bradykinin. sympathetic, splanchnic and hypogastric nerves after intra-arterial doses of

Transcription

1 Quarterly Journal of Experimental Physiology (1977) 62, EFFECTS OF BRADYKININ MEDIATED BY AUTONOMIC EFFERENT NERVES. By K. FLOYD, VERITY E. HICK, JuTHIKA KOLEY and J. F. B. MORRISON. From the Department of Physiology, University of Leeds, Leeds LS2 9JT. (Received for publication 15th April 1976) Intra-arterial injections of bradykinin cause increases in blood pressure and increased impulse rates in single sympathetic efferent fibres. The peptide also causes one or more large bladder contractions, which are associated with increased impulse rates in pelvic nerve efferent fibres whose spontaneous discharges are temporally associated with increases in intravesical pressure. These induced increases in pelvic nerve discharge and intravesical pressure could be abolished or greatly reduced by interference with conduction in pathways which mediate reflex bladder contractions. It is concluded that bradykinin has little direct action on the bladder, and that the large contractions which result from its administration are mediated by the central nervous system. Lang and Pearson [1968] showed that large intra-arterial doses of bradykinin produced hypertension after the initial vasodilator effect, and that the rise in arterial pressure could be prevented by ganglion blockade. They concluded that the pressor responses were mediated by the sympathetic nervous system and involved a central nervous component. The first part of the present study consists of observations on single sympathetic efferent fibres in the cervical sympathetic, splanchnic and hypogastric nerves after intra-arterial doses of bradykinin. In the course of some earlier experiments [Floyd, Hick and Morrison, 1975] it was found that i.a. bradykinin produced large rises in intravesical pressure, but transection of the cord at L5-6, which abolished the spontaneous activity of the bladder, substantially reduced the size of the response to bradykinin. The role of the central nervous system in this effect of the peptide was therefore investigated by recording from efferent axons in the pelvic nerves near the bladder, and observing the effects of bradykinin before, during and after cord cooling at L6 which blocked spontaneous and reflex bladder contractions. METHODS General Experiments were performed on 13 cats (1 9 to 3 5 kg) anaesthetized with a fluothane/02 gas mixture, followed by an intravenous dose of 1 % chloralose/saline solution (50-60 mg. kg-'), and maintenance doses of 10 mg. The trachea, a forelimb vein and a common carotid artery were cannulated and arterial pressure was monitored with a Devices pressure transducer and recorder. The animals were artificially ventilated with 02 enriched air; end-tidal CO2 % was measured and maintained at the desired level by alteration of ventilation or by intravenous doses of NaHCO3 solution (1 mmol.ml-1). Oesophageal temperature was monitored with a telethermometer (Yellow Springs Inst. Co. Inc.), and maintained between 37 C and 38 C. Hypotension associated with normocapnia was treated by i.v. infusion of a dextran solution (Dextran 150 inj. Fisons Pharmaceuticals Ltd.). In some experiments the bladder 11

2 12 Floyd, Hick, Koley and Morrison was cannulated via the urethra and intravesical pressure recorded using a Devices pressure transducer and recorder. Operative Procedures In experiments on sympathetic efferents the vagi were cut and the carotid sinuses denervated. The carotid arteries were stripped and the effectiveness of baroreceptor denervation was assessed using carotid clipping. (a) The cervical sympathetic nerve. This nerve was approached via the neck incision used for cannulations and baroreceptor denervations. (b) The splanchnic nerves. The chest was opened at the 7th or 8th intercostal space and the lower ribs were removed in order to expose the left splanchnic nerve above the diaphragm. (c) The hypogastric nerves. The hypogastric and lumbar splanchnic nerves were exposed retroperitoneally through a loin incision. The lower end of this skin incision was extended towards the symphysis pubis and the peritoneum and muscle layers lateral to the bladder were divided. (d) The pelvic nerves. In experiments on the pelvic nerve efferents the baroreceptors were not denervated and a pelvic nerve was approached by sectioning the attachment of the abdominal wall muscles to the inguinal ligament. The nerve was found retroperitoneally and dissected near its site of entry to the bladder wall. In some animals a laminectomy was performed to expose the spinal cord at L5-6. The cord was either tied and transected between L6 and L7, or was cooled at L6 until spontaneous bladder contractions ceased. Recording and stimulation Action potentials were recorded from nerve strands using Ag wire electrodes, Tektronix Type 122 or an Isleworth Type A101 preamplifier, and displayed on Tektronix Types 565 and 5100 oscilloscopes and fed to an audio monitor. When an action potential of distinctive size and shape was identified its spontaneous activity was recorded and 1 to 10,ug bradykinin (Synthetic Bradykinin (Sandoz)), or bradykinin triacetate trihydrate (Koch-Light Laboratories) injected intra-arterially through a polythene cannula introduced into the descending aorta from a femoral artery. Measurement of interspike intervals in single units was carried out using a PDP-12 computer after the action potentials had been recorded on a Racal Thermionic T3000 FM tape recorder. Sometimes it was not possible to count the impulses from only one nerve fibre; when this occurred action potentials from up to 4 nerve fibres were counted. The average rate per second of single and multiunit preparations was measured with an amplitude analyser and rate meter (F. Haer & Co.). Cord cooling A thermode was constructed from the cooling stage of a freeze microtome (Pelcool, M.S.E. Ltd.), in a manner essentially similar to that described by Brown [1971]. When required, the cooled thermode was brought into contact with the dura overlying L6, and the effectiveness of the cooling in blocking conduction in pathways innervating the bladder was assessed by the disappearance of spontaneous bladder contractions and of reflexly induced contractions. These movements were usually abolished within min of starting to cool the cord, and cooling was maintained for a further min. Following removal of the thermode, the cord was rewarmed by superfusion with saline at 37 C, and spontaneous and reflexly induced bladder contractions returned within 2-5 min. RESULTS Recordingsfrom sympathetic efferents Single units were recorded on 20 occasions and multiunit recordings were made from 15 strands. The recordings were made from the cervical sympathetic (3), splanchnic (12) and hypogastric (20) nerves, and the changes in

3 Bradykinin effects and the A.N.S. 13 FIG. 1. Discharge of two units in an efferent strand of splanchnic nerve. A, spontaneous discharge of one unit. B, 10 pg Bradykinin injected intra-arterially at arrow. The discharge of the spontaneously active unit increases, and the second unit is recruited. During these discharges the mean arterial pressure rose by 30 mm Hg and the intravesical pressure increased by 17 mm Hg. C, discharge of the spontaneously active unit 60 s after the injection of bradykinin. discharge rate produced by 1-10 pg bradykinin i.a. were studied. An increase in efferent discharge rate was characteristic and occurred in all but 2 of the recordings. In some cases the increased discharge began during the hypotensive phase of the vascular response while in other units the increase did not begin until the hypertensive response was well developed. In the single units the 'spontaneous' average impulse rates ranged from zero to 2-8 s-'. Fig. 1 shows the discharge in a strand of splanchnic nerve containing one spontaneously active sympathetic efferent and its response to 10 pg bradykinin i.a. The spontaneously active unit increases its rate of discharge, and another unit is recruited. Similar effects were seen in the multiunit recordings. Effects of bradykinin on the bladder Intra-aortic injection of 1 or 2 pg bradykinin produced either a very slow, small (1 or 2 mmhg) change in intravesical pressure, or one or more contractions characterized by a rapid increase in intravesical pressure of the order of 5-10 mmhg in 1 or 2 seconds. Larger doses of the peptide produced a series of bladder contractions which were of greater strength and duration than spontaneous contractions. With smaller doses within this range the response usually consisted of a few contractions separated by a short period during which intravesical pressure returned to baseline levels. Larger doses produced a series of contractions during which the rise in intravesical pressure was maintained

4 14 Floyd, Hick, Koley and Morrison for one or more minutes. The size of these changes in intravesical pressure depended on bladder volume: when the bladder was empty the response was small, and at moderate volumes (15-50 ml) it was not uncommon to record changes of 50 mmhg intravesical pressure. Modification of the response to bradykinin by cord cooling Cord transection (1 cat) and application of lignocaine at L6 (1 cat) abolished spontaneous bladder contractions and reduced the size of the response to 10 pg bradykinin to < 10% of that observed in the control. The response to lignocaine was reversible given sufficient time, but cord cooling at L6 was adopted as the best method of interruption of conduction in the cord because it was easily reversed and could be used on 7-10 occasions in one experiment with complete recovery of the earlier responses. The changes in intravesical pressure produced by 10 pg bradykinin were measured before, during and after cord FIG. 2. The recordings show changes in intravesical pressure in response to 10pg of Bradykinin i.a. on 5 occasions before, during and after cooling of the sacral cord at L6. The centre three traces indicate the response to 10 ug of the peptide 5 mins, 13 mins and 21 mins after the start of cord cooling. cooling. The pressure change produced by bradykinin during cord cooling was compared with the mean of the responses before and after this procedure using a paired t-test. Injection of 10 pg during cord cooling produced a mean increase in intravesical pressure of 6-9 mmhg (S.E.M mmhg n = 10) which was significantly less (P < 0 001) than the control (39 3 mm Hg, S.E.M mmhg). The nature of the responses during cord cooling was also modified; the rate of rise of intravesical pressure was slower and usually the response consisted of one peak rather than a series of contractions (Fig. 2). Effects of bradykinin on efferent pelvic nerve discharge In 2 cats recordings were made of efferent discharges in branches of the pelvic nerve near its entrance to the bladder wall. Seven units gave discharges which occurred in relation to spontaneous bladder contractions, and the bursts of discharge often preceded a rise in intravesical pressure. The units fitted the classification of parasympathetic efferent neurones to the bladder described by de Groat and Ryall [1969]. Injection of bradykinin intra-arterially caused a large increase in discharge rate of these units and a prominent feature of the

5 Bradykinin effects and the A.N.S. 15 discharge was that it started before the bladder contractions and became silent before the bladder relaxed. When the cord was cooled at L6, the discharge rate of these units diminished greatly and often ceased when spontaneous bladder contractions were abolished. Injection of bradykinin resulted in little or no change in either discharge rate or intravesical pressure (see Fig. 3). The response returned to the control level after rewarming the cord. u w 15 - E Ei v.e * 2 _. Ḷ... v* *0.0 **0 L en t 0. I I FIG. 3. Top, intravesical pressure. Bottom, average discharge rate per second in a pelvic efferent unit. The left panel shows the response of the unit during a spontaneous bladder contraction. The increase in impulse rate precedes the increase in intravesical pressure. In the middle panel intra-arterial administration of 10 ug bradykinin (at the arrow) caused increases in intravesical pressure and in the discharge of the pelvic efferent unit. The pressure response to the drug was less than in Fig. 2 because one pelvic nerve was sectioned. In the right hand panel the cord was cooled at L6 and intra-arterial administration of 10 jpg of bradykinin (at the arrow) caused no increase in intravesical pressure or in discharge rate of the pelvic efferent unit. The responses returned to the control values after rewarming of the cord. DISCUSSION The results of the recordings of sympathetic efferent units in the cervical sympathetic, splanchnic and hypogastric nerves of cats with denervated baroreceptors show that intra-arterial injection of bradykinin can cause discharges in pre- and post-ganglionic sympathetic pathways. Unlike the suggested central action of angiotensin [Scroop and Whelan, 1966; Smookler, Severs, Kinnard and Buckley, 1966; Morrison and Pickford, 1971] the central nervous mediation of the hypertensive response to bradykinin [Lang and Pearson, 1968] has been confirmed by single unit recording. In the cervical sympathetic and the splanchnic nerves the majority of fibres are preganglionic, and the results in these nerves indicate that the responses are mediated by the central nervous system. They do not however preclude an additional ganglionic action of the peptide, such as has been suggested by Lewis and Reit [1965]. The nature of the receptors involved is not clear; a number of afferent fibre systems can respond to bradykinin, including skin nociceptors [Burgess and Perl, 1973], low threshold mechanoreceptors in skin [Fjiillbrant and Iggo, 1961], C fibres from muscle [Mense and Schmidt, 1974] and afferents in sympathetic nerves [Lim, Guznan, Rodgers, Goto, Braun, Dickerson and Engle,

6 16 Floyd, Hick, Koley and Morrison 1964; Floyd, Hick and Morrison, 1975]. Also some central neurones in the dorsal horn of the cord appear to be directly sensitive to bradykinin applied by microelectrophoresis [Randic and Yu, 1975]. The recordings from parasympathetic efferent units in the pelvic nerve fibres close to the bladder wall show that increases in the spontaneous discharge rates of these units precede spontaneous bladder contractions. This closely parallels the results obtained by De Groat and Ryall [1969] who recorded from the cell bodies of parasympathetic neurones in the spinal cord and concluded that these cells were the efferent path involved in reflex and spontaneous bladder contractions. The large bladder contractions that occur after i.a. bradykinin were also preceded by large increases in the discharge rates of the parasympathetic efferents, which indicates that the vesical responses to the peptide were centrally mediated. This involvement of the central nervous system was confirmed by the fact that cooling the spinal cord at L6-L7 inhibited or abolished not only the spontaneous and bradykinin-induced bladder contractions bit also the preceding changes in pelvic nerve efferent discharges. It is concluded that bradykinin can activate central nervous pathways which (a) cause hypertension because of an increase in sympathetic efferent activity and (b) cause parasympathetic efferent activity which results in one or more large bladder contractions. The ability to modify the vesical response to a given dose of bradykinin by cord cooling will be used in the accompanying paper [Floyd, Hick, Koley and Morrison, 1977] to separate the mechanical and chemical components of the sensitivity of vesical afferent nerve endings to the peptide. ACKNOWLEDGMENTS We would like to thank Mr E. G. Tate for technical assistance, Mrs E. R. Baister, Sandoz Ltd., for a generous gift of synthetic bradykinin and the Medical Research Council for financial support. REFERENCES BROWN, A. G. (1971). Effects of descending impulses on transmission through the spinocervical tract. Journal ofphysiology, 219, BURGESS, P. R. and PERL, E. R. (1973). Cutaneous Mechanoreceptors and Nociceptors. In: Handbook of Sensory Physiology. Vol. II Somatosensory System. Ed. A. Iggo pp DE GROAT, W. C. and RYALL, R. W. (1969). Reflexes to sacral parasympathetic neurones concerned with micturition in the cat. Journal ofphysiology, 200, FJALLBRANT, N. and IGGo, A. (1961). The effect of histamine, 5-hydroxytryptamine and acetylcholine on cutaneous afferent fibres. Journal ofphysiology, 156, FLOYD, K., HICK, VERITY, E. and MORRISON, J. F. B. (1975). The responses of visceral afferent nerves to bradykinin. Journal ofphysiology, 247, 53-54P. FLOYD, K., HICK, VERITY, E., KOLEY, JUTHIKA and MORRISON, J. F. B. (1977). The effects of bradykinin on afferent units in intra-abdominal sympathetic nerve trunks. Quarterly Journal of Experimental Physiology, 62, LANG, W. J. and PEARSON, LEONIE (1968). Studies on the pressor responses produced by bradykinin and kalladin. British Journal of Pharmacology and Chemotherapy, 32, LEWIS, G. P. and REIT, E. (1965). The action of angiotensin and bradykinin on the superior cervical ganglion of the cat. Journal ofphysiology, 179,

7 Bradykinin effects and the A.N.S. 17 LIM, R. K. S., GuzMAN, F., RODGERS, D. W., GOTO, K., BRAUN, C., DICKERSON, G. D. and ENGLE, R. J. (1964). Site of action of narcotic and non-narcotic analgesics determined by blocking bradykinin-evoked visceral pain. Archives Internationales de Pharmacodynamie (et de Therapie), 152, MENSE, S. and SCHMIDT, R. F. (1974). Activation of group IV afferent units from muscle by algesic agents. Brain Research, 72, MORRISON, J. F. B. and PICKFORD, MARY (1971). Effects of angiotensin and nor-adrenaline on discharge in fibres of the cervical sympathetic nerve in cats and dogs. British Journal ofpharmacology, 41, RANDIC, MIRJANA and Yu, H. H. (1975). Microelectrophoresis study of cat dorsal horn neurones activated by noxious stimuli. Journal ofphysiology, 252, 23-25P. SCROOP, G. C. and WHELAN, R. F. (1966). A central vasomotor action of angiotensin in man. Clinical Science, 30, SMOOKLER, H. H., SEVERS, W. B., KINNARD, W. S. and BUCKLEY, J. P. (1966). Centrally mediated cardiovascular effects of angiotensin II. Pharmacology and Experimental Therapeutics, 153, B