ELSEVIER Neuroscience Research 24 (1996) 195 199

Rapid communication

The nitric oxide synthase inhibitor L-NAME reduces inhibitory components of somato-vesical parasympathetic reflexes in the rat

John F.B. Morrison”, Akio Sato*b, Yuko Satoc, Atsuko Suzukib aDepartment of Physiology, University of Leeds, Lee& LSA 9JT, UK

bDepartment of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho. Itabashi-ku, Tokyo 173, Japan ‘Laboratory of Physiology, Tsukuba College of Technology, Tsukuba 305, Japan

Received 6 July 1995; accepted 10 November 1995

Abstract

Reflex discharges of pelvic postganglionic parasympathetic efferent fibers on the bladder surface induced by afferent volleys in the hindlimb nerve have been recorded in anesthetized rats, and the effects of the nitric oxide synthase inhibitor, iVw-nitro-L- arginine methyl ester (L-NAME) on the reflex discharges have been investigated. Single electrical stimulation of the tibia1 nerve at intensities supramaximal for excitation of A- and C-afferents evoked a reflex discharge in the postganglionic parasympathetic efferents with four distinct components, i.e., two inhibitory components with latencies of 49 and 203 ms, respectively, and two ex- citatory components with latencies of 126 and 308 ms, respectively. These reflexes could be observed when the bladder was expand- ed, but not markedly when the bladder was empty. Intravenous administration of L-NAME resulted in (a) a reduction in the level of resting discharge, (b) a reduction in the size of the first inhibitory component, (c) the disappearance of the second inhibitory component and (d) the exaggeration of the late excitatory component. Intracisternal injection of L-NAME caused changes similar to those observed following intravenous injection. The results suggest that inhibitory components of the somato-pelvic parasym- pathetic reflex are mediated by pathways that utilize nitric oxide as a neurotransmitter or neuromodulator at the level of the brainstem.

Keywords: Pelvic nerve; Bladder; Somatic afferent; Somato-parasympathetic reflex; Nitric oxide; L-NAME; Anesthetized rat

There has been much interest in the role of nitric oxide (NO) as a neuromodulator in recent years (Knowles et al., 1989; Bredt and Snyder 1992; Morris et al., 1992). NO is known to have act as neuromodulator in nociception and nociceptive reflex transmission (Meller et al., 1992; Kolhekar et al., 1993; Coderre and Yashpal, 1994), in somato-sympathetic reflexes (Li et al., 1995), and in the peripheral autonomic system (Du et al., 1992; Sanders and Ward, 1992), including the urinary tract (Andersson et al., 1992; Vizzard et al., 1993). Its role in the central nervous system is of great

l Cor&ponding author, Tel.: +81 3 3964 3241 ext. 3087; fax: +81 3 3964 1415.

interest particularly in the physiology of the central effects of stimulation of afferent fibers. NO appears to be involved in the spinal tail flick reflex in response to a nociceptive thermal stimulus; the facilitation of this reflex by N-methyl-D-aspartate can be blocked by the intrathecal administration of NO synthase inhibitor (NOS), No-nitro+arginine methyl ester (L-NAME); however, other behavioral components of the reflex with supraspinal loops are unaffected by the blockade of NOS (Kolhekar et al., 1993). Li et al. (1995) found that a somato-cardiac supraspinal sympathetic C-reflex, in- itiated by electrical stimulation of C-fiber afferents of a tibia1 nerve, was facilitated by intracisternal administra- tion of L-NAME, indicating a role of NO in the sup- pression of this sympathetic reflex;. in this work there

0168-0102/96/%15.00 0 1996 Elsevier Science lreland Ltd. All rights reserved SSDI 0168-0102(95)00993-4

196 J. F. B. Morrison et al. /Neuroscience Research 24 (19%) 195- 199

was good evidence for a NO-dependent mechanism within the brainstem.

The present work utilizes a somatically induced parasympathetic reflex mechanism that has not been previously described in the rat, but is well known in the cat (Sato et al., 1983). In the cat with the central nervous system intact, distinct inhibitory and excitatory reflex components were identified in the pelvic vesical bran- ches after single or short tetanic electrical stimulation of the hindlimb nerves, and these somato-pelvic parasym- pathetic reflexes were modulated by bladder pressure (Sato et al., 1983). In the present experiments, the sym- pathetic innervation to the bladder, in the hypogastric nerves and lumbar sympathetic chains, was sectioned, so that the pelvic postganglionic fibers recorded from bundles dissected from the bladder, were purely parasympathetic in origin (Kuo et al., 1984). The tibia1 nerve, whose afferent fibers enter the spinal cord rostra1 to the parasympathetic outflow, were stimulated at strengths supramaximal for A- and C-fibers excitation, and the evoked activity in the postganglionic parasym- pathetic efferents to the bladder was recorded.

The paper describes a study of the effects of NOS in- hibition on the reflex responses in a somato-pelvic parasympathetic reflex: the experiments were performed to see whether NOS inhibition might alter the inhibitory and excitatory reflex components seen in the pelvic parasympathetic efferents following tibia1 afferent nerve stimulation.

Twelve female Wistar rats weighing 170-230 g (aged 3- 10 months) were anesthetized using solutions of ure- thane (1.1 g/kg, i.p.). Jugular venous, carotid arterial and tracheal cannulae were inserted, and the animal was artificially ventilated. Supplements of maintenance doses of 110 mg/kg urethane (i.v.) were given as re- quired, as judged by changes in arterial pressure, heart rate and the onset of a flexor withdrawal reflex to pin- ching. The other experimental arrangement was similar to that described previously by Morrison et al. (1995). A laparotomy was performed and the bladder was ex- posed and kept moist with warm saline. A catheter was inserted into the bladder via the urethra, and in- travesical pressure was recorded continuously on the polygraph (RM-6000, Nihon-Kohden, Tokyo). The hypogastric nerves and the lumbar sympathetic chains were sectioned bilaterally.

Efferent multiunit activity was recorded from postganglionic filaments of the pelvic nerve dissected from the surface of the bladder, using bipolar platinum-iridium wire electrodes and a preamplifier with a time constant of 0.01 s (S-0476, Nihon-Kohden). The tibia1 nerve on one side was cut and prepared for stimulation at 1 Hz using pulses of 20 V (supramaximal for A- and C-fibers) and 0.5 ms. Post-stimulus time histograms were constructed following stimuli applied to the tibia1 nerve using a computer (ATAC-3700,

Nihon-Kohden) for 60-100 trials at 1 s intervals. Each address advance of post-stimulus time histograms was 2-6 ms. The effects of a slow infusion of isotonic saline into the bladder (l-4 ml/h) were also studied, to test the modulation of the evoked responses by bladder filling.

L-NAME (Sigma, USA) was dissolved in saline, in a dose of 30 mg/kg for intravenous injection, and in a dose of 10 pg for intracisternal injection. The method of injection into the cisterna magna was same as that used by Li et al. (1995). D-NAME (No-nitro-D-arginine methyl ester, Sigma) and L-arginine (Wako Pure Chemicals, Osaka) were also dissolved in saline, in a dose of 30 mg/kg and 300 mg/kg, respectively, for intra- venous injection. The reflex responses at high bladder pressure, maintained when necessary by very slow infu- sion of saline into the bladder were then studied con- tinuously following the administration of drugs.

When the bladder was empty the pelvic post- ganglionic parasympathetic fibers were silent, but they could be recruited into activity by bladder distension, as was found previously (Mallory et al., 1989; Morrison et al., 1995). The threshold pressure of the bladder distend- ed for this response was around 100 mmH*O, and a maximum response could be seen at intravesical pressures of greater than 300 mmH*O, or during micturition contractions.

The present postganglionic parasympathetic nerve revealed tonic efferent discharge activity depending on bladder condition, whether expanded or not; there was more activity when expanded. This activity is presented as a baseline level before stimulation, as shown in Fig. 1A. Stimulation of the tibia1 nerve at 20 V evoked a reflex discharge in the postganglionic parasympathetic efferents with four distinct components when the blad- der was expanded. Fig. 1A shows an example recording. The initial response was always a depression of baseline activity refered to as I1 in Fig. 1C inset, at a latency of 49 f 3 ms (mean f S.E.M.). This early, initial in- hibitory component (11) was followed by an excitation, refered to as El in Fig. lC, at a latency of 126 f 4 ms. The initial excitatory component (El) usually did not exceed the baseline level. This initial excitatory compo- nent was followed by a second period of depression at a latency of 203 f 5 ms (refered to as 12 in Fig. lC), and this was terminated by a second period of excitatory component with a latency of 308 f 6 ms (refered to as E2 in Fig. 1C). This second excitatory component (E2) was elevated above the baseline level in 8 of 12 experi- ments. When the bladder was empty, these reflexes could not be observed markedly. The El, E2, I1 and 12 components became visible at about 100 mmH20 of the bladder pressure, and were increased when intravesical pressure was further elevated.

Intravenous administration of L-NAME resulted in a change in the resting activity of the pelvic postganglionic parasympathetic nerve: the nerve became less active,

J.F.B. Morrison et al. /Neuroscience Research 24 (19%) 195-199 191

imp/s A before L-NAME 300-

200-

B after L-NAME

0 3oo 6oo goo 300 600 900 ms 4

200r I A

- - : f

11 El 12 E2

Fig. 1. Effects of the intravenous administration of L-NAME (30 mgikg) on the somato-pelvic parasympathetic reflex responses evoked by 20 V stimulation at high intravesical pressure. A and B: post- stimulus time histograms with 6 ms/bin width of parasympathetic nerve activity before (A) and 30 min after (B) the intravenous adminis- tration of L-NAME. C: the histograms show the changes in the size of the inhibitory and excitatory responses before and after intravenous administration of L-NAME, when intravesical pressure was greater than 300 mmH20. The sizes of the reflex responses were measured by the area of the response as shown in inset diagram. The sizes of one response (consisting of 60 trials) before L-NAME administration was expressed as iOO%, and another reflex response just before administra- tion and the reflex response after L-NAME were expressed as percen- tages of the control values. The bars indicate the standard errors of the mean (n = 4). *P < 0.05, **P < 0.01; significantly different between the reflex sixes before and after L-NAME by Student’s t-test. It can be seen that the baseline activity has diminished following L-NAME, and that the inhibitory potentials II and I2 are reduced, particularly 12.

and although an increase in bladder pressure did cause an increase in resting activity, the absolute level was less than previously observed. The resting activity in the pel- vic nerve when the intravesical pressure was greater than 300 rnmH20, was reduced to 64 f 4% of the control baseline activity 20-40 min after the intravenous admin- istration of L-NAME (tested in four animals, P < 0.05).

In four experiments intravenously administered L- NAME caused a change in the reflex responses. This change began within 10 min, reached its maximum at 20 min and remained for 60 min after the injection: 11 was reduced in size, which was possibly not surprising, given that the baseline discharge was reduced (Fig. 1B). The area of I1 following L-NAME administration was 31% of the control response before L-NAME administra- tion. 12 usually disappeared, i.e. was replaced by a period of discharge at the same rate as the baseline activ- ity preceding the stimulus. The average area of the 12 re-

sponse was only 11% of the control response (Fig. 1C). The inhibitory components could not be observed even when resting discharges were increased by increasing bladder pressure. El was reduced to 85% of the control response. At the same time the period of rapid recovery from 12 appeared to merge with E2, which became exag- gerated in size and was superimposed above the baseline activity. E2 increased to 162% of the control response (Fig. 1C).

The dependence of E2 on intravesical pressure was examined in two animals. E2 with a latency of about 3 10 ms was generally absent at low intravesical pressures, and became larger at high pressure (Fig. 2). The threshold pressure of the bladder for this reflex response (E2) was variable at 50-200 mmH,O, and the reflex re- sponse became maximal at vesical pressure of 300-500 mmHzO. These pressure values were virtually unchang- ed from the controls before administration of L-NAME.

In one experiment L-arginine (300 mg/kg) was given intravenously in attempts to reverse the effects of L- NAME, but no reversal of the effect was seen. The ef- fects of D-NAME (30 mg/kg, i.v.) were studied in two other experiments, the control recordings were similar to those described above, and intravenous administra- tion of D-NAME had no effect on the reflex responses.

In three experiments, L-NAME was administered to the cisterna magna, and its effect on the somatically in- duced pelvic parasympathetic reflexes was examined. In- tracisternal injection of L-NAME caused changes in the reflex responses similar to those observed following in- travenous injection of L-NAME. The resting activity in the pelvic postganglionic parasympathetic efferent nerve

E2 response

100

F F - control

: ,i/qiy , , ,

0 100 200 300 400 500

vesical pressure (mmH20)

Fig. 2. The graph shows the changes in the sizes of the E2 responses induced by changing intravesical pressure before (solid line) and after (broken line) intravenous administration of L-NAME (30 mgikg). The sixes of E2 responses were expressed as percentages of the maximum response at 300-500 mmHzO. Note that 100% after L-NAME is not the same absolute value as before L-NAME (see Fig. 1). Data of two experiments were shown.

198 J.F.B. Morrison et al. /Neuroscience Research 24 (19%) X95-199

was reduced, and 11 and I2 were diminished, while E2 was augmented. The response of El was not clear.

In the present work, there were four components in the somato-vesical parasympathetic reflexes of rats; early inhibitory (11) and excitatory (El) components and late inhibitory (12) and excitatory (E2) components. These reflexes were easily demonstrable in the vesical branches of the pelvic nerve when pressure in the blad- der was elevated by expanding the bladder, and their amplitude was dependent on bladder pressure over the ranges of 100-500 mmH20. The dependence of the somato-vesical parasympathetic reflexes on the bladder pressure was also observed in anesthetized cats (Sato et al., 1983).

In the present work, the somato-vesical parasym- pathetic inhibitory reflex components were reduced by the intravenous administration of L-NAME, but not by its inactive isomer D-NAME. Attempts were made to reverse the effect by intravenous administration of L-

arginine; however the effects were not reversed by this treatment. Despite this, we suggest that NO pathways are involved in the mediation of the inhibitory phenom- ena in the somato-parasympathetic reflex used in these experiments. The E2 response was unmasked by treat- ment with L-NAME, probably as a result of the loss of these inhibitory influences. Another effect of L-NAME was the reduction in resting activity in the pelvic parasympathetic efferent nerve. A part of the reduction in the size of inhibitory components of the reflex re- sponse may be due to the reduction in resting activity (to 64% after intravenous administration of L-NAME), but the gross reduction of evoked inhibitory events after in- travenous administration of L-NAME suggests that, in addition, a descending’inhibitory pathway is functional- ly interrupted by the drug.

Li et al. (1995) studied the effect of L-NAME on the A- and C-reflexes in the cardiac sympathetic efferent nerves following stimulation of the tibia1 afferent nerve in rats, and found that L-NAME augmented the C-fiber somato-cardiac sympathetic reflex (C-reflex). Its action appears to be at the level of the brainstem, because in- tracisternal injection of L-NAME mimicked the effect of intravenous injection.

The present experiments on the pelvic parasym- pathetic reflexes with intracisternal administration of L-

NAME indicate that the action of this drug can be ex- plained by interference with functional connections within the brainstem: the reflexly evoked depressions of pelvic parasympathetic efferent nerve activity are reduced by the drug, as is the resting activity.

The descending pathways concerned with these effects have not been identified, but it is known that, in the cat, descending pathways from the medullary raphe nuclei and nucleus reticularis gigantocellularis have antino- ciceptive effects on nociceptive transmissions in the dor- sal horn, and depress the activity of parasympathetic

reflexes to the bladder (McMahon and Spillane 1982; McMahon et al., 1982). It is not known whether these descending pathways have nitroxergic inputs.

NO plays a part in nociceptive behavior; for example, nociceptive responses to injections of formalin are abolished by intrathecal pre-treatment with inhibitors of NOS, and the effect is reversed by administration of substrates of NOS (Moore et al., 1991; Malmberg and Yaksh, 1993; Yamamoto et al., 1993; Coderre and Yashpal, 1994). The present result adds to the growing view that NO acts as a neuromodulator or neurotrans- mitter in the central nervous system. The significance of the present study is that NO-dependent pathways appear to contribute to the somato-parasympathetic reflex at the level of the brainstem.

Acknowledgments

J.F.B.M. would like to thank Tokyo Metropolitan In- stitute of Gerontology for a visiting Professorship, and gratefully acknowledges the support of the Welcome Trust in providing a Study Leave Fellowship. This work was supported by the research fund from the Traditional Oriental Medical Science Programs of the Public Health Bureau of the Tokyo Metropolitan Government and by a Grant-in-Aid for Scientific Research (C, No. 06680820 to Y.S.) from the Ministry of Education, Science and Culture of Japan.

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