effect of nerve stimulation on those blood flows. With the bronchial circulation perfusion pump switched off, any change in the VR and VR' traces following nerve stimulation will record blood volume changes in the arterial and venous systems respectively. 323

8 324 I. DE BURGH DALY AND OTHERS TABLE 1. Pulmonary vascular resistance (PVR) responses due to nerve stimulation in isolated atropinized lung lobes perfused under 'sluice' and 'non-sluice' conditions. The values in the upper and lower lines of each observation are those before and following nerve stimulation respectively. Values during 'non-sluice' conditions are shown in heavy type, those during predominantly 'sluice' conditions in ordinary type (see text). All PVR values are calculated by formula (1) on p. 320 mm Hg PVR Obser-, LA flow increase Expt. vation PAp LAp mip A6P (ml./min) PVR (%) CVI ( CVI ( CPI ) * CPI ) Dog Expt. Wt. (kg) m2 pip ep mip t (0C) Strength /10/47 for 10 see to /10/47 for 15 see /10/47 for 15 see /10/47 for 15 see CVI, constant volume inflow perfusion; CPI, constant pressure inflow perfusion; PAp pulmonary arterial pressure; mip, mean intratracheal pressure; pip, peak inflation pressure; ep, expiratory pressure; PVR, pulmonary vascular resistance; LA, left atrial; t, blood temperature; x (of PVR formula), 6-5 in all experiments. * Perfusion of bronchial circulation temporarily interrupted during tests.

9 PULMONARY VASOMOTOR NERVE RESPONSES 325 (Table 1). (It will be noticed that the PVR response in Expt. 1, observation 2 of Table 1, is considerably less during 'non-sluice' conditions than during 'sluice' conditions of perfusion, but this difference was not confirmed in the other experiments.) TABLE 2. The table shows that the calculated PVR response due to nerve stimulation in isolated atropinized perfused left apical and cardiac lung lobes is greater during constant pressure inflow (CPI) perfusion than during constant volume inflow (CVI) perfusion when both types of perfusion are under predominantly 'sluice' conditions. The values in the upper and lower lines of each observation are those found before and following nerve stimulation respectively. The symbols have the same meaning as in Table 1 mm Hg Calcu- PVR Obser- Per- - LA flow lated increase Expt. vation Time fusion PAP AP (ml./min) PVR (%) CVI *16 CPI * CPI CVI *46 CPI CVI CVI * CVI * *05 CPI CVI *00 CPI CVI * * CVI CVI CPI In all experiments blood temperature range C; LAP, 3 7mm Hg; pip and ep, 6-2 and 2-2 mm Hg respectively; mip (calculated) 5-0 mm Hg. Stimuli in Expts. 1 and 3 10/10/47 for 15 sec, in Expt. 2; 15/10/47 for 15 sec. Expt. 1, dog, S, 19-0 kg, 0-8 M2; Expt. 2, Y, 18-0 kg, 0 77 M2; Expt. 3, X, 14-0 kg, 0-65 M2. * Bronchial circulation temporarily interrupted.

10 326 I. DE BURGH DALY AND OTHERS Constant volume inflow (CVI) and constant pressure inflow (CPI) perfusion. The effect of pulmonary nerve stimulation on the PVR increase during CVI perfusion under 'sluice' conditions was compared with that during CPI perfusion. The comparison was made either by interspersing br - 0, 28g_._ 7 26 El v X q_ El -j0 I CL80 - md 30 sec a C dc e Fig. 3. Comparison of PVR responses to nerve stimulation during CVI and CPI perfusion under 'sluice' conditions. Dog, X, 14-0 kg, 0-65 M2. Perfusion of isolated left apical and cardiac lobes (atropinized preparation). Blood temperature C; left atrial pressure 3-7 mm Hg; ventilating gas mixture 6 % CO2 in air; inspiratory pressure, 6-2 mm Hg; expiratory pressure, 2-2 mm Hg; mean intratracheal pressure (calculated), 5 0 mm Hg. Stimulation of left thoracic vagosympathetic nerve at 10/10/47 for 15 see in each of the tests a-e. The pulmonary vascular responses during constant pressure inflow (CPI) in tests a and e are compared with these during constant volume inflow (CVI) in tests b, c and d. Bronchial circulation perfusion was temporarily interrupted during test c. The pulmonary pump output was increased slightly between tests b and c. The calculated PVR responses are shown in Expt. 3 of Table 2. VOV, ventilation overflow; PAP' pulmonary arterial pressure; LA, left atrial; B.P. bronchial arterial pressure.

11 PULMONARY VASOMOTOR NERVE RESPONSES 327 TABLE 3. Pulmonary vascular resistance to nerve stimulation during forward (F) and reverse (R) perfusion in isolated atropinized lung lobes perfused under predominantly 'sluice' conditions. Expts. 1 and 3, left apical and cardiac lobes perfused; Expt. 2, left diaphragmatic lobe. The symbols have the same meaning as in Table 1 mm Hg Out- Calcu- PVR Obser- Per- Inflow flow Outflow lated increase Expt. vation fusion p p mip AP (ml./min) PVR (%) 1* 1 F CVI F F F R R F F F F F CVI F R R F F R R F F F CPI F R R 18-4 ( R R 18-0j F F Dog mm Hg _ ~ ~~~~~~~~~,,1 t Expt. Wt. (kg) m2 x pip ep mip (CC) Stimuli /10/47 for 15 sec /10/47 for 10 sec /10/47 for 15 sec Inflow p = pulmonary arterial pressure during forward perfusion, and left atrial pressure during reverse perfusion. Outflow (ml./min) = left atrial outflow during forward perfusion, and pulmonary artery outflow during reverse perfusion.

12 328 I. DE BURGH DALY AND OTHERS tests during CPI perfusion with those during CPI perfusion. It was found that the percentage increase in the PVR response during CPI perfusion was at least 11 times as great as that during CVI perfusion (Fig. 3; Table 2). The pre-stimulus pulmonary arterial pressures during CVI and CPI perfusion in each experiment were nearly similar, and since the mean intratracheal pressures were similar the pre-stimulus AP values differed only slightly from one another. Forward and reverse perfusion of the pulmonary circulation. It was found in all of eight experiments carried out at CVI perfusion under predominant 'sluice' conditions that merely changing from forward to reverse perfusion caused a rise in inflow pressure and therefore in calculated PVR. On 15 > o E X E Z 31 S a b c Reverse perfusion Forward perfusion Reverse perfusion Fig. 4. Effects of forward (F) and reverse (R) perfusion on the PVR, and on the PVR response to nerve stimulation. Dog, ci, 19-5 kg, 0-81 M2. Perfusion under 'non-sluice' conditions at CVI of left apical and cardiac lobes (atropinized preparation). Blood temperature, C; ventilation, 6% CO2 in air; peak inflation pressure, 4-4 mm Hg; expiratory pressure, 2-2 mm Hg; mean intratracheal pressure, 3-7 mm Hg; left atrial pressure, 6-7 mm Hg; B.P., bronchial arterial pressure. Stimulation of left thoracic vagosympathetic nerve in each test at 15/10/47 for 15 sec. Between a and b the pulmonary pump output was slightly increased. changing back to forward perfusion, the PVR returned to approximately its initial value (Table 3). In a single experiment performed at CPI perfusion changing over from forward to reverse perfusion diminished the outflow from the lungs and in this way increased the calculated PVR. The

13 PULMONARY VASOMOTOR NERVE RESPONSES 329 higher resistance to blood flow during reverse perfusion than during forward perfusion was observed with or without accompanying bronchial circulation perfusion (Table 3, Expt. 1). This Table also compares the effects of nerve stimulation during CVI forward and reverse perfusion. With both types of perfusion, nerve stimulation produced a rise in inflow pressure, the rise being greater during reverse than during forward perfusion in seven of the eight experiments, during or in the absence of bronchial circulation perfusion (Table 3, Expt. 1). The greater response to nerve stimulation during reverse perfusion also occurred in one experiment under 'non-sluice' conditions (Fig. 4) :& C Time Left atrium cannulated Pulmonary vein cannulated Fig. 5. The latent periods of the pulmonary inflow pressure rise following nerve stimulation during forward and during reverse perfusion in isolated, atropinized lung lobes. 0, Forward perfusion. 0O Reverse perfusion. Expt. 1, during bronchial circulation perfusion, Expts. 2-4, during temporary interruption of bronchial circulation perfusion. Ladent periods of response to pulmonary nerve stimulation. In three experiments in which the left atrium of the isolated perfused lung lobe was cannulated, the latent periods of the inflow pressure rise in response to nerve stimulation were measured during forward and reverse perfusion. The latent periods ofthe inflow pressure rise during reverse perfusion proved to be longerthan those during forward perfusion (Fig. 5, Expts. 1-3). It was suspected that this may have been due to the high compliance of the left atrium. A further experiment, however, in which the pulmonary vein was cannulated also showed longer latent periods of the inflow pressure rise during reverse than during forward perfusion (Fig. 5, Expt. 4). Blood volume and blood outflow changes. Nerve stimulation in atropinized lung lobes during CVI and 'sluice' conditions of perfusion produced a

14 330 I. DE BURGH DALY AND OTHERS diminution in lung lobe blood volume reflected by a temporary increase in blood outflow (Fig. 6). In three forward perfusion experiments the reduction in blood volume (calculated for both lungs/m2 body surface) was 8, 17 and 12 ml. In a further experiment the nerve stimulation tests were carried out during alternate forward (F) and reverse (R) perfusions. The lung blood volume reductions, calculated in the same manner, were 17 (F), 9 (R), 19 (F), 13 (R) and 12 (F) ml. Fig. 6 shows records obtained from the three earlier tests of this sequence. All the tests were made during interruption of bronchial circulation perfusion and with the inflow pressure manometers out of circuit in order to eliminate their capacity effects. Flanking tests with the manometers in circuit showed that in each case the inflow pressure increased during nerve stimulation. Thus stimulation increased the inflow pressure and decreased the blood volume of the lungs in both forward and reverse perfusions. 0- _ E > wo--. L_ L : E 2 = ca 13Qc_ I E sec a b c Fig. 6. Alterations in reservoir blood volume, and thereby in pulmonary blood volume, following nerve stimulation in an isolated, atropinized, perfused left apical lobe preparation. Dog, 6, 19-5 kg, 0-81 M2. Stimulation (during signals) during forward (a and c) as well as during reverse (b) perfusion increases, the blood volume of the reservoir indicating a diminution in lung blood volume. As shown by the B.P. trace, bronchial circulation perfusion was interrupted during nerve stimulation (see text). In support of the observations that nerve stimulation evokes a decrease in lung blood volume are two experiments carried out by I. de B. Daly, M. de B. Daly, Mary J. Scott & B. A. Waaler (unpublished experiments). Utilizing the dye-dilution technique they found in four tests that the 'appearance' and 'peak concentration' times of the dye-dilution curves became shorter by 20 % and 16 % respectively (average) following nerve stimulation. The atropinized isolated lung lobes were perfused at constant volume inflow in a forward direction and the dye (T-1824, Evans Blue) was injected into the pulmonary artery, the samples being collected from

15 PULMONARY VASOMOTOR NERVE RESPONSES 331 the left atrium. An alternative, but less likely interpretation of these dyedilution results is that the raised hydrostatic pressure produced by nerve stimulation opened up previously closed vessels with a shorter circulation time. Experiments on non-perfused isolated lung lobes (atropinized preparations). The preparations were set up as described in the legend of Fig. 2. Six experiments were carried out the results of which are given in Table 4. The values of the responses to nerve stimulation in this Table are the maximal obtained and are expressed as the volume changes in ml. of both lungs/m2 body surface of the animal from which the lung lobe preparations - _ A Fig. 7. Effect of stimulation of the left upper thoracic sympathetic chain (LSC) on the blood volume of the pulmonary vessels each side of the pulmonary capillaries which were blocked by a static intratracheal pressure of 30 mm Hg. Upward movement of the levers indicate an increase in the volume of blood in the arterial and venous vertical tubes. Atropinized left apical and cardiac lobe preparation from dog, ct, 22-5 kg m2 (see text). A, Bronchial circulation perfusion only. At signal, stimulation of LSC (15/10/47) for 17 sec. B, Repetition of stimulus at signal during interruption of bronchial circulation perfusion followed by restart of perfusion (see text). were made, using the lobe weight data of Rahn & Ross (1957) and the body weight-body surface-area relation of Meek (1879). Records from one experiment are shown in Fig. 7 in which the effect of upper thoracic sympathetic nerve stimulation on the blood displaced into the vertical tubes is shown, at A during bronchial circulation perfusion, and at B following interruption B

16 332 I. DE BURGH DALY AND OTHERS of bronchial circulation perfusion. Since in record B of Fig. 7 and in those experiments listed in Table 4 there was no blood circulating in the lung lobe it is difficult to escape the conclusion that the displacement of blood into the vertical tubes following nerve stimulation was due to constriction of pulmonary vessels each side of the capillaries and which was not evoked by a transmitter substance from circulating bronchial blood. There is however, the remote possibility that in these non-perfused lobes constriction of the bronchial vessels following nerve stimulation might lead to TA~RT. 4. Stimulation of the pulmonary nerves and reduction in the volume of blood (calculated in ml. for both lungs/m2 body surface) in the arterial (A) and venous (V) pulmonary vessels of lung lobe preparations in which the lung capillaries are blocked by high intratracheal pressures, and there is neither pulmonary nor bronchial circulation perfusion. The maximal responses in each experiment are given Expt. Decrease A- r A V -I A+V itp (mm Hg) LA cannulated * * * 2-7 2* * * *0 15 PV cannulated 5a a itp, static intratracheal pressure. * Stimulation of upper thoracic sympathetic chain; in the remaining experiments, the thoracic vagosympathetic nerve was stimulated. bronchial blood containing a transmitter substance being squeezed into the pulmonary vessels and so evoke their constriction. We regard this as extremely unlikely for the following reasons. In two of the experiments in which the lobes were statically inflated, Fig. 7 being an example of this, the communicating vessels between the bronchial vascular system and the pulmonary arterial vessels were not patent (Fig. 7A), yet nerve stimulation caused a displacement of blood into the pulmonary arterial system (Fig. 7B). Furthermore, after making allowance for the displacement of blood due to continued inflow from the bronchial circulation, the displacement of blood into the venous system following nerve stimulation was no greater during bronchial circulation perfusion than during its interruption. This finding was confirmed in a series of experiments on ventilated, collapsed and statically inflated lobes in which no evidence was obtained that the

17 PULMONARY VASOMOTOR NERVE RESPONSES 333 blood displaced from the arterial and venous systems was greater during bronchial circulation perfusion than during its interruption (Fig. 8). This Figure, as well as the data in Table 4, also show that there is a tendency for nerve stimulation to produce a greater displacement of blood from the arterial system than from the venous system. Two exceptional responses to nerve stimulation, not included in Fig. 8, deserve mention in that in one experiment two out of seven stimulation tests produced a small initial fall in the amount of blood in the arterial vertical tube of 0 5 and 09 ml./both lungs.m2 followed by an increase of 1 1 and 1 2 ml./both lungs.m2 respectively. The corresponding changes in the venous tube were an increase of 1-4 and 06 ml./both lungs.m2. 0 C _ 0 Co"E 8_ V V 0.0 A 0t > V 2.0VA ~ ~ ~ V0 -~~~~~~~ ~~ la 2 26 No perfusion of bronchial Transferred blood flow circulation (ml./min. both lungs. m2) Fig. 8. Plots relating the reduction in blood volume following nerve stimulation on the arterial system (filled symbols) and venous system (open symbols) in the absence of bronchial circulation perfusion and during bronchial circulation perfusion at different rates of transferred blood flow. Isolated, atropinized lung lobes; no pulmonary circulation perfusion. The reductions in blood volume of the venous system following nerve stimulation duringbronchial circulation perfusionand in its absence do not appreciably differ from one another. This finding militates against the possibility that the blood volume changes in the venous system are due to the presence of a bronchial transmitter substance in the transferred blood. Experiments on ventilated lobes (V, V), collapsed lobes (A, A) and statically inflated lobes (, Q). DISCUSSION The earlier results described in this paper were obtained on isolated perfused atropinized lung lobes lying in a horizontal position in order to minimize differences in hydrostatic pressure gradients in blood vessels at different levels in the lung lobe. The lobes were perfused at constant

18 334 I. DE BURGH DALY AND OTHERS volume inflow and ventilated with positive pressure by a Starling 'Ideal' pump. Sluice and non-sluice conditions ofperfusion. As mentioned in the section on Methods, perfusion in ventilated lungs alternated between 'sluice' and 'non-sluice' conditions, unless the left atrial pressure exceeds the peak inflation pressure, when 'non-sluice' conditions prevail. We have arbitrarily chosen the mean intratracheal pressure (mip) for assessing the downstream pressure and regarded perfusion as being predominantly under ' sluice' conditions when the mip exceeds the left atrial pressure, and predominantly 'non-sluice' when the left atrial pressure exceeds the mip. It may be suggested that the selection of the mean intratracheal pressure for the purpose of assessing the downstream pressure under ' sluice' conditions of perfusion may be invalid and that the peak inflation pressure should have been used in the calculations of PVR. If, however, this alternative is adapted, it can be calculated from the data given in Table 1 that the PVR responses to nerve stimulation during 'sluice' and 'nonsluice' still do not consistently differ from one another. It may be reasonably argued that because the calibre of the alveolar capillaries may possibly be smaller, and thus their resistance greater, during 'sluice' than 'non-sluice' conditions of perfusion, any increase in capillary resistance following nerve stimulation should be greater during ' sluice' than during 'non-sluice' conditions of flow. Assuming the validity of this argument, our failure to detect any quantitative differences in the resistance increase following nerve stimulation under the two differing kinds of perfusion suggests that the capillaries play no active part in the production of an increase in resistance to nerve stimulation. It should be mentioned, however, that electronmicroscope studies by Fillenz (1969) have revealed that, not infrequently, non-myelinated fibres run between the smooth muscle layers of the smaller pulmonary arteries and capillaries which are characterized by being surrounded by pericytes. The axon bundles lie between the capillary endothelium and the process of a pericyte. The axons contain vesicles characteristic of noradrenergic and cholinergic terminal fibres. Fillenz suggested that the pericytes may respond to released transmitters. However, there is no direct evidence from our experiments suggesting that the capillaries are actively involved in the increase in pulmonary vascular resistance following nerve stimulation. Constant inflow and constant pressure perfusions. The most likely explanation of the larger responses to nerve stimulation during CPI perfusion than during CVI perfusion is that in CVI perfusion the rise in inflow hydrostatic pressure not only opposes the reduction in calibre of the vessels responsive to nerve stimulation but also may open up previously closed vascular channels and passively dilate vessels not participating in the nervous

19 PULMONARY VASOMOTOR NERVE RESPONSES 335 response. There is also the possibility that during CPI perfusion the vascular smooth muscle contraction due to release of the sympathetic transmitter substance following nerve stimulation may lead to critical closure of some vessels (Burton & Stinson, 1960). Forward and reverse perfusion; site of action of pulmonary vasomotor nerves. At first sight the observation that nerve stimulation produced a greater rise in inflow pressure and in PVR during reverse than during forward perfusion suggested that the nervous control of the resistance vessels on the venous side of the pulmonary vascular bed was stronger than that on the arterial side. Our confidence in this interpretation was shaken by finding that merely changing over from forward to reverse perfusion increased the PVR (Table 3). This raised the suspicion that the perfused vascular channels during forward and reverse perfusion may not be precisely similar and/or the abnormal direction of blood flow during reverse perfusion through vessels architecturally designed for forward perfusion may give rise to turbulence in the blood stream and thus to an increase in vascular resistance. The pulmonary blood flows and PVR values in our experiments fell within the normal range for dogs (see Daly, 1961 a). In contrast to our results Brody et al. (1968) reported that PVR was less during reverse than during forward perfusion. The blood flows in their experiments were approximately one fifth of those in our experiments. It is possible that the higher blood flows that we used during reverse perfusion may produce turbulence. Another point of interest is that during reverse perfusion the blood flow in the bronchopulmonary veins opposes the direction of blood inflow in the pulmonary circulation whereas in forward perfusion it is in the same direction. It was found, however, that this was not a factor in determining the relative response to nerve stimulation during forward and reverse perfusion (see Expt. 1, Table 3). Although such haemodynamic changes cannot be dismissed, perhaps another factor in the production of a greater PVR during reverse than during forward perfusion may be due to the lower pulmonary arterial pressure in the former type of perfusion. By the application of LaPlace's Law this would lead to a diminution in calibre and an increase in resistance to blood flow of the downstream arterial vessels (see Burton, 1952, 1954; Bayliss, 1966). If this were so, the greater arterial resistance during reverse perfusion must override any reduction in PVR that may occur as a consequence of the veins being maximally dilated by the high inflow pressures (see Caro et at. 1967). On this hypothesis a constrictor effect of nerve stimulation on the arteries already with a reduced calibre during reverse perfusion would cause a greater increase in their resistance. This would lead to a greater increase in PVR following nerve stimulation during

20 336 I. DE BURGH DALY AND OTHERS reverse perfusion as compared with stimulation during forward perfusion. This resistance increase in the downstream arteries could only influence the mean inflow pressure during the shorter 'non-sluice' perfusion period of the ventilation cycle (see Methods). This is in all probability the explanation of our results and is a view which receives support from other considerations: the first, that the latent periods of the inflow pressure rises due to nerve stimulation during reverse perfusion were longer than those during forward perfusion. It is also relevant that the latent periods of the pulmonary pressor responses to adrenaline injected into the blood inflow stream of isolated lungs are longer during reverse than during forward perfusion (Daly et al. 1940); the second, that it seems unlikely that the longer period of the responses during reverse than during forward perfusion could be accounted for by the maximally dilated pulmonary venous system having a greater compliance than an arterial system which is under a relatively low pressure; the third, that maximally dilated veins would be expected to be irresponsive to nerve stimulation. We are of the opinion therefore that during reverse perfusion pulmonary vasomotor nerve responses on the venous side of the capillaries are attenuated or abolished, and on the arterial side accentuated. If this is so then with both types of perfusion the inflow pressure responses to nerve stimulation mainly reflect constriction of the pulmonary arterial vessels. Our experiments on atropinized non-perfused lung lobes at high intratracheal pressures have shown that nerve stimulation diminishes the volume of the vessels each side of the blocked capillary bed, the response of the venous side of the bed being weaker than that of the arterial side. But these experiments do not give any indication as to the size of the responsive vessels, which may be resistance and/or larger vessels. Our results, however, both on the perfused and non-perfused lung lobe preparations suggest that the functional activity of sympathetic adrenergic fibres, stimulation of which produces vasoconstriction, is more marked on the arterial than on the venous vessels. The recent studies by Fillenz (1966, 1969, 1970) on the innervation of dog lung are of particular interest for our problems. She used the fluorescent method of Falck, Hillarp, Thiema & Torp (1962) and electronmicroscopy, and found that noradrenergic axons supply the pulmonary hilar arteries and the smaller arteries and veins of Mu in diameter. Bundles of fluorescent fibres were seen encircling muscular vessels of I in diameter at their point of origin at right angles from arteries of It in diameter. The terminal axons contained accumulations of vesicles; these were characteristic of both cholinergic and noradrenergic terminal fibres. The small arterial side branches finally supplied networks of alveolar capillaries. Fillenz suggested that the innervation of these side branches

21 PULMONARY VASOMOTOR NERVE RESPONSES 337 may serve as a point for controlling the blood flow through portions of the alveolar capillary networks. Bronchial transmitter substances. The results of the experiments on nonperfused lungs in which nerve stimulation diminishes the volume of blood in both the pulmonary arterial and venous vessels are regarded, for reasons previously given, as supporting the view that a bronchial transmitter substance is not responsible for the pulmonary vascular response. In lung preparations perfused through the pulmonary circulation nerve stimulation increases the PVR temporary interruption of bronchial circulation (see Daly & Hebb, 1966). Under these conditions it is conceivable that some blood from the pulmonary circulation may reach bronchial vessels via anastomoses between the two vascular systems and after taking up bronchial transmitter substances be returned via the pulmonary veins to the left atrium. This eventuality arises from the fact that in the absence of bronchial circulation perfusion the pressure on the bronchial side of the vessels common to both circulations would be low with a pressure gradient falling from the pulmonary to the bronchial side of these vessels. An aberrant circulation of this kind might lead to pulmonary venous constriction by a bronchial transmitter substance, although to a lesser degree than if the transmitter were carried by bronchial blood during bronchial circulation perfusion. Against this hypothesis is that nerve stimulation may produce a PVR increase following a short interruption of bronchial circulation perfusion that differs little from that during bronchial circulation perfusion (Daly, 1961b) or may even be larger (Allison et al. 1961), although, as shown by the last-named investigators, the pulmonary vasomotor response to nerve stimulation is extinguished by prolonged interruption of bronchial circulation perfusion. We wish to express our thanks to Sir Lindor Brown, F.R.S., for granting us laboratory facilities; to Dr D. H. Bergel and Dr Marianne Fillenz for helpful discussions, and Miss Susan Beere and Mrs Pamela Slingsby for skilled technical assistance. The investigation was supported by a United States Public Health Service Research Grant H E from the National Heart Institute. REFERENCES ALLISON, P. R., DALuY, I. DE B. & WAATLTia, B. A. (1961). Bronchial circulation and pulmonary vasomotor nerve responses in isolated perfused lungs. J. Physiol. 157, ATrRzE, V. H. (1950). An electronic stimulator for biological research. J. 8cient. In.strum. 27, BANiSTER, JEAN & ToRRANcE, R. W. (1960). The effects of the tracheal pressure upon flow: pressure relations in the vascular bed of isolated lungs. Q. JL exp. Phy8iol. 45,

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