THE NATURE OF THE ATRIAL RECEPTORS RESPONSIBLE FOR A REFLEX INCREASE IN ACTIVITY IN EFFERENT CARDIAC SYMPATHETIC NERVES

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1 Quaterly Journal of Experimental Physiology (1982), 67, Printed in Great Britain THE NATURE OF THE ATRIAL RECEPTORS RESPONSIBLE FOR A REFLEX INCREASE IN ACTIVITY IN EFFERENT CARDIAC SYMPATHETIC NERVES R. J. LINDEN, D. A. S. G. MARY AND D. WEATHERILL* The Department of Cardiovascular Studies, University of Leeds, Leeds LS2 9JT (RECEIVED FOR PUBLICATION 23 JANUARY 1981) SUMMARY In dogs anaesthetized with chloralose, distension of small balloons in the right upper and middle pulmonary vein-atrial junctions, to stimulate left atrial receptors, caused an increase in heart rate and an increase in activity in efferent sympathetic nerve fibres in cardiac branches of the right stellate ganglion. Cooling of the cervical vagi in steps reduced the magnitude of the responses in these sympathetic nerve fibres. In four dogs, the response in six preparations of sympathetic nerves was slightly reduced with the vagi at 18 C and markedly reduced or abolished at 12 IC. In these nerves there was no significant response to distension of the balloons when the cervical vagi were cooled to 9 IC. The effect of cooling the vagi was the same as the previously shown effect of cooling on the increase in activity in myelinated afferent vagal fibres and the increase in heart rate during stimulation of atrial receptors. It is concluded that the increase in activity in efferent sympathetic cardiac nerve fibres during distension of small balloons in the pulmonary vein-atrial junctions involves receptors discharging into myelinated vagal nerve fibres; receptors which discharge into non-myelinated vagal nerves or afferent sympathetic nerve fibres are not involved in this response. Thus, the efferent sympathetic nerve fibres studied are likely to represent the efferent pathway of the response of an increase in heart rate to distension of the small balloons. INTRODUCTION Distension of small balloons at the pulmonary vein-atrial junctions to stimulate left atrial receptors causes a reflex increase in heart rate (Ledsome & Linden, 1964a; Kappagoda, Linden & Mary, 1975), and an increase in activity in efferent sympathetic nerves in branches from the right stellate ganglion (Karim, Kidd, Malpus & Penna, 1972). The afferent limb of the reflex effecting the increase in heart rate lies in the vagal nerves and the efferent limb solely in the sympathetic nerves to the sino-atrial node (e.g. Linden, 1976). Distension of the small balloons has been shown to stimulate three types of receptors in the left atrium, receptors which discharge into myelinated vagal nerve fibres, receptors which discharge into non-myelinated vagal nerve fibres and receptors whicb discharge into afferent sympathetic nerve fibres (Holton, 1977; Kappagoda, Linden & Sivananthan, 1979). Kappagoda et al. (1979) showed that the reflex increase in heart rate during distension of the balloons involves only atrial receptors attached to myelinated fibres. Kappagoda et al. (1979) used graded cooling of the vagi to show that the increase in activity in myelinated fibres and the increase in heart rate during distension of the balloons were blocked over the same narrow range of temperature in the nerve, which was different from the range of temperatures required to block the increase in activity in non-myelinated fibres from the same stimulus. * M.R.C. Research Fellow, Present address: Department of Anaesthetics, Western Infirmary, Glasgow.

2 144 R. J. LINDEN, D. A. S. G. MARY AND D. WEATHERILL However, the effects of stimulating receptors in the atria attached to non-myelinated vagal nerve fibres and sympathetic afferent nerve fibres on the activity in efferent sympathetic cardiac nerves are unknown. In addition, the positive correlation between the response of the heart rate and the response in myelinated vagal fibres (Kappagoda et al. 1979, commented on above) may not be strictly reflected in the response of efferent cardiac sympathetic nerves (see Discussion). Therefore, the present investigation was designed to resolve these two problems; the reflex response of an increase in activity in right efferent sympathetic nerves in branches supplying the heart was studied using the cooling technique described by Kappagoda et al. (1979) in an attempt to examine a potential correlation between the response in the efferent cardiac sympathetic nerves and the responses in heart rate and afferent myelinated vagal fibres. METHODS Dogs weighing kg were given a subcutaneous injection of morphine sulphate (0-5 mg. kg-'). Subsequently the dogs were anaesthetized with chloralose and artificially ventilated with oxygenenriched air as described by Kappagoda et al. (1979). During recording of the activity in the efferent sympathetic nerves, the animals were given gallamine triethiodide (initial dose 2 mg. kg-'; subsequent doses 1 mg. kg-') as described by Linden, Mary & Weatherill (1980); periodically the doses ofgallamine were omitted and the animal allowed to recover from its effects so as to gauge the depth of the anaesthesia. The chest was opened on the right side with the removal of the second to the fifth ribs. Small balloons were inserted in the right upper and middle pulmonary vein-atrial junctions, and distended with warm saline as described by Ledsome & Linden (1964a). Pressures in the femoral artery and left atrium were recorded through cannulae inserted in the right femoral artery and a pulmonary vein, together with tracheal pressure, end-tidal Pco2 and e.c.g.; the oesophageal temperature and ph, Pco2 and Po2 ofarterial blood were monitored and maintained within normal limits using techniques described previously (Kappagoda, Linden & Snow, 1972). Both cervical vagi were carefully dissected free and cooling thermodes were applied as described by Kappagoda et al. (1979) and Linden, Mary & Weatherill (1981 b). Efferent cardiac nerves were identified in branches from the right stellate ganglion (usually the inferior cardiac nerve). Activity in fine strands of these nerves was recorded, monitored and counted using techniques described by Linden, Mary & Weatherill (1981 a). Having observed a reflex increase in heart rate in response to distension of the small balloons (Ledsome & Linden, 1964a), this response was then prevented during subsequent distension of the balloons by giving bretylium tosylate (dose 10 mg. kg-' i.v.) as described by Linden et al. (1980). The increase in activity in cardiac nerves in response to distension of the small balloons was examined by recording the activity in preparations of the cardiac nerves for at least 1 min before, 2 min during and 1 min after distension of the balloons. The response was calculated as the increase in mean spike frequency during the second minute of distension expressed as a percentage of the mean spike frequency during the two steady-state 1 min control periods. In each animal the cut distal end of the nerve from which recordings were made was electrically stimulated using techniques described by Linden et al. (1981 b); stimulation of these nerves always caused an increase in heart rate which was abolished by giving the bretylium tosylate, as was previously shown in detail by Ledsome & Linden (1964b). RESULTS In four dogs recordings commenced approximately 3-5 h after the initial dose of anaesthetic had been given. The initial heart rate was beats. min-' (mean; range ), the femoral artery pressure was kpa (mean; range ) and the left atrial pressure was 0O80 kpa (mean; range ). The ph, Pc02 and Po, of arterial blood were (mean; range ), 5-35 kpa (mean; range ) and kpa (mean; range ) respectively. The haematocrit was 43 3 % (mean; range 40-47). Distension

3 ATRIAL RECEPTORS AND ACTIVITY IN CARDIAC SYMPATHETIC NERVES 145 A B C is Fig. 1. Examples of experimental records, taken from records before (A), during (B) and after (C) distension of the balloons, demonstrating the increase in activity in an efferent sympathetic nerve fibre during distension of the balloons, with the vagi at 37 C. From above downwards are shown records of tracheal pressure (Resp. F), expired PC0, (E. Pcoy). femoral artery pressure (Fem. P), mean left atrial pressure (M.l.a. F), action potential (A. Pot.) and electrocardiogram (e.c.g.). The response during distension of the balloons was unaccompanied by changes in Fem. P, M.l.a. P or heart rate. of the small balloons in all four dogs led to an increase in heart rate of 13 1 beats. min1 (mean; range 6-36); this response was abolished after giving bretylium tosylate. A total of twenty preparations of efferent cardiac nerves were investigated and, of these, six preparations were examined that responded to distension of the small balloons. An example of records obtained during one experiment is shown in Fig. 1; changes in activity in the nerve during distension of the small balloons were unaccompanied by significant changes in mean femoral artery and left atrial pressure or heart rate, calculated over the 4 min experimental period. The increase in activity in one efferent nerve preparation in response to distension of the balloons is shown in Fig. 2. The mean frequency over a stable 1 min control period before distension of the balloons was 5*80 spikes. s'l, 6*98 spikes. - during the second minute of distension, and 5 76 spikes. s-l during the final stable 1 min control period; in this example the response to distension of the balloons was therefore an increase of 1 ~2O spikes. s-l, i.e. an increase of 20*8 / relative to the mean frequency of spikes during the control periods. The pattern of immediate increase and decrease in activity in the nerve on distension and release of distension respectively was consistent in all responding units. In the six nerve preparations that showed an increase in activity in response to distension of the balloons, this response was studied with the vagi warm (35-37 C), with the vagi cooled to 18 or 12 C, and finally with the vagi rewarmed. To facilitate comparison of the effects of vagal cooling on the responses obtained from different preparations of cardiac nerves the responses obtained with the vagi cooled were expressed as a percentage of the mean of the average of the two responses obtained with the vagi warm; this percentage was named the relative response (e.g. Linden et al. 1980).

4 146 R. J. LINDEN, D. A. S. G. MARY AND D. WEATHERILL Balloon distension 8-0 r 5 80 r a) " 4-0 ce Time (s) Fig. 2. Histogram showing mean spike frequency. s-i in an efferent sympathetic nerve fibre during successive 5 s intervals. The values indicated above the records are the mean spike frequencies during the respective periods. The mean spike frequency during the second minute of distension of the balloons was compared with the mean frequency during two 1 min control periods before and after distension of the balloons. There was a 20-8% increase in activity in the sympathetic nerve during distension of the balloons with the vagi at 37 OC. a) el 100 r 80 k 60 F CIF a,.5i U u) ct W, 40 F L Temperature ( C) 10 0 Fig. 3. The effect of cooling the vagi on the response in efferent sympathetic nerves during stimulation of atrial receptors. The ordinate shows the magnitude of the response expressed as a percentage of the mean control responses with the vagi warm; the abscissa shows temperature of the vagi. The oblique lines are the same as those in Fig. 5 in Kappagoda et al. (1979), enclosing most responses in afferent myelinated vagal fibres. Each symbol represents results from one preparation of sympathetic nerves. The response was slightly reduced by cooling the vagi to 18 OC; on cooling to 12 IC the response was markedly reduced or abolished.

5 ATRIAL RECEPTORS AND ACTIVITY IN CARDIAC SYMPATHETIC NERVES 147 Table 1. The effect of cooling the vagi to 12 C on the control frequency of activity in six preparations ofefferent cardiac nerves, femoral artery pressure, left atrial pressure and heart rate Mean Femoral left atrial Nerve activity artery pressure pressure Heart rate (spikes. s-1) (kpa) (kpa) (beats. min-') Vagi C Vagi 12 C P >070 >070 >080 >070 All values are mean + 1 S.E. of mean. The P values were calculated using two-tailed paired Student's t tests. In the six nerve preparations examined the response to distension of the balloons with the vagi warm was an increase in activity of 17-8% (mean; range 10-25). The relative response observed with the vagi cooled to 18 C was 95-5% (mean; range ) and at 12 IC was 117% (mean; range 0-34). The individual results for the six nerve preparations are shown in Fig. 3. Thus the increase in activity in the nerve was slightly reduced with the vagi at 18 IC; at 12 IC the response was greatly reduced or abolished; all remaining responses were abolished by cooling the vagi to 9 IC. This effect of vagal cooling on the response was the same as that described by Kappagoda et al. (1979) on the increase in activity in afferent myelinated vagal fibres in response to distension of the small balloons. In the present experiments cooling the vagi to 12 IC had no significant effect on heart rate, mean femoral artery pressure, mean left atrial pressure or the mean control frequency of activity in the efferent cardiac nerves (Table 1). DISCUSSION Electrophysiological studies in the dog have located atrial receptors with myelinated afferent vagal fibres to the atrial endocardium (e.g. Kappagoda, Linden & Mary, 1977). In addition receptors with non-myelinated vagal afferent fibres and with sympathetic afferent fibres have also been demonstrated in the pulmonary vein-atrial junctions (Malliani, Recordati & Schwartz, 1973; Kappagoda et al. 1979). Localized distension of the pulmonary vein-atrial junctions with small balloons causes an increased discharge in each of the three types of receptors (Holton, 1977; Kidd, Ledsome & Linden, 1978; Kappagoda et al. 1979). Such distension of the small balloons has been shown to result in an increase in activity in efferent sympathetic nerve fibres in branches from the right stellate ganglion (Karim et al. 1972). However, the effects of distending the small balloons on the activity in the efferent cardiac nerves are known only for those receptors in the atria attached to myelinated vagal afferent fibres; the effects of stimulation by the same technique of receptors discharging into non-myelinated vagal fibres and those discharging into sympathetic afferent fibres are not known. The study of Karim et al. (1972) was not designed to investigate which of the vagal afferents were involved; sectioning the vagus or cooling to 5 'C would also block the increased activity in non-myelinated fibres. Again sectioning the vagi or cooling to 5 IC are procedures likely to alter the baseline state of the animal and may prevent any possible changes being observed; thus a role for sympathetic afferents would not be excluded by this technique.

6 148 R. J. LINDEN, D. A. S. G. MARY AND D. WEATHERILL It has been shown that during stimulation of atrial receptors there is a positive correlation between the response of a change in heart rate to cooling of the vagi and the response of a change in myelinated afferent vagal fibres to the same cooling of the vagi (Kappagoda et al. 1979). However it does not follow that the same correlation will be obtained in the response of efferent cardiac sympathetic fibres. First, from the study of Karim et al. (1972) it could not be concluded with any certainty that the efferent sympathetic fibres studied were representative of those connected with the sino-atrial node. Secondly, it is possible that the stimulation of the sympathetic afferents or non-myelinated vagal fibres was such that they were affecting efferent sympathetic fibres, but at either end of the stimulus-response relationship such that there were no observable effects on heart rate. That there can be changes in efferent sympathetic fibres to the sino-atrial node during supramaximal stimulation of cardiac efferent nerves without changes in heart rate has been previously demonstrated (Rushmer, 1958; Furnival, Linden & Snow, 1973). The present investigation, therefore, was planned to determine the relationship between the stimulation of the three types of receptor in the atria and the response of an increased activity in the efferent nerves using the technique of graded cooling of the vagi. Kappagoda et al. (1979) found that the response of an increase in activity in afferent myelinated vagal fibres during distension of small balloons at the pulmonary vein-atrial junctions was blocked by a range of temperatures in the nerve different from that at which increase in activity in non-myelinated fibres was affected (see Figs. 5 and 11, Kappagoda et al. 1979). They showed that in each of twelve myelinated fibres examined, regardless of the magnitude of the initial response with the vagus warm, the response of an increase in activity during distension of the balloons was slightly reduced at 18 C, whilst at 12 IC the response was markedly reduced or abolished; almost all responses were abolished at 9 IC. In contrast, responses in twelve non-myelinated fibres were attenuated over a wider range of temperature. In four fibres the increase in activity resulting from distension of the balloons was blocked above 18 'C and in the remaining eight fibres the increase in activity was still present at 9 IC. Kappagoda et al. (1979) showed that their technique of graded cooling did not cause significant changes in the baseline data and their results allowed the conclusion that only atrial receptors attached to myelinated fibres were involved in a reflex increase in heart rate observed in response to distension of the balloons. The same technique of cooling of the vagi was used by Linden et al. (1980) to show that only atrial receptors attached to myelinated fibres were involved in the reflex inhibition of activity in renal nerves in response to distension of the balloons (Karim et al. 1972). The results of the present investigation, for similar reasons to those of Kappagoda et al. (1979) detailed above, allow the conclusion that atrial receptors attached to myelinated fibres were involved in the reflex increase in activity in efferent sympathetic nerves in branches from the right stellate ganglion; the responses in these fibres to distension of the balloons were present at 18 'C but markedly attenuated at 12 'C. The attenuation of the reflex response by cooling the vagi to 12 'C was not attributable to a change in the control level of activity in the sympathetic fibres since such cooling had no effect on the basal frequency of activity in the efferent sympathetic nerve fibres. Furthermore, the abolition of the response with the vagi cooled to 12 and 9 'C, in the present investigation, provides evidence that the sympathetic afferent nerves were not involved in the reflex increase of activity in the efferent sympathetic nerves resulting from distension of the small balloons. That the efferent sympathetic nerve fibres studied, in the branches from the right stellate ganglion, were likely to be those which innervated the sino-atrial node is suggested by two facts. First, electrical stimulation of the efferent nerves from which the single fibres were

7 ATRIAL RECEPTORS AND ACTIVITY IN CARDIAC SYMPATHETIC NERVES. 149 obtained resulted in an increase in heart rate; a response which was abolished by bretylium tosylate (Ledsome & Linden, 1964a). Secondly, the effect of graded cooling of the cervical vagus on the response of an increase in activity in these efferent nerve fibres to distension of the small balloons correlated positively with the effect on the response of an increase in heart rate to distension of the balloons. Such a finding shows that the nerves studied involve the same type of atrial receptors and suggests that these efferent sympathetic nerves are likely to represent the efferent pathway of the reflex response of an increase in heart rate. The evidence presented therefore demonstrates that first, the reflex response of an increase in activity in cardiac sympathetic nerves resulting from distension of small balloons at the pulmonary vein-atrial junctions involves stimulation of atrial receptors attached to myelinated fibres in the vagi; non-myelinated fibres and sympathetic afferent nerve fibres are not involved in this response. Secondly, the cardiac sympathetic nerve fibres studied are likely to be the same fibres involved in the response of an increase in heart rate to stimulation of atrial receptors attached to vagal myelinated nerve fibres. The authors wish to express their gratitude to the Medical Research Council, British Heart Foundation and the Wellcome Trust for financial assistance and to Messrs D. Kaye and J. Haigh for technical assistance. REFERENCES FURNIVAL, C. M., LINDEN, R. J. & SNOW, H. M. (1973). Chronotropic and inotropic effects on the dog heart of stimulating the efferent cardiac sympathetic nerves. Journal ofphysiology 230, HOLTON, A. (1977). Characteristics of cardiac receptors with fibres in the rami communicantes. Ph.D. thesis, University of Leeds. KAPPAGODA, C. T., LINDEN, R. J. & MARY, D. A. S. G. (1975). Gradation of the reflex response from atrial receptors. Journal of Physiology 251, 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. (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 junction on right atrial receptors in the dog. Journal of Physiology 227, KARIM, F., KIDD, C., MALPUS, C. M. & PENNA, P. E. (1972). The effects of stimulation of the left atrial receptors on sympathetic efferent nerve activity. Journal of Physiology 227, KIDD, C., LEDSOME, J. R. & LINDEN, R. J. (1978). The effect of distension of the pulmonary vein-atrial junction on activity of left atrial receptors. Journal of Physiology 285, LEDSOME, J. R. & LINDEN, R. J. (1964a). A reflex increase in heart rate from distension of the pulmonary vein-atrial junctions. Journal of Physiology 170, LEDSOME, J. R. & LINDEN, R. J. (1964b). The effect of bretylium tosylate on some cardiovascular reflexes. Journal of Physiology 170, LINDEN, R. J. (1976). Reflexes from receptors in the heart. Cardiology 61, (Suppl. 1), LINDEN, R. J., MARY, D. A. S. G. & WEATHERILL, D. (1980). The nature of the atrial receptors responsible for a reflex decrease in activity in renal nerves in the dog. Journal of Physiology 300, LINDEN, R. J., MARY, D. A. S. G. & WEATHERILL, D. (1981 a). The responses in renal nerves to stimulation of atrial receptors, carotid sinus baroreceptors and carotid chemoreceptors. Quarterly Journal of Experimental Physiology 66, LINDEN, R. J., MARY, D. A. S. G. & WEATHERILL, D. (1981 b). The effect of cooling on transmission of impulses in vagal nerve fibres attached to atrial receptors in the dog. Quarterly Journal of Experimental Physiology 66, MALLIANI, A., RECORDATI, G. & SCHWARTZ, P. J. (1973). Nervous activity of afferent cardiac sympathetic fibres with atrial and ventricular endings. Journal of Physiology 229, RUSHMER, R. F. (1958). Autonomic balance in cardiac control. American Journal of Physiology 192,