Injection of adenosine into the renal artery activates spontaneous activity of renal afferent nerve fibers

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1 192 vascular resistance, glomerular filtration rate, renin release, epithelial transport, intrarenal inflammation, and the growth of mesangial and vascular smooth muscle cells [4]. Afferent renal nerve fibers generated from renal sensory receptors carry out physiological functions such as mediating painful stimulation from the kidney, regulation of cardiovascular activity, and renorenal reflex [5, 6]. In addition, via their connections to specialized centers in the brain, the afferent renal nerve fibers are a part of an integrated neurohormonal system that participates in the regulation of body fluid volume and electrolyte homeostasis and the regula- Acta Physiologica Sinica, April 25, 2004, 56(2): Research Paper Injection of adenosine into the renal artery activates spontaneous activity of renal afferent nerve fibers MA Hui-Jie, MA Hui-Juan, LIU Yi-Xian, WANG Qing-Shan * Department of Physiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang Abstract: The effects of injection of adenosine into the renal artery on multi- and single-unit spontaneous discharges of renal afferent nerve fibers were investigated in anesthetized rabbits. The results obtained are as follows: (1) injection of 50, 100, and 200 nmol/kg adenosine into the renal artery increased the renal afferent nerve activity (ARNA) in a dose-dependent manner with unchanged arterial pressure; (2) pretreatment with 8-cyclopenthl-1,3-dipropylxanthine (DPCPX, 160 nmol/kg), an adenosine A 1 receptor antagonist, partly abolished the effect of adenosine; and (3) pretreatment with a nitric oxide synthase inhibitor N ω -nitro-l-arginine methylester (L-NAME, 0.1 mmol/kg) significantly enhanced the ARNA response to adenosine. The results suggest that injection of adenosine into the renal artery activates ARNA via adenosine receptors in anesthetized rabbits and that nitric oxide may be involved in regulating the activity of renal sensory nerve fibers as an inhibitory neurotransmitter. Key words: adenosine; renal afferent nerve; unit-activity *, , : (1) 50, nmol/kg, (2) A 1 DPCPX (160 nmol/kg), (3) L-NAME (0.1 mmol/kg),,, : ; ; ; : Q463 Adenosine, a metabolite of adenine nucleotides, is one of the major neuromodulators [1,2]. It is now generally accepted that adenosine is capable of regulating a wide range of physiological functions [3]. It has been shown that extracellular adenosine exerts its actions via high affinity to the adenosine receptors which are the members of G protein coupled receptor family. Four adenosine receptor subtypes were found and cloned: A 1, A 2A, A 2B and A 3, which are all found in the kidney [4]. Previous studies have shown that adenosine participates in the regulation of preglomerular and postglomerular Received Accepted * Corresponding author. Tel: ; wangqs@ hebmu.edu.cn

2 MA Hui-Jie et al: Adenosine and Renal Afferents tion of arterial pressure [6]. Two classes of renal sensory receptors have been identified neurophysiologically: renal mechanoreceptors responding to increases in intrarenal pressure, and renal chemoreceptors responding to renal ischemia and changes in the chemical environment of the renal interstitium. Two types of renal chemosensitive neurons have been identified, R1 and R2 chemoreceptors [6]. Some of the mechanoreceptors and R2 chemoreceptors have spontaneous discharges under control conditions [6, 7]. There is little information about the mechanisms of adenosine on renal receptors. It has been shown that the function of adenosine may be mediated by the release of nitric oxide (NO) via A 1 receptor in the heart [8-10]. NO has been described as an inhibitory neurotransmitter regulating the activity of renal sensory nerve fibers [11,12]. Our previous studies have shown that intracarotid injection of adenosine can affect the activity of central neurons such as rostral ventrolateral medullary neurons and area postrema neurons [13,14]. All the above-mentioned experimental results promote us to investigate whether injection of adenosine into the renal artery activates the spontaneous activity of renal afferent nerve fibers in anesthetized rabbits. If it does, are adenosine receptors and NO involved in the process? 1 MATERIALS AND METHODS 1.1 General surgical preparation and procedures. Sixtyfive rabbits of either sex, weighing 2.5~3.0 kg, were anesthetized with intravenous sodium pentobarbital (30.0 mg/ kg). Maintenance doses of sodium pentobarbital (2.0~3.0 mg/kg h -1 ) were given intravenously when needed. The trachea was cannulated for ventilation. The right carotid artery was cannulated and connected to a pressure transducer (MPU-0.5, Nihon Kohden), and the signals were fed into a carrier amplifier (AP-620G, Nihon Kohden) for recording blood pressure (BP). 1.2 Intra-arterial injections. After the aorta and the left renal artery were exposed retroperitoneally, the drug was administered into the proximal part of left renal artery using the technique similar to that previously described by Ma et al. [15]. 1.3 Recording of multi-unit activity of the afferent renal nerve. Left postganglionic sympathetic renal nerves were approached retroperitoneally. A branch of left renal nerve was found at the angle between the aorta and the left renal artery and dissected under a dissecting microscope as we have described elsewhere [16,17]. The exposed nerve was cut centrally to prevent efferent input and covered with warm 193 (37ºC) liquid paraffin. Afferent activity was picked from the distal end of the nerve with a bipolar platinum electrode to a biophysical amplifier (AVB-11, band-pass width: 50 Hz~1 khz, Nihon Kohden), the output of which was fed into an integrator (EI-600G, Nihon Kohden) with an integrating time of 1.0 s. The raw afferent renal nerve activity (ARNA) and integrated multi-unit activity of renal afferents were continuously monitored on a polygraph system (RM-6000, Nihon Kohden) with a thermal array recorder (WS-682G, band-pass width: 0~2.8 khz, Nihon Kohden). At the end of the experiment, the nerve was clamped distally for recording the noise level of ARNA. 1.4 Recording of single-unit activity of the afferent renal nerves. A branch of renal nerves was cut proximally and desheathed gently under a dissecting microscope. The desheathed distal end of the nerve was carefully dissected to pick up the single unit. A bipolar platinum electrode connected to a biophysical amplifier (AVB-11, Nihon Kohden) was used to record single-unit activity. The amplified bioelectrical signals were recorded along with BP on a polygraph system with a thermal array recorder. In this experiment, the single-unit afferent nerve discharge was determined by directly counting the number of action potentials from the neurogram. Discharge rate during the control period was determined by averaging the numbers of impulse over at least 60 s. If the rate was irregular or intermittent, a longer period was used. 1.5 Experimental protocols. A period of 15~20 min was allowed for stabilization after the operation and then the ARNA was recorded. The experimental animals were divided into the following groups: In group 1, after a stable recording was obtained, adenosine (50, 100, or 200 nmol/ kg) was administered into the renal artery, and the changes in BP and multi-unit discharge of renal afferent were examined. In group 2, following injection of adenosine into the renal artery (100 nmol/kg), the changes in single-unit discharge along with BP were observed. In group 3, after repeating the experimental protocol for group 2, the adenosine receptor antagonist 8-cyclopenthl-1,3- dipropylxanthine (DPCPX, 160 nmol/kg), was administered into the renal artery, and adenosine (100 nmol/kg) was injected into the renal artery 2 min later. The effect of DPCPX on the actions of adenosine was observed. About 40 min later, when the action of DPCPX disappeared, adenosine was administered again to observe the recovery of the effect. In group 4, after repeating the experimental protocol for group 2, the nitric oxide synthase inhibitor L- NAME (0.1 mmol/kg) was administered intravenously, and

3 194 Acta Physiologica Sinica, April 25, 2004, 56(2): adenosine (100 nmol/kg) was injected into the renal artery 10 min later. About 40 min later, when the action of L- NAME disappeared, the injection of adenosine was repeated. 1.6 Statistics. All data are presented as means±sd. The significance of differences between groups was determined by one-way ANOVA and Student s t test. Statistical significance was accepted when P< RESULTS 2.1 Effects of adenosine on multi-unit discharge of renal afferents (n=30) Injection of adenosine (50, 100, 200 nmol/kg) into the renal artery resulted in a dose-dependent increase in ARNA in 30 rabbits. The effect occurred 1~3 min after the injection, and lasted for 2~6 min. As compared with the control values, 50, 100, and 200 nmol/kg adenosine increased the ARNA to ±7.16%, ±21.85%, and ±23.81%, respectively (P<0.001 vs control), while the mean arterial pressure (MAP) showed no change (Figs. 1 and 2A). 2.2 Effects of adenosine on single-unit discharge of renal afferent (n=23) Injection of adenosine (100 nmol/kg) into the renal artery increased single-unit discharges of renal afferent fibers from 0.19±0.03 to 0.42±0.09 impulse/s (P<0.001). Fig. 1. Histograms showing the increase in multi-unit discharge of renal afferents following injection of different doses of adenosine into the renal artery. * P<0.001 vs control, # P<0.001 vs 50 nmol/kg adenosine, + P<0.001 vs 100 nmol/kg adenosine. The latent period was 1~3 min, and the discharge lasted for 2~5 min in 23 units. The MAP showed no significant change throughout the experiment. The results are shown in Fig. 2B and Table Effects of DPCPX on adenosine responses (n=6) Intravenous administration with DPCPX (160 nmol/ kg) did not affect the baseline single-unit activity, but partly blocked the effects of adenosine on single-unit activity from 0.45±0.04 to 0.29±0.03 impulse/s (P<0.001, n=6) while Fig. 2. Original records showing the responses of multi-unit and single-unit activity of renal afferents and BP to injection of adenosine into the renal artery. A: Responses of multi-unit activity to injection of adenosine (50 nmol/kg) into the renal artery. B: Responses of singleunit activity to injection of adenosine (100 nmol/kg) into the renal artery., injection of adenosine.

4 MA Hui-Jie et al: Adenosine and Renal Afferents 195 Table 1. Effects of adenosine (100 nmol/kg) on MAP and singleunit discharge of renal afferent (n=23) Control Adenosine MAP (mmhg) ± ±8.41 ARNA (impulses/s) 0.19± ±0.09 *** *** P<0.001 vs control. MAP was unaltered (Fig. 3 and Table 2). 2.4 Effects of L-NAME on adenosine responses (n = 6) Intravenous administration of L-NAME (0.1 mmol/ kg) did not affect the single-unit activity, but increased the effect of adenosine in single unit activity from 0.45 ± 0.10 to 0.64 ± 0.09 impulse/s (P<0.001, n=6), and the response of renal afferents to injection of adenosine into the renal artery are also prolonged from 2~5 min to Fig. 3. Original records showing the effect of DPCPX (160 nmol/kg) on the changes in BP and single-unit activity of renal afferent induced by adenosine (100 nmol/kg). A: Effect of adenosine on single-unit activity. B: Effect of DPCPX. C: Effect of adenosine on single-unit activity after DPCPX administration., injection of adenosine., injection of DPCPX. Table 2. Effects of DPCPX (160 nmol/kg) and L-NAME (0.1 mmol/kg) on the changes in MAP and single-unit activity of renal afferent induced by injection of adenosine into the renal artery (100 nmol/kg) (n=6) MAP (mmhg) Single-unit activity (impulses/s) Control After treatment Control After treatment Adenosine 99.67± ± ± ±0.04 *** DPCPX ± ± ± ±0.01 DPCPX+adenosine 99.72± ± ± ±0.03 ***### Adenosine 99.78± ± ± ±0.10 *** L-NAME 99.33± ± ± ±0.02 L-NAME+adenosine 99.39± ± ± ±0.09 ***## *** P<0.001 vs control. ## P<0.01 vs adenosine. ### P<0.001 vs adenosine.

5 196 Acta Physiologica Sinica, April 25, 2004, 56(2): Fig.4. Original records showing the effect of L-NAME (0.1 mmol/kg) on the changes in BP and single-unit activity of renal afferent induced by adenosine (100 nmol/kg). A: Effect of adenosine on single-unit activity. B: Effect of intravenous administration of L-NAME. C: Effect of adenosine on single-unit activity after L-NAME administration., injection of adenosine., injection of L-NAME. 3.5~7.5 min (Table 2 and Fig. 4). 3 DISCUSSION The present study demonstrated that injection of adenosine into the renal artery significantly activated the ipsilateral ARNA in a dose-dependent manner in anesthetized rabbits. Available studies indicate that four subtypes of adenosine receptors, A 1, A 2A, A 2B and A 3, have all been found in the kidney [4]. Adenosine easily gets to the tissues where its receptors exit, because many cell types contain bi-directional nucleoside transporters that facilitate diffusion of adenosine [18]. We hypothesized that the increase in ARNA induced by adenosine might be mediated by adenosine receptors. Pretreatment with the selective A 1 adenosine receptor antagonist DPCPX partially attenuated the effect of adenosine. This result indicates that adenosine may stimulate the renal sensory terminals through several subtypes of adenosine receptors including A 1 receptor. The underlying mechanisms are probably: (1) vasoconstrictor effect of adenosine on renal vessels; and (2) activation of renal afferent afferent fibers by adenosine in renal pelvis. Experiments in laboratory animals clearly show that adenosine acts as a vasoconstrictor metabolite in the kidney [19]. Thus adenosine may increase the renal hydrostatic pressure, enhance the stimuli acting on the renal mechanoreceptors, and then increases the renal afferent nerve discharges [20]. It has been shown that adenosine-sensitive nerve endings are located within the renal pelvis [21]. To further determine whether NO is involved in this process, L-NAME, a NO synthase inhibitor was administered intravenously. Pretreatment with L-NAME did not alter the baseline of ARNA, but prolonged and increased the response of renal afferents to the injection of adenosine into the renal artery. Increasing evidence has been accumulated to suggest that NO is an inhibitory factor of renal afferents [11,12]. It has been demonstrated that the effect of adenosine may be mediated by the release of NO via A 1 receptor in the heart as we described previously [8-10]. In vascular smooth muscle cells, adenosine may also contribute to the increase in NO synthesis via activation of A 2B adenosine receptors [22]. We hypothesized that when adenosine was administered into the renal artery, both activating factors of the renal afferents and inhibiting ones are elicited, but the former is predominant. However, when L-NAME was administered intravenously, the inhibitory factor by NO was abolished, so the subsequent application of adenosine resulted in an increased and prolonged activation of ARNA. Since the MAP showed no significant changes throughout the experiment, the possibility that the response of renal afferents was secondary to BP disturbance might be

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