THE NATURE OF ATRIAL RECEPTORS RESPONSIBLE FOR THE INCREASE IN URINE FLOW CAUSED BY DISTENSION OF THE LEFT ATRIUM IN THE DOG

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1 Quarterly Journal of Experimental Physiology (1981) 66, Printed in Great Britain THE NATURE OF ATRIAL RECEPTORS RESPONSIBLE FOR THE INCREASE IN URINE FLOW CAUSED BY DISTENSION OF THE LEFT ATRIUM IN THE DOG N. SIVANANTHAN*, C. T. KAPPAGODAt AND R. J. LINDEN Department of Cardiovascular Studies, New Medical School, University of Leeds, Leeds, LS2 9JT (RECEIVED FOR PUBLICATION 21 MAY 1980) SUMMARY In dogs anaesthetized with a-chloralose the effects of distension of a large balloon in the lumen of the left atrium on the discharge of action potentials in vagal fibres and on urine flow were studied with the cervical vagi cooled at 18 and 12 IC. Distension of the balloon in seven dogs resulted in an increase in urine flow. Cooling the cervical vagi to 18 'C reduced the response to 70% of that obtained at 37 IC, and at 12 IC the response was reduced to 28%. In a second group of dogs, the effect of distension of the balloon on atrial receptors which discharged into myelinated nerve fibres of the vagi, i.e. Paintal type A and type B receptors, was examined. The increase in the activity of these receptors was reduced to 68% when the vagus nerve was cooled to 18 'C and was reduced further to 25% at 12 IC. In a third group of dogs, the effect of distension of the balloon on receptors which discharged into non-myelinated nerve fibres in the vagi was examined. Cooling of the cervical vagi also reduced the evoked increase in activity in these fibres; this reduction in activity occurred over a wider range of temperature, unlike the effect of cooling on the response in myelinated vagal fibres. It is concluded that the increase in urine flow caused by distension of a balloon in the left atrium is mediated solely by the Paintal-type atrial receptors which discharge into the myelinated fibres in the vagi. INTRODUCTION In anaesthetized dogs distension of a balloon in the lumen of the left atrium results in an increase in urine flow (e.g. Henry & Pearce, 1956). This phenomenon has been attributed to stimulation of the complex unencapsulated nerve endings in the atrial endocardium which discharge into the myelinated fibres in the vagus. The main evidence supporting this claim is the finding that application of a discrete stimulus to areas of the atrium which contain a preponderance of these nerve endings (e.g. the pulmonary vein-atrial junctions) also results in an increase in urine flow which is qualitatively similar to that obtained by distending a large balloon in the left atrium (e.g. Ledsome & Linden, 1968). However, it has been shown recently in the cat (Thoren, 1976) that there is a second type of nerve ending in the atrial wall which increases its activity during a rise in pressure in the atrium and which discharges into non-myelinated fibres ('C' fibres) in the vagi. In the dog these fibres have previously been described by Sleight & Widdicombe (1965) and Coleridge, Coleridge, Dangel, Kidd, Luck & Sleight (1973). It is therefore possible that these non-myelinated fibres are involved in an increase in urine flow which results from distension of a balloon in the left atrium. Recently, using a technique of differential cooling of the vagi, it has been shown that the afferent limb of the reflex increase in heart rate caused by the distension of small balloons * Present address: Department of Radiology, General Hospital, Nottingham. t Present address: Division of Cardiology, Department of Medicine, Clinical Sciences Building, University of Alberta, Edmonton 61, Alberta, Canada /81/ $ The Physiological Society

2 52 N. SIVANANTHAN, C. T. KAPPAGODA AND R. J. LINDEN at the pulmonary vein-atrial junction, consists. solely of the myelinated fibres (Kappagoda, Linden & Sivananthan, 1979). Using the same technique the present investigation was undertaken to discover which fibres, myelinated, non-myelinated or both, were involved in the reflex increase in urine flow. A preliminary report of this investigation has already been given to the Physiological Society (Kappagoda, Linden & Sivananthan, 1978). METHODS Dogs weighing kg were given an injection of morphine sulphate (dose 0 5 mg. kg-') and half an hour later were anaesthetized with an intravenous infusion of a-chloralose (dose 0 1 g. kg-'; Etablissements Kuhlman, Paris); the chloralose solution consisted of 0 9 g NaCl and 1 g a-chloralose dissolved in 100 cm3 distilled water. After induction of anaesthesia the chest was opened through the fifth left intercostal space and a latex balloon mounted on a nylon cannula was inserted into the left atrium through the left atrial appendage. The mitral orifice was partially obstructed by distending this balloon with 125 cm3 saline (mean; range 10-14). The pressure in the femoral artery was recorded through a nylon cannula (Portex surgical quality no. 2, Portland Plastics Ltd., Hythe, Kent) inserted through the right femoral artery and that in the left atrium was obtained through a nylon cannula inserted through the left atrial appendage. In addition, the tracheal pressure, end-tidal Pco, and the electrocardiogram were recorded. The temperature and the acid-base status of the animal were monitored and maintained within the normal limits. Both ureters were cannulated through a mid-line incision in the abdomen, and urine was collected and measured every 10 min. The basic experimental protocol for the demonstration of the response of an increase in urine flow and the techniques for obtaining experimental records have been described previously (Ledsome, Linden & O'Connor, 1961). Recording action potentials Action potentials were recorded in slips of the cervical vagus. Dissection of nerve fibres in the left cervical vagus was continued until a single functional unit was obtained. The balloon was then distended to determine whether the frequency of impulses in this fibre was increased and such units were studied further. In addition to recording the action potentials on paper sensitive to ultraviolet light they were continuously monitored, through a display screen coupled to an 'on-line' computer (PDP- 12, Digital Equipment Corporation, Maynard, U.S.A.), in the form of time-frequency histograms. Pressures, electrocardiogram and action potentials were also recorded on magnetic tape for subsequent analysis. The conduction velocity of the fibres stimulated by distension of the balloon was determined. The techniques used for recording action potentials and for measuring conduction velocities have been described previously (Kappagoda, Linden & Snow, 1972; Kappagoda et al. 1979). Cooling the cervical vagus The cervical vagi were cooled using silver thermodes (2 5 cm wide) attached to 'Frigistor' cooling modules (De La Rue Frigistor Ltd., Canal Estate, Langley, Bucks.) (see Kappagoda et al. (1979) for details). Experimental protocol Three groups of dogs were studied. In each group of dogs the balloon was distended in the lumen of the left atrium partially to obstruct the mitral orifice and the effects of the following manoeuvres were studied: the effect of cooling both vagi on the induced increase in urine flow (group I); the effect of cooling the vagus on the response of atrial receptors which discharged into myelinated fibres (Group II) and which discharged into non-myelinated fibres (group III). The fibres were allocated to each group according to conduction velocity. In each group the vagi were cooled to 18 and 12 IC. Group I. The increase in urineflow The response to atrial distension was calculated as the urine flow measured during the 30 min experimental period expressed as a percentage of the average of the urine flow during a 30 min period before, and a 30 min period after, the experimental period (Ledsome et al. 1961); the experimental period was defined as the final 20 min of distension and the first 10 min immediately afterward.

3 ATRIAL RECEPTORS AND URINE RESPONSE 53 First the response to distension of a balloon in the left atrium was determined whilst the vagi were maintained at 37 C (first control response). After demonstration of this response the vagi were cooled to either 18 IC or 12 IC and the procedure was repeated. Finally the vagi were re-warmed to 37 C and a second control response was obtained. Group II. Myelinated fibres The responses to distension of the balloon were determined with the vagi maintained at 37 C (initial control response), 18 C, 12 C and again at 37 C (final control response). At each temperature the following protocol was adopted. Records for an initial control period were obtained for 3-5 min; the balloon was distended and further records obtained for a period of 20 min followed by a final control period of 3-5 min. From the records of action potentials the mean frequency (spikes. s-1) was obtained by counting, during a steady state, the spike potentials over 1 min and a constant number of respiratory cycles regardless of heart rate. The response was then expressed as a percentage increase in mean frequency in a manner similar to that in Group I. Group III. Non-myelinated fibres The protocol adopted was similar to that used in Group II. In the three non-myelinated fibres where the control discharge was almost zero the response was taken to be the mean frequency during the test periods as expressed in spikes. s-. RESULTS When the recording commenced at the end of surgical procedures in 15 dogs the ph, Pco, and Po2 of the arterial blood were 7 41 (mean; range ), 5 1 kpa (mean; range ) and 25-6 kpa (mean; range ), respectively. The heart rate was 60 beats. min-' (mean; range ), the arterial pressure 17-6 kpa (mean; range ) and the left atrial pressure 0 7 kpa (mean; range ). Response of an increase in urineflow The responses of an increase in urine flow to distension of a large balloon were examined in three dogs with vagi cooled to 18 IC and in four others with vagi cooled to 12 IC. In all these dogs at least one response was obtained with vagi at IC after re-warming the vagi. In the three dogs in which three responses at 18 IC were determined nine responses were obtained at 37 IC. One of the above experiments is illustrated in Fig. 1. Distension of the balloon was accompanied by a fall in mean arterial pressure, an increase in heart rate, left atrial pressure and urine flow. The magnitude of the increase in urine flow was calculated in the manner of Ledsome et al. (1961), and briefly described in the Methods. It was found that before cooling the vagi, the increases in urine flow (i.e. initial control responses) were 167% and 198% of the average of the control values. When the vagi were cooled to 18 'C, the increase in urine flow (experimental response) was 135% and after re-warming the vagi the increase in urine flow (i.e. final control response) was 217%. The response obtained when the vagi were cooled to 18 IC, when expressed as a percentage of the control responses was found to be 65% (increase in urine flow as a percentage of average control response). In seven dogs a total of seventeen responses were obtained with the vagi at IC; in three of these dogs two responses were obtained in the initial control periods (e.g. see Fig. 1). For the purposes of this analysis the values for the heart rates, cardiovascular pressures and the urine flow obtained during the control period and after distension were averaged to yield a single control value. This value was compared to that obtained during distension of the balloon. In these seventeen experiments during the control periods the heart rate, mean left atrial pressure and mean arterial pressure were 78 beats. min-' (mean; range ), 0-8 kpa (mean; range ) and 17-3 kpa (mean; range ) respectively.

4 54 N. SIVANANTHAN, C. T. KAPPAGODA AND R. J. LINDEN C 37 0C 18 0C F C 1 "-G-s - E ra ,- A-A-A-A A.A &.A. L.A f+a-- *~-- X,*-. 6., 0-. a I,-.-. *.O-', C) E ;E I O L Time in 10 min intervals Fig. 1. Changes occurring in measured variables during distension of a large balloon in the lumen of the left atrium in one dog, before, during and after cooling the vagi to 18 IC. From above down vagal temperature, mean left atrial pressure (L.a.p.), heart rate, femoral arterial pressure (F.a.p.) and urine flow. The four pairs of vertical lines enclose periods during which the balloon was distended. The numbers above urine flow indicate the responses of an increase in urine flow (as percentage of the average of the control values). The urine flow was 0-7 cm3. min-' (mean; range ). During the distension of the balloons, the heart rate, mean left atrial pressure and mean arterial pressure were 125 beats. min-' (mean; range ), 2-0 kpa (mean; range ) and 17-4 kpa (mean; range ) respectively. The urine flow increased to 1-84 cm3. min-' (mean; range ). In these seventeen experiments an increase in urine flow was observed in each instance and the average increase (expressed as a percentage of the control values) was 157% (range ).

5 O3 A 100 -n"0 V ATRIAL RECEPTORS AND URINE RESPONSE 55 5i80 0E ~ 0, Temperature ( C) Fig. 2. The increase in urine flow resulting from distension of a balloon in the lumen of the atrium, with the vagi at 37 + I C and to cooling to 18 and 12 IC. Each point represents the increase in urine flow at each temperature expressed as a percentage of the control responses obtained with the temperature at 37 IC; the control responses were obtained before and after the cooled responses. Each symbol represents the results from one dog. The responses at 37 'C were rated 100% response. The continuous lines are the same as Figs. 3 and 4 and drawn by eye to aid comparison. Responses qualitatively similar to those described above were obtained with the vagi at 18 and 12 IC respectively. In three dogs, with vagi at 18 IC, the responses were consistently smaller than those obtained with vagi at 37 IC; the average response (expressed as percentage ofcontrol response) obtained at 37 'C was 69 8% (range ). In four dogs with the vagi at 12 'C the responses were reduced further to 27.9% (mean; range ). The individual responses are shown in Fig. 2. Group II. Myelinatedfibres (conduction velocity more than 5 m.s-1) Seven atrial receptors discharging into myelinated fibres of the vagus nerve were studied in four dogs. The conduction velocity of the fibres was 17-6 m. s-' (mean; range ). The receptors were identified as atrial receptors from their pattern of discharge and their response to distension of the balloon. For the purpose of this analysis the receptors will be classified as Paintal type B receptors which discharge in time with the 'v' wave of the atrial pressure pulse and Paintal type A receptors which discharge mainly in time with the 'a' wave (e.g. Paintal, 1972). A fuller description of the patterns in, and origins of, fibres from types A, B and intermediate atrial receptors has been published (Kappagoda, Linden & Mary, 1977). Six of them were type B and one was type A. All of them increased their frequency and duration of discharge during distension of the balloon. The distension of the large balloon increased the left atrial pressure from 09 kpa (mean; range 05-13) to 2 3 kpa (mean; range ). The mean frequency of discharge during the control periods was 8-4 spikes. s- (mean; range ) and it increased during

6 56 N. SIVANANTHAN, C. T. KAPPAGODA AND R. J. LINDEN C 80 0 a) 0 60 Q 0U <40 -\ i U Temperature ( C) Fig. 3. Response of the increase in action potentials in myelinated fibres resulting from distension of balloons in the lumen of the left atrium, with the vagus at IC and to cooling to 18 and 12 'C. Each point represents the increase in action potentials (mean frequency) expressed as a percentage of the response with the vagus at 37 IC. Each symbol represents the response in one nerve fibre. Seven fibres with conduction velocities (CV) of m. s- were examined in four dogs. The two continuous lines were drawn by eye and are the same as in Fig. 2. distension of the balloon to 37 9 spikes, s-1 (mean; range ). In all seven receptors the major increase in the discharge occurred during the 'v' wave of the atrial pressure pulse. The receptor with the type A discharge was converted to give a type B discharge and the discharge during the 'a' wave was lost; when the distension of the balloon was released, the pattern of discharge reverted back to type A. All atrial receptors reached their peak rate of discharge during the injection of saline into the balloon (i.e. the period of increasing left atrial pressure). Once the left atrial pressure reached a steady level, the receptors discharged at a steady rate following a period of 'adaptation' which lasted s. For the following 20 min they maintained their rate of discharge with minimal change. Immediately after the release of the balloon the receptors exhibited a phase of post-stimulatory inhibition during which the discharge ceased. Over the next 1 or 2 min the activity returned to its original control value. When the vagus was cooled to 18 and 12 'C the pattern of discharge in the seven receptors remained unchanged but the mean frequency was diminished. At 18 IC the control frequency was 6-9 spikes. s-1 (mean; range ) and it increased to 23-8 spikes.s-1 (mean; range ) during distension of the balloon. When this response at 18 'C was calculated and expressed as a percentage of the response obtained at C, it was found to be 68 2% (mean; range ). When the vagus was cooled to 12 IC the control frequency was 4 5 spikes. s-1 (mean; range ) and the frequency increased to 8-0 spikes. s- (mean; range ). When the response was calculated and expressed as a percentage of the response obtained at 37+1 C it was 25.4% (mean; range ). The individual results from the seven fibres are shown in Fig. 3. This change in the pattern of response to cooling is the same as the change in pattern of the response of urine flow.

7 ATRIAL RECEPTORS AND URINE RESPONSE Group III. Non-myelinatedfibres (conduction velocity less than 2 m.s-') Six receptors discharging into non-myelinated afferent axons of the left vagus were studied in four dogs. The responses were calculated as in Group II. The conduction velocity was 1-3 m. s-i (mean; range ). Since most of these receptors were silent or only discharged sparsely at normal left atrial pressure, each slip of vagus dissected was placed on the recording electrodes and the balloon was distended each time to detect any increase in activity. In this process often several Paintal-type receptors were found and discarded before a receptor discharging into non-myelinated axons was obtained. Five of the six receptors had a control discharge of 0-3 spikes. s-1 (mean; range 0-1 3) at a left atrial pressure of 0-7 kpa (mean; range 0-3-0*9). During distension of the balloon the left atrial pressure increased to 2-4 kpa (mean; range ) and the mean frequency of discharge increased to 3-4 spikes. s- (mean; range 1 2-6*0). Like the receptors discharging into myelinated fibres, the peak rate discharge was seen during the injection of saline into the balloon. Once the pressure in the atrium reached a steady state the receptors showed a rapid 'adaptation' to reach a steady state during which the measurements were taken. During distension of the balloon, they also showed a cardiac rhythm which was modulated by changes in respiratory pressure. When considered as a group, cooling the vagus to 12 IC did not affect the response in these five units. At 12 IC the discharge during the control periods was 0-2 spikes. s-' (mean; range ) and that during the test period was 3 5 spikes. s-i (mean; range ). The responses at 12 IC were 96% (mean; range ) of those obtained at 37+1 C. Cooling the vagus to temperatures less than 12 IC (to 8 and 6 IC) resulted in a decrease in percentage response (% average of control responses) in three of the five receptors, one to 70% at 8 IC and 20% at 6 IC and the other two to 63% and 52% at 6 IC. In contrast to the behaviour of these five receptors the sixth receptor discharged at 5*5 spikes. s-i during the control periods; the left atrial pressure being 0'8 kpa. When the balloon was distended the left atrial pressure increased to 2 5 kpa and the frequency of discharge increased to 113 spikes.s-'. When the vagus was cooled to 18 IC, the response in this fibre was reduced to 40% of the value at 37 IC. Cooling the vagus to 17 IC abolished the response. The individual results of all fibres are summarized in Fig. 4. The change in the pattern of responses to cooling is quite different from that of the myelinated fibres. 57 DISCUSSION The atrial endocardium of the mammalian heart contains complex unencapsulated nerve endings which discharge into the myelinated fibres of the cervical vagus (e.g. Paintal, 1972) and nerve endings which discharge into non-myelinated fibres of the vagus (e.g. Thoren, 1976). In addition a third group of atrial receptors discharging into the sympathetic afferent fibres have also been demonstrated (see Kappagoda et al. (1979) for references). It seemed likely that the technique of distending a balloon in the lumen of the left atrium, which has been widely used to investigate the role of the complex unencapsulated nerve endings in the regulation of urine flow, provided a relatively 'crude' stimulus and thus was likely to stimulate any or all of the three types of receptor. The present investigation has confirmed that distension of a balloon in the lumen of the left atrium stimulates atrial receptors which discharge into both myelinated axons (Paintal type) and non-myelinated axons of the vagus.

8 58 N. SIVANANTHAN, C. T. KAPPAGODA AND R. J. LINDEN A U' raa VkV '40 0) Temperature ( C) Fig. 4. Response of the increase in action potentials in non-myelinated fibres resulting from distension of balloons in the lumen of the left atrium, with the vagus at 37 IC and to cooling to 18 and 12 IC. Each point represents the increase in action potentials (mean frequency) expressed as a percentage of the response with the vagus at 37 IC. Each symbol represents the response in one nerve fibre. Six fibres with conduction velocities (CV) < 2 m. s-i were obtained in four dogs. The continuous lines were drawn by eye and are the same as in Fig. 3. Note all the responses to cooling lie outside the two lines. Response in Paintal type receptors. It was seen that both type A and type B receptors were stimulated by the distension of a large balloon in the left atrium in a manner comparable to that previously reported when the atrial receptors were stimulated discretely by distending small balloons at the pulmonary vein-atrial junctions (Kappagoda et al. 1979). These responses of type A and type B receptors to distension of a large balloon resembled their responses to large intravenous infusions (Kappagoda et al. 1977). This increase in activity observed in the Paintal type receptors by distension of the balloons was attenuated by cooling the cervical vagi. At a temperature of 18 IC the response was reduced to 68% of the value obtained with the vagi at 37 'C while at 12 IC the response was further reduced to 25%. These findings are again similar to those observed by Kappagoda et al. (1979) when using the smaller balloon without obstructing the mitral orifice and where pulmonary vein-atrial junctions were stretched for relatively short periods of time (2 min). Response in non-myelinated vagal afferents Cooling the cervical vagus resulted in a reduction in the response in non-myelinated fibres to distension of the balloon but the temperature at which this reduction occurred was different to that in the myelinated fibres. These findings are also in accord with the previous report relating to the effect of cooling on the transmission of impulses in non-myelinated vagal afferents (see Kappagoda et al. 1979).

9 ATRIAL RECEPTORS AND URINE RESPONSE 59 Effect of cooling the vagi on the response of an increase in urine flow The present investigation has confirmed that the increase in urine flow produced in response to the distension of the balloon in the left atrium is reduced by cooling the vagi but in addition there was a graded effect of cooling. Cooling the cervical vagi to 18 OC resulted in a reduction in the response to 70% of that obtained at 37 OC. When the vagi were maintained at 12 OC the response was reduced to 28%. It is evident that there is a strong similarity between effect of cooling the vagi on the 'urine response' and on the responses in myelinated fibres. No such similarity exists between the 'urine response' and the response in non-myelinated fibres. Thus, cooling the vagi in this manner links the response of an increase in urine flow to the increased activity in myelinated vagal afferents during distension of the balloon and thus to Paintal type atrial receptors. Receptors discharging into non-myelinated fibres are not involved. Since, as well as the similarity of the urine response and response in myelinated fibres to vagal cooling, it is known that the urine response is abolished by section of the vagi (e.g. Ledsome et al. 1961) it is possible to conclude that receptors attached to the sympathetic afferent fibres are unlikely to be involved in this reflex. Thus the results are entirely similar to those of the previous investigation (Kappagoda et al. 1979) which showed that the increase in heart rate caused by distending small balloons at the pulmonary vein-atrial junctions results from stimulation of atrial receptors attached to myelinated fibres in the vagi; non-myelinated fibres are not involved. The authors wish to express their thanks to members of the department, in particular Dr D. A. S. G. Mary for helpful discussion and D. Kaye for technical assistance. Gratitude is also expressed to the British Heart Foundation, the Medical Research Council and the Wellcome Trust for their support. N. S. was a W.H.O. Research Fellow. REFERENCES COLERIDGE, H. M., COLERIDGE, J. C. G., DANGEL, A., KIDD, C., LUCK, J. C. & SLEIGHT, P. (1973). Impulses in slowly conducting vagal fibres from afferent endings in the veins, atria and arteries of dogs and cats. Circulation Research 33, HENRY, J. P. & PEARCE, J. W. (1956). The possible role of cardiac atrial stretch receptors in the induction of changes in urine flow. Journal of Physiology 131, KAPPAGODA, C. T., LINDEN, R. J. & MARY, D. A. S. G. (1977). Atrial receptors in the dog and rabbit. Journal of Physiology 272, KAPPAGODA, C. T., LINDEN, R. J. & SIVANANTHAN, N. (1978). Atrial receptors and the urine response. Journal of Physiology 282, 49P. KAPPAGODA, C. T., LINDEN, R. J. & SIVANANTHAN, N. (1979). The nature of the atrial receptors responsible for a reflex increase in heart rate in the dog. Journal of Physiology 291, KAPPAGODA, C. T., LINDEN, R. J. & SNOW, H. M. (1972). The effect of stretching the superior vena caval-right atrial junctions on right atrial receptors in the dog. Journal ofphysiology 277, LEDSOME, J. R. & LINDEN, R. J. (1968). The role of the left atrial receptors in the diuretic response to left atrial distension. Journal of Physiology 198, LEDSOME, J. R., LINDEN, R. J. & O'CONNOR, W. J. (1961). The mechanism by which distension of the left atrium produces diuresis in anaesthetized dogs. Journal of Physiology 159, PAINTAL, A. S. (1972). Cardiovascular receptors. In Handbook ofsensory Physiology, Enteroreceptors, vol. 3/1, ed. NEIL., E. Berlin: Springer Verlag. SLEIGHT, P. & WIDDICOMBE, J. G. (1965). Action potentials in fibres from receptors in the epicardium and myocardium of the dogs left ventricle. Journal of Physiology 181, THOREN, P. N. (1976). Atrial receptors with non-medullated vagal afferents in the cat. Discharge frequency and pattern in relation to atrial pressure. Circulation Research 38,