Acta Physiologica Sinica,   April  25, 2003, 55(2): 225-231

Received 2002-08-15

Accepted 2002-10-10

Corresponding author. Tel: +86-311-6062490;  Fax: +86-311-6062490;   E-mail: syho@hebmu.edu.cn

 

 Research  Paper

Intrarenal artery injection of L-arginine inhibits  spontaneous activity of renal afferent nerve fibers

MA Hui-Juan, LIU Yi-Xian, WU Yu-Ming, HE Rui-Rong*

Department of Physiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017

 

Abstract:  The purpose of this study was to determine the effect of intrarenal artery injection of L-arginine on multi- and single-unit spontaneous discharges of renal afferent nerve fibers in anesthetized rabbits. The results obtained are as follows: (1) intrarenal artery injection of L-arginine (0.05, 0.24, and 0.48 mmol/kg) decreased the renal afferent nerve activity (ARNA)  in a dose-dependent manner with arterial pressure unchanged; (2) pretreatment with a nitric oxide synthase inhibitor L-NAME (N6-nitro-L-arginine methylester, 0.11 mmol/kg), completely abolished the effect of L-arginine;  and (3) intrarenal artery injection of a nitric oxide donor SIN-1 (3-morpholinosydnonimine, 3.75 ¦Ìmol/kg) also resulted in an inhibition of ARNA. The results suggest that intrarenal artery injection of NO precursor (L-arginine) and donor (SIN-1) can inhibit ARNA in anesthetized rabbits.

 

Key words: physiology; L-arginine; unit-activity; renal afferent nerve

 

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 L-arginine, a nitric oxide (NO) precursor, can generate NO by NO synthases (NOS) in many systems. NO is an important signaling and effector molecule that plays critical roles in numerous essential physiological processes in virtually every organ[1]. It is not only a regulator of vascular tone but also a neuromodulator in the central and peripheral nervous system. There are three different types of NOS, referred to as neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), which have all been found in the kidney[1,2]. Previous studies have shown that L-arginine influences the regulation of renal hemodynamics and  glomerular microcirculation in isolated perfused  kidney[1,3]. At the same time NO is known to affect vasomotor responses of both pre-glomerular and post-glomerular arterioles[2]. In addition, NO functions as an inhibitory neurotransmitter regulating the activity of renal afferent arteriole in rats[3]. The renal afferent  nerve fibers are  generated from   the renal sensory receptors. The study of renal afferent fibers most likely began with the work of  Pines on cats[4]. Since then 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[5].Four patterns of the single-unit discharge have been recorded from the renal afferents: (1) no spontaneous discharge; (2) regular spontaneous discharge; (3) spontaneous discharge in bursts, and (4) irregular discharge[6,7]. The aims of the present study were to examine the effects of NO precursor L-arginine and NO donor SIN-1 on spontaneous activity of afferent renal fibers and the possible underlying mechanism in anesthetized rabbits. The multi- and single-unit spontaneous activities of afferent renal nerve were adopted to observe the response.

 

1  MATERIALS AND METHODS

1.1 General surgical preparation and procedures. Fifty-two rabbits of either sex, weighing 2.5-3.5 kg, were anesthetized with intravenous pentobarbital sodium (30.0 mg/kg). Maintenance doses of pentobarbital sodium (2.0-3.0 mg/kg¡¤h-£±) 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). A polyethylene catheter was advanced into the proximal part of the left renal artery for drug administration.

1.2 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[8]. The exposed nerve was cut centrally to prevent efferent input and covered with warm (37¡æ) 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 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.

1.3 Recording of single-unit activity of the afferent renal nerve. A branch of renal nerve was cut proximally and desheathed gently under a dissecting microscope. The desheathed distal end of nerve was carefully dissected to pick up the single unit. A mono-polar 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 potential 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.

1.4 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: group 1, in which after a stable recording was obtained, L-arginine (0.05, 0.24 or 0.48 mmol/kg) was administered into the renal artery, and the changes in BP and multi-unit discharge of renal afferent were examined; group 2,  in which following intrarenal L-arginine (0.24 mmol/kg), the changes in single-unit discharge along with BP were observed;   group 3,  in which after repeating the experimental protocol for group 2, the NOS inhibitor N6-nitro-L-arginine methylester (L-NAME, 0.11 mmol/kg) was administered intravenously, and L-arginine (0.24 mmol/kg) was injected intrarenally 10 min later. About 40 min later, when the action of L-NAME disappeared, L-arginine (0.24 mmol/kg) was administered again to observe the recovery of the effect.   In  group 4,    after a stable recording of single-unit discharge of renal afferent and BP, an NO donor, 3-morpholinosydnonimine (SIN-1, 3.75 ¦Ìmol/kg) was injected into the renal artery, and the changes in BP and single-unit discharge of renal afferent were observed.

1.5 Drugs.   L-arginine (Sigma) and L-NAME (Sigma) were dissolved in normal saline. SIN-1 (Cassella, Germany) was dissolved in a acidified solution (normal saline plus 0.1 mol/L hydrochloric acid in a volume ratio 10¡Ã1.25).

1.6 Statistics.   All data are presented as means¡ÀSD. The significance of group differences was determined by one-way ANOVA or unpaired t test. Statistical significance was accepted when P<0.05.

 

2  RESULTS

2.1 Effects of L-arginine on multi-unit discharge of renal afferents

Injection of L-arginine (0.05, 0.24 or 0.48 mmol/kg) into the renal artery resulted in a dose-dependent decrease in ARNA in 30 rabbits. The effect occurred 1-3 min after the injection of L-arginine, and lasted for 2-6 min. As compared with the control values, L-arginine (0.05, 0.24, 0.48 mmol/kg) decreased the ARNA to 83.58¡À2.06%, 37.29¡À4.10%, and 15.33¡À2.70%, respectively (P<0.001 vs control), while mean arterial pressure (MAP) showed no change  (Figs. 1 and 2). Intrarenal normal saline (n=5) had no significant effects on ARNA.

Fig.1.Original recording showing the responses of multi-unit activity of renal afferents and BP to intrarenal L-arginine (0.48 mmol/kg).    ¡ý,  injection of L-arginine.

Fig.2.Histograms showing the decrease in multi-unit discharge of ARNA following the intrarenal L-arginine of different doses.  *P<0.001 vs control, #P<0.001 vs L-arginine (0.24 mmol/kg) and L-arginine (0.48 mmol/kg), +P<0.001 vs L-arginine (0.48 mmol/kg).

2.2 Effects of L-arginine on single-unit discharge of renal afferents

Intrarenal injection of L-arginine (0.24 mmol/kg) decreased single-unit discharge of ARNA from 0.19¡À0.05 to 0.07¡À0.02 impulse/s (P<0.001), lasting 3.8¡À0.3 min in 12 units from 10 rabbits. The MAP showed no significant change throughout the experiment. The results are shown in Fig. 3 and Table 1.

2.3 Effects of L-NAME on L-arginine responses 

 Intravenous administration with L-NAME (0.11 mmol/kg) did not affect the  single-unit afferent renal nerve activity, but completely blocked the effects of L-arginine on ARNA while MAP was unaltered (Fig.4 and Table 2).

 

Table¡¡£±.Effects of L-arginine (0.24 mmol/kg) and SIN-1 (3.75 ¦Ìmol/kg) on MAP and   single-unit activity of renal afferent

	n	MAP  (mmHg)		ARNA  (impulses/s)
		Control	After treatment	Control	After treatment
L-arginine	12	100.05¡À6.42	99.86¡À6.07	0.19¡À0.05	0.07¡À0.02***
SIN-1	6	100.12¡À2.55	99.95¡À2.35	0.19¡À0.06	0.05¡À0.01***

***P<0.001 vs control.

Fig.3.Original records showing the effects of intrarenal injection of L-arginine (0.24 mmol/kg) and SIN-1 (3.75 ¦Ìmol/kg) on BP and single-unit activity of renal afferent.   A:  Effect of L-arginine on ARNA.  B:  Effect of SIN-1 on ARNA.  ¡ý,  injection of L-arginine;   -¡ý,  injection of SIN-1.

 

2.4 Effects of SIN-1 on single-unit discharge of renal afferents

 Intrarenal artery injection of SIN-1 (3.75 ¦Ìmol/kg) significantly decreased the single-unit discharge of renal afferents from 0.19¡À0.06 to 0.05¡À0.01 impulse/s (P<0.001) without any changes in  MAP (Fig. 3, Table 1). The inhibition occurred immediately after SIN-1 administration, and lasted for 5.1¡À0.3 min. The vehicle (acidified normal saline) of SIN-1 had no significant effects on ARNA (n=5).

Table¡¡2.Effects of L-NAME (0.11 mmol/kg) on the changes in MAP and single-unit activity of renal afferent induced by intrarenal L-arginine (0.24 mmol/kg)

	MAP  (mmHg)		ARNA  (impulses/s)
	Control	After treatment	Control	After treatment
L-arginine	99.45¡À4.81	99.38¡À3.92	0.19¡À0.05	0.07¡À0.02***
L-NAME	99.45¡À4.81	100.71¡À3.77	0.18¡À0.05	0.18¡À0.04
L-NAME+L-arginine	100.72¡À3.77	100.17¡À3.60	0.18¡À0.04	0.18¡À0.04
L-arginine	100.17¡À3.60	99.5¡À3.08	0.18¡À0.04	0.07¡À0.01***

n=6. ***P<0.001 vs control.

Fig.4.Original records showing the effect of L-NAME (0.11 mmol/kg) on the changes in  BP and single-unit activity of renal afferent induced by L-arginine (0.24 mmol/kg).  A: Effect of L-arginine on ARNA. B: Effect of intravenous administration of L-NAME.   C: Effect of L-arginine on ARNA after L-NAME administration.  ¡ý,  injection of L-arginine;     -¡ý, injection of L-NAME.

 

3  DISCUSSION

   The present study demonstrates that intrarenal L-arginine significantly inhibits ipsilateral ARNA in a dose-dependent manner in anesthetized rabbits. Considering that all three types of NOS are located in the kidney, we hypothesized that the decrease in ARNA might be due to the action of NO generated from L-arginine by NOS. Pretreatment with the NOS inhibitor L-NAME could completely abolish the effect of L-arginine. This result is in accordance with our hypothesis. To further determine this hypothesis, a NO donor SIN-1 was injected into the renal artery and exhibited the similar effect to that of L-arginine.  It is therefore concluded that the inhibitory effect of L-arginine on ARNA is due to NO generated form L-arginine by NOS in the kidney.

   With regard to the underlying mechanism, it is reasonable to suggest that the vasodilating action of NO might be involved. NO acting as a vasodilator may reduce the renal microcirculation pressure, weaken the stimuli acting on the renal mechanoreceptors, and then decrease the renal afferent nerve discharges. In our experiments,  L-arginine injected into the renal artery can generate NO via eNOS which is the predominant isoform involved in the regulation of renal blood flow under basal conditions[9]. It is reported that NO might induce vasorelaxation through two different pathways: cGMP-dependent and cGMP-independent mechanisms. NO diffuses rapidly through the interstitial space to activate the soluble guanylate cyclase of the adjacent smooth muscle cells[10], maintaining high levels of cGMP[11]which in turn activates protein kinase G that reduces Ca2+ influx. Consequently, low intracellular Ca2+ may result in a relaxation of vascular smooth muscle[12]. Also, NO can induce vasodilation by activating large conductance Ca2+-activated K+ channels in smooth muscle cells via cGMP in rabbit coronary artery [13]. As to the cGMP-independent mechanisms, NO has been shown to directly affect ion channel activity such as activating large conductance Ca2+-activated K+ channels in rabbit aortic smooth muscle cells[14] and smaller K+ channels in colonic smooth muscle[15], and inhibiting voltage-dependent Ca2+ channels in the preglomerular microvasculature of  kidney in rabbits[12]. In addition, recent studies have demonstrated that the relaxant effects of the SIN-1 on the rat mesenteric artery were relatively insensitive to cGMP[16].In our study, NO generated from L-arginine or SIN-1, worked as an exogenous factor, might elicit renal arteriolar vasodilation through both cGMP-dependent and cGMP-independent mechanisms[2]. 

   It is demonstrated that NO is released from the nerve endings and diffuses toward its target cells instead of being stored and released from the synaptic vesicles[2, 10]. In the present study exogenous NO, as a liposoluble molecule, may diffuse to the sensory nerve terminals and function  like a neurotransmitter. NO is involved in the desensitization of the sensory nerve via cGMP[10]. And it has been reported NO  mediates hyperpolarization of the cell membrane by decreasing sodium current in dorsal root ganglion neurons[17]. Furthermore,  endogenous NO as well as exogenously added NO donors have been shown to inhibit tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ currents in the baroreceptor neurons[18]. Interestingly, NO can also deactivate the renal pelvic mechanosensory nerves by a mechanism related to substance P receptors[2]. Taken together, all the above-mentioned physiological actions of NO may contribute to the decrease of ARNA in our study. Since the MAP showed no significant changes after intrarenal L-arginine or SIN-1, the possibility that the response of renal afferents secondary to BP disturbance might be ruled out.

   Renal afferent  inputs participate in the reflex control system. It contributes significantly to the neural regulation of cardiovascular system[5, 19]. The changes in ARNA produce renorenal reflex that enables renal function to be self-regulated and balanced between the two kidneys[5], and can induce the release of vasopressin[20], cortisol[21] and atrial natriuretic peptide[22].

   In summary, our work has revealed that intrarenal artery injection of L-arginine can inhibit ARNA, which is due to the generation of NO via NOS.

 

REFERENCES

 

[1]Kone BC, Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol 1997;272(5 Pt 2):F561-F578.

[2]Kopp UC, Cicha MZ, Smith LA,  H¢‰kfelt T. Nitric oxide modulates renal sensory nerve fibers by mechanisms related to substance P receptor activation.  Am J Physiol  2001;281:R279-R290.

[3]Greg T, Triggle CR, O¡äNeill SK, Loutzenhiser R.  Cyclic GMP-dependent and cyclic GMP-independent action of nitric oxide on the renal afferent arteriole.  Brit J Pharmacol 1998;125:563-569.

[4]Pines IL.  Electrophysiological investigation on the mechanoreceptors in the intrarenal vessels.  Fiziol Zh SSSR Im  I M Sechenova 1959;45:1339-1347.

[5]Dibona GF, Kopp UC.  Neural control of renal function.  Physiol Rev 1997; 77:75-197.

[6]Ma G (Âí¸ê), Ho SY.  Observation on the afferent nerve activity induced by stimulation of renal receptors in the rabbits.  Acta Physiol Sin (ÉúÀíѧ±¨), 1990, 42(3):269-276 (Chinese,    English abstract).

[7]Moss NG. Renal function and renal afferent and efferent nerve activity. Am J Physiol 1982; 243:F425-F433.

[8]Wu YM (ÎäÓîÃ÷), Ho SY. Biphasic activation of renal afferent by intrarenal artery injection of bradykinin in  anesthetized rabbits. Acta Physiol Sin (ÉúÀíѧ±¨) 1999;51(6):651-659.

[9]Kakoki M, Zou AP,  Mattson DL. The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney.  Am J  Physiol  2001;281:R91-R97.

[10]Summers BA, Overholt JL, Prabhakar NR.  Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid.  J Neurophysiol 1999;81:1449-1457.

[11]Brophy CM, Knoepp L, Xin JD, Pollock JS.  Functional expression of NOS 1 in vascular smooth muscle. Am J Physiol  2000;278:H991-H997.

[12]Kramp RA, Fourmanoir P, Ladri¨¨re L, Joly E, Gerbaux C, Hajjam A, Caron N.  Effects of Ca2+ channel activity on renal hemodynamics during acute attenuation of NO synthesis in the rat. Am J Physiol 2000; 278:F561-F569.

[13]Li P, Zon A, Cambell WB.  Regulation of potassium channels in coronary arterial smooth muscle by endothelium-derived vasodilators.  Hypertension 1997;29:262-267.

[14]Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA.  Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 1994;368:850-853.

[15]Koh SD, Campbell JD, Carl A, Sanders KM.  Nitric oxide activates multiple potassium channels in canine colonic smooth muscle.  J Physiol 1995;489:765-743.

[16]Plane F, Hurrell A, Jeremy JY, Garland CJ.  Evidence that potassium channels make a major contribution to SIN-1-evoked relaxation of rat isolated mesenteric artery.  Br J Pharmacol 1996;119:1557-1562.

[17]Renganathan M, Cummins TR, Hormuzdiar WN, Black JA, Waxman SG. Nitric oxide is an autocrine regulator of Na+ currents in axotomized c-type DRG neurons.  J Neurophysiol  2000;83:2431-2442.

[18]Li Z, Chapleau MW, Bates JN, Bielefeldt K, Lee HC, Abboud FM.  Nitric oxide as an autocrine regulator of sodium currents in baroreceptor neurons.  Neuron 1998;20:1039-1049.

[19]Ma G (Âí ¸ê), Ho SY. Hemodynamic effects of renal interoreceptor and afferent nerve stimulation in rabbit.  Acta Physiol Sin (ÉúÀíѧ±¨)  1990;42(3):262-268 (Chinese, English abstract).

[20]Shiotsu T, Yamamoto A, Kagawa S, Tamaki T, Abe Y, Reid IA.  Effect of moderately increased intrapelvic pressure on renal tissue pressure and vasopressin release in rabbits.  Hypertens Res 1995;18(3):197-202.

[21]Zhan CD  (Õ²²ýµÂ), Pan JY. Effect of stimulation of renal afferent nerves on plasma cortisol concentrations.  Acta Physiol Sin (ÉúÀíѧ±¨) 1993;18(3):305-309  (Chinese,  English abstract).

[22]Antunes-Rodrigues  J, Machado  BH,   Andrade  HA, Mauad H, Ramalho MJ, Reis LC, Silva-Netto CR, Favaretto AL, Gutkowska J, McCann SM. Carotid-aortic and renal baroreceptors mediate the atrial natriuretic peptide release induced by blood volume expansion. Proc Natl Acad Soc USA  1992;89:6828-6831.