University of Birmingham, Birmingham B15 2TJ

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1 J. Phy8iol. (1972), 226, pp With 5 text-figures Printed in Great Britain EFFECT OF RENAL NERVE STIMULATION, RENAL BLOOD FLOW AND ADRENERGIC BLOCKADE ON PLASMA RENIN ACTIVITY IN THE CAT BY J. H. COOTE, E. J. JOHNS, VALERIE H. MACLEOD AND BERTHA SINGER From the Department of Physiology, the Medical School, University of Birmingham, Birmingham B15 2TJ (Received 26 January 1972) SUMMARY 1. The effect of electrical stimulation of the distal cut ends of the renal nerves of unilaterally nephrectomized, anaesthetized cats was studied. Using stimulation parameters of 15 pulses per second (pps), 15 V and -2 msec duration, there was an immediate sharp drop in renal blood flow, as determined by an electromagnetic flowmeter, which was maintained for about 2 min. Flow gradually returned to control values over approximately the next 1 min in spite of continued stimulation for up to 3 min. 2. Plasma renin activity (PRA) increased markedly after 1 min of stimulation but 2 min later fell towards pre-stimulation values whether stimulation was maintained or not. 3. Phentolamine, an az-adrenergic-receptor antagonist, abolished both the blood flow and PRA responses to a 1 min period of renal nerve stimulation. 4. When the renal artery was constricted in order to produce blood flow changes similar to those found with renal nerve stimulation, the rise in PRA was similar to that observed with renal stimulation. 5. In phentolamine-blocked animals, renal artery constriction, as described, produced the same effect on PRA as was observed with renal nerve stimulation. 6. Propranolol, a fl-adrenergic-receptor antagonist, did not block the blood flow response to renal nerve stimulation, but did block the rise in PRA normally associated with renal nerve stimulation. 7. It is suggested that the effect of renal nerve stimulation on PRA is mediated, primarily, by changes in renal blood flow and that one of the steps leading to renin release following stimulation is sensitive to propranolol. This step must be distal to the effect on vascular smooth muscle.

2 16 J. H. COOTE AND OTHERS INTRODUCTION Although there is much evidence indicating that a completely intrarenal mechanism exists which can alter renin release, there is also some evidence to indicate that the sympathetic nervous system can significantly modify renin secretion (Vander, 1967). This evidence is based on the effects of renal denervation (Brubacher & Vander, 1968; Mogil, Itskovitz, Russell & Murphy, 1969), infusion of catecholamines (Vander, 1965; Wathen, Kingsbury, Strouder, Schneider & Rostorfer, 1965; Gordon, KUchel, Liddle & Island, 1967), induction of catecholamine release (Otsuka, Assaykeen, Goldfein & Ganong, 197), use of adrenergic blocking agents (Winer, Chokshi, Yoon & Freedman, 1969; Assaykeen, Clayton, Goldfein & Ganong, 197), reflex stimulation of renal nerves (Bunag, Page & McCubbin, 1966; Gordon et al. 1967), stimulation of pressor areas of the medulla oblongata (Passo, Assaykeen, Otsuka, Wise, Goldfein & Ganong, 1971 b), pretreatment with reserpine (Birbari, 1971), and ganglion blockade or local anaesthesia of renal nerves (Hodge, Lowe & Vane, 1966; Bunag et at. 1966). A more direct approach was taken by Vander (1965) who electrically stimulated the renal nerve-artery complex and found an increased renin concentration in renal vein blood. In these experiments, the renal nerves were not cut but loop electrodes were placed around the renal artery and accompanying nerves. It is not clear whether the rise in renal vein renin concentration was maintained throughout the period of stimulation, whether the rise was sufficiently great to produce a significant increase in circulating renin concentration, and what proportion of the changes observed was due to contraction of the renal artery and what part to excitation of the renal nerves. The present study was undertaken to answer some of these questions. This was done, in the cat, by studying the effect of electrical stimulation of the distal cut ends of renal nerves on renal blood flow and plasma renin activity. In addition, we have examined the mechanisms by which neural stimuli caused the release of renin by the use of adrenergic receptor blocking agents and by constriction of the renal artery. METHODS E86tiration of renin Except for some minor modifications described below, the method developed by Ryan, McKenzie & Lee (1968) and Ryan & McKenzie (1968), for the rabbit, was found suitable for use in cats. This method is based on the in vitro incubation of plasma with added substrate, in the presence of angiotensinase and kallikrein inhibitors. The angiotensin I generated on incubation is estimated by the blood

3 NERVE STIMULATION AND PLASMA RENIN 17 pressure assay in the rat. In this bio-assay male rats (25-35 g), anaesthetized with pentobarbitone (6 mglkg i.p.) and ganglion-blocked with pentolinium (5 mg/kg s.c.) were used. Angiotensin II (Hypertensin, Ciba) was used as a standard in early studies and was replaced by angiotensin I (1-asp-5-isoleu angiotensin I, Schwarz Bioresearch, Orangeburg, U.S.A.). Kallikrein and angioteminase inhibitors. The soybean trypsin inhibitor recommended by Ryan et al. (1968) was replaced by trasylol (Aprotinin, F.B.A. Pharmaceutical Ltd., Sussex) as a kallikrein inhibitor (Skinner, 1967). The dimercaprol used in the original procedure was omitted. The effectiveness of EDTA and trasylol in blocking angiotensinase and kallikrein activity in cat plasma was tested as follows: a mixture of 1 ml. plasma, 5 ml. 4 nlm-edta (disodium salt, in phosphate buffer) and -5 ml. trasylol (5 KIU/ml.) was made. -5 ml. aliquots of this mixture were added to -4 ml. angiotensin II (44g/ml.) and made up to 1-5 ml. with phosphate buffer (-1 M, ph 6 containing -1 % Hibitane (ICI)). Incubation at 42 C for up to 24 hr showed no measurable loss of angiotensin activity. Preparation of renal renin. The method employing DEAE cellulose adsorption for the preparation of renal renin in rabbits, described by Ryan & McKenzie (1968), required some modification for use in cats, as angiotensinase activity was still present in the final eluate. Angiotensinase activity was destroyed by acidification of the eluate to ph 3-3 for 25-3 min. Depressor substances, formed during this final acidification, were removed by dialysis against -15 M-NaCl at 4 C for 24 hr. The preparation was freeze-dried and stored in this state at 4 C. When required it was reconstituted in an appropriate volume of water. Renal renin thus prepared was found to be angiotensinase-free for a 24 hr incubation period at 42 C both in the absence and presence of inhibitors. Preparation of renin substrate. This was prepared from the plasma of male cats nephrectomized 48 hr previously. Dialysis of the plasma, as recommended by Ryan & McKenzie ( 1968), was found to result in a loss of activity and was therefore omitted. In order to avoid the possible interference of heparin with the renin-renin substrate reaction, the blood of nephrectomized cats was collected into tubes containing EDTA giving a final concentration of 3 mm. Plasma was obtained by centrifugation at 3 rev/min for 2 min at 4 C. Substrate concentration of the plasma was determined by mixing 1 ml. of this plasma with 5 ml. 4 mm-edta, -5 ml. trasylol (5 KIU/ml.) and -5 ml. concentrated renal renin. -6 ml. aliquots were pipetted into separate tubes and incubated for, 12 and 24 min for the release of all of the angiotensin. The incubations were terminated by the addition of 2-4 ml.. 1 % saline followed by heating on a boiling-water bath for 5 min. The substrate preparations routinely gave values either between 4 and 6 ng of angiotensin II equivalents per ml. of original plasma, or approximately double this value of angiotensin I equivalent. Renin-renin substrate reaction in cat plasma. In the study of Ryan & McKenzie (1968) the renin-renin substrate reaction in the rabbit obeyed first order kinetics to substrate concentrations of at least 1 ng angiotensin I/ml. of incubate, i.e. the rate of angiotensin generation depended upon substrate concentration at all substrate concentrations studied. On the other hand, Lever, Robertson & Tree (1964) found that the reaction between rabbit renin and ox substrate obeyed zero order kinetics at considerably lower substrate concentrations. Similar results were reported by Pickens, Bumpus, Lloyd, Smeby & Page (1965) with human renin and substrate. To establish the nature of the kinetics of the reaction in the present study, small quantities of renal renin were incubated for 3 hr at 42 C (in the presence of the usual inhibitors) with different concentrations of substrate. Dilutions of the cat substrate preparations were made with -1 m phosphate buffer, ph 6. The results

4 18 J. H. COOTE AND OTHERS indicate that the reaction of cat renin and substrate obeyed zero order kinetics at relatively modest substrate concentrations. Under these conditions ofincubation, using angiotensin II as the standard, the Michaelis constant was calculated to be 5 ng/ ml. of incubation medium. Thus, so long as the pre-incubation substrate concentration exceeds this value by at least threefold, and the post-incubation concentrations by at least twofold, the reaction should obey zero order kinetics. In practice pre-incubation substrate concentrations were found to range from 267 to 4 ng angiotensin II equivalents/ml. The applicability of the data obtained from these studies with renal renin to plasma renin was confirmed using two pools of plasma renin, one having a moderate and the other a high value. These two pools were 3 5 ml. blood sample taken from animal. Centrifuged at C for 1 min and plasma removed Plasma (1.5 ml.) + 4 mm-edta (.75 ml.) + 5 KIU trasylol (.75 ml.) Mix. 5 ml. aliquots Substrate Substrate Substrate Angiotensin II Angiotensin II (1 ml.) (1 ml.) (1 ml.) (.2 ml. ( 2 ml. 25 ng/ml.) 25 ng/ml.) I ~~~~+ + Buffer (.8 ml.) Buffer (.8 ml.) Incubation at42c hr 2 hr 2 hr hr 2hr Blank Rate duplicates Angiotensinase test Fig. 1. Flow sheet for plasma renin measurements in the cat. Plasma is mixed with EDTA and trasylol, added to substrate or angiotensin II, and incubated at 42 C to generate sufficient angiotensin to assay with the rat blood pressure assay. The reaction is stopped by snap-freezing in liquid nitrogen. incubated with undiluted substrate and the same substrate preparation diluted to half-concentration with phosphate buffer. Measured plasma renin activity in both pools of plasma was similar regardless of the substrate preparation used. Summary of procedure. Blood samples of 3-5 ml. were taken from the carotid artery of heparinized animals and immediately centrifuged at C for 2 min at 3 rev/ min. The plasma was stored in the deep-freeze until required for estimation. A summary of the final procedure adopted for the estimation of renin is presented in Fig. 1. For each determination of PRA, 1-5 ml. plasma was required. To this was added 75 ml. 4 mrm-edta and 75 ml. trasylol containing 5 KIU/ml. -5 ml. aliquots of this mixture were transferred to five tubes (nos. 1-5). Renin substrate (1 ml.) was added to tubes 1-3 and 5 ng of angiotensin II to tubes 4 and 5. Tubes 1 and 4 were kept frozen until assayed. Tubes 2, 3 and 5 were incubated at 42 C for 2 hr. The reaction was stopped by snap-freezing in liquid nitrogen. The use of added

5 NERVE STIMULATION AND PLASMA RENIN angiotensin was to confirm the effectiveness of the inhibitors. Each batch of substrate was calibrated before use to ensure that a sufficient excess was present. Results are expressed in terms of generation of angiotensin I/ml. hr (except when it is indicated that angiotensin II was used as standard in the bio-assay), and referred to as plasma renin activity (PRA). The period of 2 hr has been chosen taking into account the levels of renin found in cat plasma and technical convenience. It has been confirmed in incubation studies at 15, 2 and 24 hr that the rate of generation of angiotensin is constant during this period. Reproducibility and reliability of the renin estimates. The angiotensin content of each incubation tube was determined by bracketing the pressor response between two known doses of standard (Lever et al. 1964). At least two bracket assays were performed on each of the duplicate assay tubes and the upper and lower limits for each estimation are given in the tables. The percentage variation of the estimate varied, being about 2 % for samples with low renin values and less than 1 % for samples with high renin content. In order to determine between-assay variability, a pool of cat plasma was used. Six 1-5 ml. aliquots of the plasma were treated and incubated as described above and tested on separate assay animals. The upper and lower limits of the replicate estimates of renin were used in computing a mean value and its S.D. The renin activity of this plasma, calculated in this manner, was (S.D.) (ng angiotensin I/ml. hr). Renal renin was added to some of this plasma and five aliquots of the mixture were incubated and assayed in separate animals, as were six aliquots of renal renin equivalent to the amount added to each aliquot of plasma. The mean value of angiotensin I/ml. hr, computed as described above, for the added renin was and the mean value for the plasma plus exogenous renin was Surgical techniques and experimental procedures. Thirty-four experiments were performed on twenty male cats in the weight range kg. They were anaesthetized with sodium pentobarbitone, 42 mg/kg i.p., and further small doses were administered i.v. as required. Blood pressure was recorded from the carotid artery using a Statham pressure transducer (P23Dc) or a Consolidated-electrodynamics transducer (type L 221). Renal blood flow was measured using a non-cannulating electromagnetic flow probe on the renal artery connected to a flowmeter (Medical and Biological Instrumentation Ltd, Kent). In all experiments right nephrectomy was performed using the retroperitoneal approach. A similar dissection was used to expose the left kidney. With the aid of a dissecting microscope, the renal nerves were cut close to the coeliac ganglion and dissected out until they were of sufficient length to place over silver wire stimulating electrodes. The area around the renal vein and artery was closely inspected and all other small nerves to the kidney were cut. Heparin (1 u./kg) was administered i.v., 3-45 min after completion of dissection. Stimulation of renal nerves was not performed less than 2 hr after the end of the major dissection. If any other procedure was performed which might affect the release of renin, such as obtaining zero flow on the flowmeter by brief occlusion of the renal artery, at least 15 min were allowed to elapse before any sample was taken. Square wave stimuli were available from a Grass S 8 Stimulator. All recordings were made with a Grass (model 7) or Devices polygraph. Sequential sampling. In preliminary studies with intact animals it was found that repeated removal of 5 ml. blood samples from the carotid artery at the rate of 4 ml./ min and at 2 min intervals did not result in a rise of PRA until a total of 1 ml./kg had been removed. It was confirmed that removal of seven 3-5 ml. blood samples, taken at intervals of 2 min or greater between sampling, in the unilaterally nephrectomized and renally denervated cat did not produce a significant rise in PRA. In the definitive experiments, therefore, blood samples of 3-5 ml. were taken at intervals of 19

6 2 J. H. COOTE AND OTHERS 2 min or more and, usually, no more than seven samples were taken. If it was necessary to reduce the time between sampling or if adrenergic antagonists were used, either the cells were reinfused intravenously in saline or blood from a donor cat was used to replace the blood taken. RESULTS The effect of prolonged stimulation of renal nerves on renal blood flow Preliminary experiments were carried out to find the stimulus parameters necessary to obtain a maximum reduction in renal blood flow when renal nerves were stimulated. The nerves were dissected out as described 4-3 o 2 _o I ~~~ / 1 _C 15 V c 5 / 'U ) II PPs Fig. 2. The effect of stimulation of distal out ends of the renal nerves on renal vascular resistance (pressure/flow) using different stimulus frequencies at two different voltages of -2 msec pulse duration. Values plotted represent maximal responses obtained at each frequency. above and stimulated for a short period of time sufficient for the maximum effect on renal blood flow and the renal vascular resistance to be measured. An indication of renal vascular resistance was obtained by dividing the blood pressure by the blood flow at any one time. Fig. 2 shows the effect of increasing square-wave frequency at two different voltages of -2 msec pulse duration on renal vascular resistance in one experiment. A maximum effect was obtained with approximately 15 pps at 15 V. Higher voltages and shorter or longer pulse durations gave no greater effect. The effect of stimulating the renal nerves for a period of 14 min is illus-

7 NERVE STIMULATION AND PLASMA RENIN 21 trated in Fig. 3. At the start of stimulation there was a dramatic fall in renal blood flow which lasted about 2 min and the calculated renal vascular resistance increased by a factor of 2'5. The flow then gradually returned toward control values over the next 1 min. These results were consistently seen whenever these stimulus parameters were used and renal blood flow Stimulation period t4 15 E E F ff 5_ Time (min) a, _ L. 3.-.=.2 I % increase Fig. 3. Effect of a 14 min period of renal nerve stimulation, 15 V, 15 pps of -2 msec duration, on blood pressure, renal blood flow and calculated renal vascular resistance (blood flow given in arbitrary units). measured. Blood flow was reduced to a minimum of times the initial value on different occasions and the return to pre-stimulation levels generally took between 8 and 12 min. This pattern of response was also observed with submaximal voltages and with lower frequencies. The possibility that the decline in response was due to the continuous nature of the stimulation was examined by interrupting the stimulation at regular intervals. Rest periods of up to 9% of the stimulation time were ineffective

8 22 J. H. COOTE AND OTHERS in preventing the decay in the response. The possibility that this phenomenon was due to damage of the renal nerve during stimulation was ruled out by the observation that, if a suitable time was allowed between each stimulation period (usually 2 min), it was possible to repeat the procedure of prolonged stimulation of the renal nerves several times in the same cat and obtain the same response. In addition, the character of the response was unchanged when the electrodes were moved peripherally, by up to 1 cm, during stimulation. Effect of renal nerve stimulation on plasma renin activity. The effect of a 1 min period of stimulation of the renal nerves on PRA was studied in eight experiments on six cats (Table 1). In four of these experiments the stimulation period was extended to 3 min. In experiments in which stimulation was continued for 1 min, blood samples were taken before stimulation was begun (usually 1 min), after 1 min of stimulation, and 2 min after the end of stimulation. When stimulation was continued for 3 min, blood samples were taken 1 min before, after 1 and 3 min of stimulation and 2 min after the end of stimulation. Most of the prestimulation values for PRA were between 1 and 2 ng/ml. hr except in one cat (no. 5), where the value was 9-5 mg/ml. hr. Renal nerve stimulation for 1 min resulted, in all cases, in a rise in PRA. The degree of response varied considerably, extending from an increase to 1F54 times in one case, to one greater than 12 times the initial value in another. In view of the reproducibility of the bioassay, and the small effect on PRA of the intermittent removal of small volumes of blood in these experimental conditions, it was concluded that the changes in PRA were a consequence of stimulation of the renal nerve. The variability of the response was attributed, primarily, TABTm 1. Effect of prolonged renal nerve stimulation on plasma renin activity PRA (ng angiotensin I/ml. hr) Experi.- A Cat no. ment Pre- After 1 min After 3 min 2 min posi- (wt. kg) no. stimulation stimulation stimulation stimulation 1 (3.3) ' (3.1) * (2.9) *26 1* (3.2) 5 1* * * * (3.) * (2-9) * * * - + Indicates the limits of the bracket assay for each estimation. * Indicates blood sample replaced with red blood cells of the previous sample suspended in saline.

9 NERVE STIMULATION AND PLASMA RENIN 23 to the different amounts of nerve fibre which could be dissected out in adequate length to be placed on the electrodes. This difficulty may, in part, also be responsible for the observed variation in the degree of reduction in blood flow in response to renal nerve stimulation. When stimulation of the nerve was stopped at 1 min and the PRA studied 2 min later the values had, in all cases, returned toward prestimulation levels. In four experiments, despite continuing stimulation for 3 min, the PRA values measured after this time had also returned toward pre-stimulation levels and there was no further reduction 2 min after the end of stimulation. -=12.E E _ E 4 E tw~ M - v8 C, _L 75 4' C.w o'uc o E C- O~~~~~~~ I I I e5 5-~lp min Renal nerve stimulation Timing of sampling and stimulation Fig. 4. Changes in plasma renin activity and renal blood flow in response to a 3 min period of renal nerve stimulation in a 2-9 kg male cat (cat no. 6). Stimulus parameters as in Fig. 3. The pre-stimulation renin value is projected to the start of stimulation assuming no change. In one of the experiments in which the renal nerves were stimulated for 3 min (no. 8) renal blood flow was measured at the same time. The results are illustrated in Fig. 4. Plasma renin activity was estimated before and at 5, 1 and 3 min of stimulation. (In this experiment, the blood cells of the first few samples were suspended in saline and infused, i.v., as soon as possible.) Plasma renin activity was moderately elevated at 5 min, reached a peak at 1 min and, in the subsequent 2 min of stimulation, fell toward the pre-stimulation value. Renal blood flow decreased abruptly at the start of the stimulation reaching, for a short period, 415 times the pre-stimu-

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11 NERVE STIMULATION AND PLASMA RENIN 25 lation level. Flow gradually returned to the control value over approximately 15 min. The peak of reduction in renal blood flow preceded the peak in PRA by about 1 min. It is of interest that the increase in PRA in this animal was one of the greatest observed and that the length of time taken for the blood flow to return to normal was the longest we have recorded. Effect of reduction of renal blood flow by renal artery constriction on PRA. The renal blood flow changes normally observed following renal nerve stimulation were mimicked by constriction of the renal artery distal to the flow probe on the renal artery. Seven experiments were performed in. 5 - Catno.15 W E 4 - ~j2 'u.2 I E co C o 4 R.A. constriction R.A. constriction R.A. constriction 4~~~~~~~~~~~m Fi5 Fig min Effect of renal artery constriction on plasma renin activity and renal blood flow (cat no. 15) four unilaterally nephrectomized renally denervated cats. Results are presented in Table 2. The degree of constriction was regulated as required by observation of the record obtained from the flowmeter. This procedure resulted in a momentary stoppage of blood flow, but this lasted only a few seconds before a flow level of about -15 times the control was achieved (Fig. 5). It was found that such momentary stoppage of blood flow did not produce a measurable change in PRA 1 min later. The renal blood flows achieved bythis procedure ranged from -9 to -17 times the control. These maximal reductions were maintained for approximately 2 min. Flow was gradually allowed to return over the next 8-12 min until the constriction was fully relaxed. Blood samples for estimation of PRA were obtained before constriction, 1 min after the start of constriction and 2 min

12 26 J. H. COOTE AND OTHERS later. The increases in PRA obtained by renal artery constriction were similar to those observed with renal nerve stimulation, varying from 1*73 to 5-5 times the initial value. Values had returned toward pre-constriction levels 2 min later in all cases. In none of these experiments was the increase in PRA as great as that observed in cats nos. 1 and 6 following renal nerve stimulation (Table 1). In the present experiments the blood flow was returned towards normal by 1 min, mimicking the most frequent pattern ofblood flow change obtained with renal nerve stimulation. This pattern was not followed in cat no. 6 where renal blood flow was still significantly reduced after 1 min of stimulation (Fig. 4). It is possible that the latter accounts for the somewhat smaller increase in PRA following mechanical reduction of renal blood flow to that following nerve stimulation in that animal. Blood flow changes were not followed in cat no. 1. Effect of c-adrenergic-receptor blockade on the response to renal nerve stimulkion. The effect of renal nerve stimulation was studied during a- adrenergic-receptor blockade in six experiments in three cats (Table 3). Phentolamine (Rogitine, Ciba, 1 mg/ml. in saline) was infused slowly into the renal artery in two cats and into the jugular vein in the third, and the drop in renal perfusion pressure which this tended to produce was counteracted, partially, by constriction of the aorta below the renal artery. Blockade was considered satisfactory when no reduction in renal blood flow occurred following a 15 sec period of renal nerve stimulation. The amount of phentolamine required to achieve this varied from animal to animal and was not dependent on the route of administration. Blockade was maintained by regular administration of the drug (Table 3). After completion of each experiment (i.e. after the 2 min post-stimulation blood sample was removed and before the start of the next experiment) the effectiveness of the blockade was checked as before. In none of these experiments was there a reduction in renal blood flow during the 1 min period of renal nerve stimulation and in none of them was there evidence of an increase in PRA in response to renal nerve stimulation. The PRA values found in these animals are presented in Table 3. Two features of these experiments merit comment. First, in two animals, an increased base line in PRA was observed after phentolamine blockade. This is likely to be attributable to the reduced perfusion pressure to which the kidneys were exposed despite the measures taken to counteract it. Secondly, removal of blood for PRA estimation in these ac-blocked animals tended to accentuate the effect of the already reduced perfusion pressure. Small quantities of blood, removed from a donor cat as required, and infused into the experimental animal immediately after blood sampling, prevented this effect, as in cat no. 9 (Table 3).

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14 28 J. H. COOTE AND OTHERS Effect of reduction of renal blood flow by renal artery constriction on PRA in a-blocked cats. Six experiments were performed in three cats in which the renal artery was constricted in phentolamine-blocked animals. This was to determine whether mechanical reduction of renal blood flow, in the a-blocked animal, could reverse the effects of phentolamine on the response to renal nerve stimulation. Phentolamine was administered and the effectiveness of blockade was checked by renal nerve stimulation as described above. Constriction of the renal artery was performed as described in the previous section. Blood flow was reduced to a minimum of times the initial value. Flow was allowed to return to unrestricted levels after 8-12 min. Results of these experiments are presented in Table 4. In every case there was a clear-cut response to the reduction in renal blood flow. Increases in PRA 1 min after the start of constriction ranged from a 2*3 to a ten-fold increase which was well within the range obtained following renal nerve stimulation. Values had returned toward pre-constriction levels 2 min later. Effect of fi-adrenergic-receptor blockade on the response to renal nerve stimulation. The effect of propranolol (DL-propranolol hydrochloride, IC1, 2 mg/ml. in saline) on the response to a 1 min period of renal nerve stimulation was studied in seven experiments in four cats (Table 5). The propranolol was infused slowly, i.v., until the response to 8,uglkg (1- #ug/kg in cat no. 2) of isoprenaline (i.v.) on the heart rate was blocked. Before infusion of the antagonist, I Iug/kg (i.v.) of isoprenaline produced a measurable increase in the heart rate. Small amounts of propranolol were administered continuously throughout the rest of the experiment and it was confirmed, at the end of each experiment, that the blockade was maintained. The amount of propranolol required to produce blockade and the amount infused during the rest of the experiments are presented in Table 5. The drop in blood pressure which this treatment tended to produce was again partially counteracted by constriction of the aorta below the renal artery. In three of these cats, blood taken for PRA estimation was replaced with red cells suspended in saline, while in a fourth, donor blood was used (Table 5). In none of these experiments was there any evidence that the propranolol had any influence on the extent of reduction in blood flow observed following renal nerve stimulation or in the timing of the return to pre-stimulation flow rates. On the other hand, no evidence was found to suggest that renal nerve stimulation resulted in a rise in PRA in any of these experiments. In experiments 1, 2, 3, 5, 6 and 7 PRA remained fairly steady throughout the experimental period, with all the values falling below 2 ng angiotensin I/ml. hr. In the fourth experiment, PRA tended to drift upward,

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17 NERVE STIMULATION AND PLASMA RENIN 31 although no evidence of a peak can be detected after 1 minutes of renal nerve stimulation. In this animal the diastolic blood pressure had been dropping as the experiment progressed and had reached 55 mm Hg when the last blood sample was taken. DISCUSSION The present studies indicate that the method for estimation of renin in plasma developed for use in the rabbit by Ryan et al. (1968) can, with minor modifications, be used for the assay of renin in cat plasma. The method was found to be reproducible as measured between assays and reliable as indicated from recovery studies. The variability of individual estimates ranged between 1 and 2% which is general in bio-assays of this type. The sensitivity of the renin-releasing system in the cat, in response to haemorrhage. was found to be similar to that reported in the dog (Hodge et al. 1966) as removal of blood over the value of 1 ml./kg resulted in a significant increase in plasma renin activity. In unilaterally nephrectomized cats with the remaining kidney denervated, the PRA was usually between 1 and 2 ng angiotensin I generated/ ml. hr. In view of methodological differences in different laboratories, it is difficult to make comparisons between species. However, it is of interest to note that Passo et al. (1971 b) reported a value of 3 ng/ml. hr (angiotensin II equivalents) in intact anaesthetized dogs. Boucher, Menard & Genest (1967) reported a value of 12-5 ng/ml. hr (angiotensin II) in conscious rats, while Pickens et al. (1965) obtained a value of '65 ng/ml. hr (angiotensin II) in normal human subjects. The present investigation has clarified some of the questions which we considered were left unanswered by Vander (1965). First, in view of the stimulation parameters used by Vander (2-25 V, 3-6 pps and msec) it is likely that the rise in renal vein renin concentration and the changes in renal blood flow were not constant throughout the period of stimulation (2-3 min). Secondly, our results indicated that stimulation of the nerve alone was sufficient to produce a marked increase in renal renin release. And, finally, our results clearly indicated that the effect of renal nerve stimulation on renin release was sufficiently great to produce a marked increase in the level of PRA in the general circulation. Renal nerve stimulation. This study has also revealed that the maximal reduction in renal blood flow produced by stimulation of renal nerves could not be maintained for more than about 2 min regardless of which stimulation parameters were used. In view of the fact that it was possible to demonstrate the effect of stimulation on blood flow repeatedly in the same animal, it appears highly unlikely that the decay in response observed with prolonged stimulation was related to permanent damage of the nerve fibres 2 PHY 226

18 32 J. H. COOTE AND OTHERS or terminals. A similar phenomenon has been reported by Folkow, Lewis, Lundgren, Mellander & Wallentin (1964) in intestinal blood vessels following splanchnic nerve stimulation. These authors could not demonstrate this effect in skin or resting skeletal muscle. They referred to it as ' autoregulatory escape' and attributed it to a local vasodilator mechanism. This 'escape' response in the intestine has been confirmed many times although it is still uncertain how it occurs (Ross, 1971). Much less information is available regarding renal blood flow; however, DiSalvo & Fell (1971) have reported a similar escape phenomenon in the dog following renal nerve stimulation. They found that this occurred, within seconds of stimulation, at frequencies of 5 pps or greater. They did not demonstrate it at frequencies of 1 pps or less but stimulation was maintained for only 2-3 sec in all experiments. With physiological stimulation of renal nerves, the blood flow changes may be less intense but considerably more prolonged. Bunag et al. (1966), using bilateral common carotid artery occlusion as a stimulus, did find this type of change in renal blood flow. The drawback to using this type of preparation to study the effect of renal nerve stimulation on renin release is that the response may, at least in part, be due to the rise in adrenal catecholamines produced by the stimulus. In the present investigation, as the timing and pattern of the response to electrical stimulation is quite reproducible, this method of increasing activity within the renal nerves has proved to be useful. The observation that renal nerve stimulation resulted in a dramatic decrease in renal blood flow which was not maintained in spite of continued stimulation, and that this was followed, approximately 1 min later, by an increase in PRA which was also not maintained, strongly suggested that the reduction in blood flow was responsible for the increase in PRA. The increase in PRA was interpreted as a reflexion of an increase in renin release by the kidney. The time lag between the maximum response in flow and in maximum increase in PRA may be related to the time required for the increased release of renin to have an effect on the renin level in the general circulation, to a 'wash-out' effect as the renal blood flow returns toward normal, or to a combination of both of these factors. Renal blood flow and renin release. The present experiments have demonstrated not only that renal nerve stimulation results in a decrease in renal blood flow and a rise in PRA but also that both phenomena can be blocked by the a-receptor antagonist phentolamine. Again, this suggests that the rise in PRA is a consequence of the reduction in renal blood flow. Of course, the possibility exists that reduction in renal blood flow is not an obligatory, or the only, step in the sequence of events initiated by renal nerve stimulation leading to the release of renin. Renal nerve stimulation might also stimulate the renin-containing cells directly, thereby causing the release

19 NERVE STIMULATION AND PLASMA RENIN 33 of renin. Phentolamine could, theoretically, block both of these effects. We have explored the relationship between the reduction in blood flow and the increase in PRA which follow stimulation in several ways. The possibility of blocking the blood flow response to renal nerve stimulation with the smooth muscle relaxant, papaverine, was examined. With papaverine infused into a renal artery, the dose necessary to block the blood flow response to renal nerve stimulation for long enough for an experiment to be carried out also caused a great disturbance of respiration. These experiments were therefore abandoned. In a recent brief abstract, Martin & White (1971) reported that papaverine infusion into the renal artery did not prevent the increase in renin secretion following renal nerve stimulation in the dog. This evidence would suggest that the release of renin is unrelated to a decrease in renal blood flow. However, in view of our experience with papaverine, it is essential to confirm that the smooth muscle response to nerve stimulation was, in fact, blocked before accepting this conclusion. We altered our approach to the problem by studying the effect of reduction of renal blood flow by constriction of the renal artery in the renally denervated animal. When the degree and timing of the reduction of the renal blood flow were kept within the limits observed with renal nerve stimulation, the responsesin measured PRA were comparable to those observed with renal nerve stimulation. Therefore it is clear that the reduction in renal blood flow caused by renal nerve stimulation can account for virtually its entire effect on PRA. It is of interest that the reduction in blood flow produced by two such different means led to similar increases in PRA. The foregoing conclusion is further supported by the results obtained in the studies on the effect of renal artery constriction in phentolamine-blocked animals. In these experiments, renal artery constriction produced a rise in PRA comparable to that observed with renal nerve stimulation, suggesting that the effect of phentolamine in preventing the response to renal nerve stimulation can be attributed to its effect on renal blood flow. Renin-release and fi-adrenergic-receptors. The present experiments indicate that fl-adrenergic-receptor blockade with propranolol prevents the increase in PRA normally seen with renal nerve stimulation in spite of the fact that the reduction in blood flow is still observed. Clearly, in order to obtain a full response to renal nerve stimulation the blood flow response must not be prevented and neither must propranolol-sensitive receptors be blocked. The results indicate that in the chain of events leading to renin release following renal nerve stimulation this receptor must be distal to the response of the vascular smooth muscle. The possibility that the rise in PRA in response to renal nerve stimulation may be due to two effects, one 2-2

20 34 J. H. COOTE AND OTHERS of which is mediated by an action on vascular smooth muscle and sensitive to a-blockade, and another due to direct innervation ofthe renin-containing cells which is sensitive to fl-blockade, does not appear to be tenable on the basis of the present experiments. The work of Michelakis, Caudle & Liddle (1969) using dog renal cell suspensions suggests that cyclic-amp may play a role as an intracellular mediator of renin release. If this is so then propranolol, which is believed to act by blocking stimulation of adenyl cyclase (Robison & Sutherland, 197), may be having a similar effect in the present experiments. Several recent studies tend to support this idea. A study which is comparable, at least partially, to the present investigation is that of Passo et al. (1971 a) who studied the effect of adrenergic-receptor blocking agents on the release of renin in dogs following electrical stimulation of pressor areas within the medulla oblongata. The authors had previously reported that this increase in PRA was mediated by the renal nerves (Passo et al b). They reported that phenoxybenzamine did not affect the increase in renin secretion, propranolol reduced it, while both, together, abolished it. Unfortunately, the authors were unable to determine whether, at the time of electrical stimulation, there were any changes in renal blood flow due to renal nerve activity, and thus to determine the effectiveness of a-blockade. Winer et al. (1969) reported that both phentolamine and propranolol blocked the increase in PRA observed in humans following such stimuli as upright posture or the administration of diazoxide, ethacrynic acid or theophylline. Superficially, our results appear to agree with theirs. It could be argued that in their experiments propranol was acting on the f,- receptors of the renin-containing cells. It could also be suggested that the effect of upright posture might be mediated by renal nerve activation and that phentolamine should block this action. However, it is not at all clear that the action of the three drugs used is mediated by an action on renal nerves. In fact, in a more recent study, the authors have suggested quite a different explanation for their results (Winer, Chokshi & Walkenhorst, 1971). They reported that infusion of cyclic-amp into the renal artery of dogs resulted in an increase in the release of renin which could be blocked by both propranolol and phentolamine. This was interpreted to mean that these blocking agents can act at a step distal to cyclic-amp formation. However, if reduction in renal blood flow, as described in the present communication, results in an increased formation of cyclic-amp, and if Winer et al. (1971) are correct regarding the site of action of phentolamine, we should have observed no response to renal artery constriction in the phentolamine-treated animals. As we invariably did observe a response, our results seem to be in disagreement with these workers, at least in regard to this site of action of phentolamine.

21 NERVE STIMULATION AND PLASMA RENIN 35 Although the present experiments do not shed any light on the mode of action of other factors which influence renin release, such as diuretics and circulating catecholamines, they clearly indicate that renal blood flow changes are of prime importance in mediating the action of renal nerves on rein release, and that a propranolol-sensitive step is involved. The full sequence of events leading to the release of renin by increased activity in the renal nerves has not yet emerged. We wish to thank Mr H. K. Richards for able technical assistance, and ICI Ltd (Macclesfield) for a supply of propranolol. This work was supported by a grant from the Medical Research Council. RESULTS ASSAYKEEN, T. A., CLAYTON, P. L., GOLDFEIN, A. & GANONG, W. F. (197). Effect of alpha and beta adrenergic blocking agents on the renin response to hypoglycemia and epinephrine in dogs. Endocrinology 87, BIRBARI, A. (1971). Effect of sympathetic nervous system on renin release. Am. J. Physiol. 22, BOucHER, R., MENARD, J. & GENEST, J. (1967). A micromethod for measurement of renin in the plasma and kidney of rats. Can. J. Physiol. Pharmac. 45, BRUBACHER, E. S. & VANDER, A. J. (1968). Sodium deprivation and renin secretion in unanaesthetized dogs. Am. J. Physiol. 214, BUNAG, R. D., PAGE, I. H. & MCCUBBIN, J. W. (1966). Neural stimulation of release of renin. Circulation Res. 19, DiSALvo, J. & FELL, C. (1971). Changes in renal blood flow during renal nerve stimulation. Proc. Soc. exp. Biol. Med. 136, FOLKOW, B., LEWIS, D. H., LUNDGREN, O., MELLANDER, S. & WALLENTIN, W. (1964). The effect of graded vasoconstrictor fibre stimulation on the intestinal resistance and capacitance vessels. Acta physiol. scand. 61, GORDON, R. D., KuCHEL, O., LIDDLE, G. W. & ISLAND, D. P. (1967). Role of sympathetic system in regulating renin and aldosterone production in man. J. clin. Invest. 46, HODGE, R. L., LowE, R. D. & VANE, J. R. (1966). The effects of alteration of bloodvolume on the concentration of circulating angiotensin in anaesthetized dogs. J. Physiol. 185, LEVER, A. F., ROBERTSON, J. I. S. & TREE, M. (1964). The estimation of renin in plasma by an enzyme kinetic technique. Biochem.. J. 91, MARTIN, D. M. & WHITE, F. N. (1971). Evidence for direct neural release of renin. Fedn Proc. 3, abst MiciELAxis, A. M., CAUDLE, J. & LIDDLE, G. W. (1969). In vitro stimulation of renin production by epinephrine, norepinephrine and cyclic AMP. Proc. Soc. exp. Biol. Med. 13, MOGIL, R. A., ITSKOVITZ, H. D., RUSSELL, J. H. & MURPHY, J. J. (1969). Renal innervation and renin activity in salt metabolism and hypertension. Am. J. Physiol. 216, OTSUXA, K., ASSAYKEEN, T. A., GOLDFEIN, A. & GANONG, W. F. (197). Effect of hypoglycemia on plasma renin activity in dogs. Endocrinology 87, PASSO, S. S., ASSAYKEEN, T. A., GOLDFEIN, A. & GANONG, W. F. (1971a). Effect of alpha and beta-adrenergic blocking agents on the increase in renin secretion produced by stimulation of the medulla oblongata in dogs. Neuroendocrinology 7,

22 36 J. H. COOTE AND OTHERS PAsso, S. S., ASSAYKEEN, T. A., OTSUKA, K., WHITE, B. L., GoLDuEIN, A. & GANONG, W. F. (1971 b). Effect of stimulation of the medulla oblongata on renin secretion in dogs. Neuroendocrinology 7, 1-1. PICKENS, P. T., BuMrus, M., LLOYD, A. M., SMEBY, R. R. & PAGE, I. H. (1965). Measurement of renin activity in human plasma. Circulation Res. 17, ROBISON, G. A. & SUTHERLAND, E. W. (197). Sympathin E, sympathin I and the intracellular level of cyclic AMP. Circulation Res. 27, Suppl. I, Ross, G. (1971). The regional circulation. A. Rev. Physiol. 33, RYAN, J. W. & MCKENZIE, J. K. (1968). Properties of renin substrate in rabbit plasma with a note on its assay. Biochem. J. 18, RYAN, J. W., MCKENZIE, J. K. & LEE, M. R. (1968). A rapid simple method for the assay of renin in rabbit plasma. Biochem. J. 18, SKINNER, S. L. (1967). Improved assay methods for renin 'concentration' and 'activity' in human plasma. Circulation Res. 2, VANDER, A. J. (1965). Effect of catecholamines and the renal nerves on renin secretion in anaesthetized dogs. Am. J. Physiol. 29, VANDER, A. J. (1967). Control of renin release. Physiol. Rev. 47, WATHEN, R. L., KINGSBURY, W. S., STROUDER, D. A., SCHNEIDER, E. G. & ROSTORFER, H. H. (1965). Effects of infusion of catecholamines and angiotensin II on renin release in anaesthetized dogs. Am. J. Physiol. 29, WINER, N., CHOKSHI, D. S. & WALKENHORST, W. G. (1971). Effects of cyclic AMP, sympathomimetic amines, and adrenergic receptor antagonist on renin secretion. Circulation Res. 29, WINER, N., CHOKSHI, D. S., YooN, M. S. & FREEDMAN, A. D. (1969). Adrenergic receptor mediation of renin secretion. J. clin. Endocr. Metab. 29,