- Email: [email protected]
Nitric oxide-dependent and -independent neurogenic relaxation of isolated dog urethra
European Journal of Pharmacology, 231 (1993) 209-214
209
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 52893
Nitric oxide-dependent and -independent neurogenic relaxation of isolated dog urethra Shigeki H a s h i m o t o , S h i g e r u Kigoshi a n d I k u n o b u M u r a m a t s u Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11, Japan Received 27 July 1992, revised MS received 28 October 1992, accepted 10 November 1992
In the presence of adrenergic and cholinergic blocking agents, transmural electrical stimulation evoked a relaxation in isolated dog urethra precontracted with histamine. The response was abolished by tetrodotoxin, indicating its neurogenic origin. The non-adrenergic and non-cholinergic relaxation developed rapidly and was transient at low stimulation frequencies (< 1 Hz). However, at higher frequencies (> 5 Hz) the recovery phase of the relaxation became slow and often showed a notch, suggesting the presence of transient and slow components. NO-Monomethyl-L-arginine, a nitric oxide synthase inhibitor, inhibited the transient relaxation but did not affect the relaxation evoked at high stimulation frequencies. N~-Nitro-L-arginine, a more potent nitric oxide synthase inhibitor, abolished the transient relaxation produced at low stimulation frequencies and markedly attenuated the transient component at high frequencies. However, NG-nitro-L-arginine did not affect the slow component. The inhibition by NG-monomethyl-L-arginine and NG-nitro-L-arginine was reversed by the addition of L- but not D-arginine. Exogenously applied vasoactive intestinal polypeptide (VIP) produced a slowly developing relaxation. The slow relaxation induced by transmural electrical stimulation and VIP was not affected by [4-C1-D-Phe6,Leu17]VIP, a reportedly competitive VIP antagonist. NG-Nitro-L-arginine did not affect the relaxation induced by VIP and sodium nitroprusside. These results suggest that the non-adrenergic and non-cholinergic relaxation induced by transmural electrical stimulation is composed of nitric oxide-dependent and -independent components in the isolated dog urethra. Urethra (dog); Non-adrenergic, non-cholinergic (NANC) relaxation; Nitric oxide (NO) synthase inhibitor; VIP receptor antagonists
1. Introduction
Relaxations mediated by non-adrenergic and noncholinergic (NANC) nerves have been demonstrated in the urethra isolated from rabbits (Andersson et al., 1983; Ito and Kimoto, 1985; Mattiasson et al., 1990, 1985), pigs (Klarskov et al., 1983) and humans (Andersson et al., 1983). In the isolated dog urethra, we have also demonstrated NANC relaxation in response to transmural electrical stimulation (Hashimoto et al., 1992). There are several reports indicating that vasoactive intestinal polypeptide (VIP) causes relaxation, and that VIP-containing nerves are distributed in the urethra of several species (Aim et al., 1980; Fahrenkrug, 1979; Hashimoto et al., 1992; Larsen et al., 1981; Mattiasson et al., 1990; Sj6gren et al., 1985). The peptide is therefore considered a candidate NANC transmitter, although there are no reports on the ef-
Correspondence to: I. Muramatsu, Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11, Japan.
fects of VIP antagonists or VIP antiserum on the NANC relaxation in the urethra. Nitric oxide (NO) or a NO-like substance was recently suggested to be a mediator of NANC relaxation in several smooth muscles including mouse and rat anococcygeus muscle (Gibson et al., 1990; Gillespie et al., 1989), rat gastric fundus (Li and Rand, 1990), guinea-pig trachea (Tucker et al., 1990), dog ileocolonic junction (Bult et al., 1990) and dog cerebral artery (Toda and Okamura, 1991). This suggestion is based on the finding that NG-mono methyl-L-arginine (L-NMMA) and NC-nitro-L-arginine (L-NOARG) inhibit the synthesis of NO from Larginine in several types of cells (Moncada et al., 1989; Miilsch and Busse, 1990) and also block the NANC relaxation in the tissues mentioned above. Recently, Andersson et al. (1992, 1991), Dokita et al. (1991) and Garcia-Pascual et al. (1991) reported that the NANC relaxation in response to electrical stimulation was inhibited by L - N O A R G in isolated rabbit, sheep and human urethras. In the present study, we evaluated the NANC relaxation in response to transmural electrical stimulation by using L-NMMA, L - N O A R G and [4-C1-
210 D-Phe6,LeuI7]VIP, a competitive VIP antagonist (Pandol et al., 1986), in isolated circular muscle preparations of the dog urethra.
nitro-L-arginine (L-NOARG), prazosin hydrochloride, [4-CI-D-Phe6,Leu17]VIP (Sigma, MO, U.S.A.), D,Lpropranolol hydrochloride (Sumitomo, Osaka, Japan), tetrodotoxin (Sankyo, Tokyo, Japan) and VIP (Peptide Institute, Osaka, Japan).
2. Materials and methods Female mongrel dogs, weighing 8 to 12 kg, were anesthetized with sodium thiopental (20 m g / k g i.v.) and exanguinated from the common carotid arteries. The proximal and middle portions of the urethra were cut longitudinally, and the connective tissue and mucosal layer were removed under a dissecting microscope (Hashimoto et al., 1992). Transverse muscle strips (approximately 3 mm in width, 10 mm in length and 2 mm in thickness) were mounted vertically in an organ bath containing 10 ml (5 ml when the VIP antagonist was used) of Krebs-Henseleit solution of the following composition (mM): NaC1 112, KCI 5.9, MgCI 2 1.2, CaC12 2.0, N a H C O 3 25, N a H 2 P O 4 1.2 and glucose 11.5. The bathing medium was maintained at 37°C, pH 7.4, and was equilibrated with a gas mixture consisting of 95% 0 2 and 5% CO 2. An initial resting tension of 1.0 g was applied; responses were recorded isometrically with force-displacement transducers. Indomethacin (3 x 10 -6 M), prazosin (10 -7 M), propanolol (10 -6 M) and atropine (10 -6 M) were present in the bath solution to block spontaneous contractile activity, a 1and/3-adrenoceptors and muscarinic receptors, respectively. All preparations were equilibrated for at least 60 min before the experiments were started. The preparations were exposed to histamine (10 -5 M), which produced an active tension. Transmural electrical stimulation was applied through a pair of platinum-wire electrodes. Stimulus parameters were 0.3 ms in duration, single pulse or trains at a frequency of 1, 5 or 10 Hz for 5 s and supramaximal voltage (10 V). Drugs were added directly to the bath. The relaxation in response to transmural electrical stimulation and agonists was investigated before and after the tissue was exposed to VIP antagonist or NO synthase inhibitors, which were left in contact with the preparation for 30 or 15 min, respectively. Experimental values are expressed as means _ S.E. The relaxation responses were normalized against the maximal relaxation induced by papaverine ( 1 0 - 4 M ) . The statistical significance of differences between groups was determined by paired Student's t-test or an analysis of variance followed by ScheffCs multiple comparison test. Differences were considered to be significant when P < 0.05. The following drugs were used: L- and D-arginine, atropine sulfate (Wako, Tokyo, Japan), histamine dihydrochloride, sodium nitroprusside, papaverine hydrochloride (Nacalai, Kyoto, Japan), indomethacin, N Gmonomethyl-L-arginine acetate salt (L-NMMA), N °-
3. Results
3.1. Relaxing responses to transmural electrical stimulation Histamine (10 -5 M) produced a sustained contraction (2.5 + 0.2 g, n = 49), which corresponded to about 65% of the maximal contraction elicited by histamine (10 -4 M) in all urethra preparations. Transmural electrical stimulation with a single pulse or a train at 1, 5 or 10 Hz produced a rapid relaxation (figs. 1A and 2A). The amplitude of relaxation was frequency dependent and the maximal relaxation was reached at 5 Hz (table 1). The relaxations produced with a single pulse and at 1 Hz were transient: the tension recovered to the original level within 1 min (figs. 1A and 2A). However, the recovery phase of the relaxations elicited at 5 and 10 Hz was slow and often showed a notch (figs. 1A and 2A). These relaxations were reproducible upon repeated stimulation and were abolished by tetrodotoxin (3 X 10 -7 M).
3.2. Effects of L-NMMA and L-NOARG on the relaxing responses to transmural electrical stimulation In the preparations precontracted with histamine (10 -5 M), application of L-NMMA (10 -5 and 10 -4 M) further increased tension to 3.5 _ 1.5 (n = 7) and 16.4 _+ 2.9% (n = 18) of the contraction elicited by his-
Single
1Hz
5Hz
10Hz
A L-NMMA 10"4M
B lml
1.0g[
Fig. 1. Representative recordings of the effect of L-NMMA on the NANC relaxation induced by transmural electrical stimulation in isolated dog urethra. (A) Control; (B) in the presence of L-NMMA (10 -4 M). The preparation was contracted with histamine (10-5 M) and was electricallystimulated with a single pulse or at 1, 5 or 10 Hz for 5 s. Indomethacin (3 × 10 -6 M), atropine (10 -6 M), propranolol (10-6 M) and prazosin (10-7 M) were present throughout the experiment.
211 Single
1Hz
SHz
10Hz
L-NOARG
10-4M
L-NOARG
10"4M ÷ L-Arg 3x10-3M
I
B !
i
(3
,,j
"' ~
1rain 1.0g[-Fig. 2. Representative recordings of the effect of L-NOARG on the NANC relaxation induced by transmural electrical stimulation and of its reversal by L-arginine in isolated dog urethra. (A) Control; (B) in the presence of L - N O A R G (10 -4 M); (C) in the presence of LN O A R G (10 -4 M) and L-arginine (3 x 10 .3 M). Other experimental conditions were the same as in fig. 1.
tamine (10 -s M). The contraction induced by LNMMA was not affected by tetrodotoxin (3 x 10 -7 M, n = 3). L-NMMA (10 -5 and 10 -4 M) inhibited the relaxation induced by a single pulse of transmural electrical stimulation in a concentration-dependent manner (fig. 1B, table 1). The inhibitory effects of L-NMMA (1.0 -4 M) were reversed by L- but not Darginine (10 -3 M) (table 1). On the other hand, no
inhibitory effects of L-NMMA (10 -4 M) were observed at stimulation frequencies of 5 and 10 Hz (fig. 1B, table 1). In the absence of L-NMMA (10 -4 M), neither relaxations induced by transmural electrical stimulation nor contractions induced by histamine were affected by L- and D-arginine (10 -3 M). However, in the presence of L-NMMA (10 -4 M), L- but not D-arginine (10 -3 M) produced a decease in muscle tension (25.2 + 4.4% reduction, n = 7). L-NOARG (10 -5 M) also inhibited the relaxations induced by transmural electrical stimulation with a single pulse and at a frequency of 1 Hz. The inhibitory potency of L-NOARG (10 -5 M) was the same as that of L-NMMA (10 -4 M) (tables 1 and 2). At 10 -4 M the inhibitory effects of L-NOARG became more pronounced (table 2, fig. 2B). In addition, L-NOARG (10 -4 M but not 10 -5 M) partially inhibited the relaxations induced at 5 and 10 Hz (table 2, fig 2B). Close inspection revealed that the inhibitory effect of LN O A R G (10 -4 M) was evident only on the initial rapid phase of the relaxation and that the slow recovery phase was not affected (fig. 2B). The inhibitory effects of L-NOARG (10 -4 M) were reversed by Lbut not D-arginine (3 × 10 -3 M) at all stimulation frequencies tested (table 2, fig. 2C). In addition to these inhibitory effects, L-NOARG itself (10 -5 and 10 -4 M) increased muscle tension in preparations precontracted with histamine (data not shown).
TABLE 1
Effects of L-NMMA and L- and D-arginine on the NANC relaxation induced by transmural electrical stimulation of the dog urethra. Transmural electrical stimulation
Drugs
Control L-NMMA L-NMMA L-NMMA L-NMMA
(10 -5 (10 -4 (10- 4 (10 -4
M) M) M) + D-arginine (10- 3 M) M ) + L-arginine (10 -3 M)
Single pulse
1 Hz
5 Hz
10 Hz
53.1±3.6 37.9±3.7 14.7±2.0 a'b 14.6±3.6 a 51.4±4.6 c,d
75.1±3.7 74.0±7.0 55.9±7.3 _
91.4±2.2 86.4±2.5 _ _
90.4±1.4 92.0±2.4 _ _
Each value is the mean ± S.E. (n = 4-13) and is expressed as a percentage of the maximal relaxation induced by papaverine (10 -4 M). a p < 0.01 versus control, b p < 0.05 versus L-NMMA (10 -5 M). c p < 0.01 versus L-NMMA (10 -4 M). d p < 0.01 versus L-NMMA (10 -4 M ) + D-arginine (10 -3 M). ( - ) not tested. The experimental conditions are the same as in fig. 1.
TABLE 2
Effects of L-NOARG and L- and D-arginine on the NANC relaxation induced by transmural electrical stimulation of the dog urethra. Transmural electrical stimulation
Drugs
Control L-NOARG L-NOARG L-NOARG L-NOARG
(10 -5 (10 -4 (10 -4 (10 -4
M) M) M ) + D-arginine (3 × 10-3 M) M ) + L-arginine (3 X 10 -3 M)
Single pulse
1 Hz
44.0±2.3 15.4±1.8 0.9±0.5 a 0.7±0.7 a 20.1±2.8
79.5±1.6 47.1±1.9 4.8±2.5 3.1±1.3 66.3±3.6
a a'b mb a'b'c'd
5 Hz
10 Hz
88.3± 1.0 78.3± 5.3 50.2± 9.4 a 45.5±12.7 a 87.6± 2.4
89.2± 1.1 85.7± 5.7 70.9±12.9 69.5±14.2 ~ . 3 ± 2.2
Each value is the mean + S.E. (n = 4-12) and is expressed as a percentage of the maximal relaxation induced by papaverine (10 -4 M). a p < 0.01 versus control, b p <0.01 versus L - N O A R G (10 -5 M). c p <0.01 versus L - N O A R G (10 -4 M). d p <0.01 versus L - N O A R G (10 -4 M ) + D arginine (3 x 10 -3 M). The experimental conditions are the same as in fig. 2.
212 100-
DControl
o o0
50
1114-CI - D - Phe6, Leu '7] -VIP (10"%1)
i
i
ETS 10Hz
i
VIP 10"aM
10TM
Fig. 3. Effect of [4-CI-D-Phe6,Leu17]VIP (10 -5 M) on the relaxation induced by transmural electrical stimulation (ETS) and by exogenously applied VIP. L-NOARG (10 -5 M) was present throughout the experiment. Other experimental conditions were the same as in fig. I. The results are the means + S.E. of four to five experiments.
3.3. Effects of VIP antagonist on the relaxing responses to transmural electrical stimulation Application of [4-CI-D-Phe6,Leu17]VIP (10 -5 M) did not change the amplitude of the contraction elicited by histamine (10 -5 M). The agent also did not affect the relaxation induced by transmural electrical stimulation at any of the stimulation frequencies tested (data not shown). In the presence of L-NOARG (10 -4 M), [4-C1D-Phe6,LeulT]vIP (10 -5 M) did not affect the other components of the contraction induced by transmural electrical stimulation (10 Hz) (fig. 3).
3.4. Effects of L-NMMA, L-NOARG and VIP antagonist on the relaxing responses to nitroprusside and VIP Exogenously applied sodium nitroprusside (10 -810 -4 M) caused a concentration-dependent relaxation, with the maximum relaxation being reached at 10 -4 M (99.4 + 0.5%, n = 5). The ECs0 value of the agent was 2.3 + 0.3 × 10 -7 M (n = 5). L-NMMA and L-NOARG (10 -4 M) did not affect the relaxation induced by sodium nitroprusside (data not shown). VIP (10 -8 and 10-7 M) produced a slowly developing relaxation which was not affected by L-NOARG (10 -4 M). In the presence of L-NOARG (10 -4 M), the VIP-induced relaxation was not changed by [4-CI-D-Phe6,Leu 17]VIP (10-5 M) (fig. 3).
4. Discussion
Recently, we reported that, in isolated dog urethra precontracted with prostaglandin F2,~ transmural electrical stimulation produced relaxations which were sensitive to tetrodotoxin but which were not affected by
adrenergic and cholinergic blocking agents (Hashimoto et al., 1992). In the present study these NANC relaxations were observed in preparations contracted with histamine. The relaxations patterns were similar under the different conditions, indicating that NANC responses are not influenced by either contractile agent. The relaxation elicited at low stimulus frequencies ( < 1 Hz) was transient, but at higher frequencies a notch was seen in the slowly decaying recovery phase, suggesting the involvement of at least two distinct neurogenic components in the dog urethra (Hashimoto et al., 1992). In contrast, only a transient relaxation has been reported in rabbit, sheep and human urethras (Andersson et al., 1992, 1991, 1983; Dokita et al., 1991; Garcia-Pascual et al., 1991). L-NMMA and L-NOARG prevent the synthesis of NO from L-arginine in several cell types, including vascular endothelial cells, macrophages and brain cells (Moncada et al., 1989; Miilsch and Bussue, 1990), and also inhibit the NANC relaxation in various smooth muscles (see Introduction). These reports suggest a possible involvement of NO or a NO-like substance in NANC relaxation. In the present study, L-NMMA also inhibited the relaxation of dog urethra evoked by low stimulation frequencies (< 1 Hz). A similar but more potent inhibition was produced by L-NOARG. The inhibitory effects of L-NMMA and L-NOARG were reversed by L- but not D-arginine, and neither LNMMA nor L-NOARG inhibited the relaxation induced by sodium nitroprusside. These results strongly suggest that the NANC relaxation produced at low stimulation frequencies is predominantly mediated by NO derived from L-arginine. The fact that the inhibitory effects of L-NOARG were more potent than those of L-NMMA may reflect a difference in the potency of the two compounds to inhibit NO synthesis (Gibson et al., 1990). In addition, L-NMMA and LNOARG by themselves increased the tension of preparations precontracted with histamine, and this increase in muscle tension was reversed by L-arginine. In preliminary experiments we could not observe any effects of L-NMMA and L-NOARG on the resting tension of dog urethra preparations. These results suggest that NO (or a NO-like substance) may be released continuously from contracted, but not resting, urethral muscle. Since the increase in muscle tension produced by LNMMA and L-NOARG was not affected by tetrodotoxin, the enhanced basal release of NO (or a NO-like substance) from contracted muscle may not be associated with Na ÷ channel-dependent mechanisms. L-Arginine per se did not affect either the active tension induced by histamine or the relaxation induced by transmural electrical stimulation. In contrast to these results. Andersson et al. (1992) reported that, in isolated rabbit urethra, L-arginine enhanced the relaxation induced by electrical stimulation without chang-
213
ing the noradrenaline-induced contraction. The causes for this difference are unclear. The relaxation produced at high stimulation frequencies (5 and 10 Hz) was not inhibited by L-NMMA but was partially inhibited by L-NOARG. The LNOARG-induced inhibition was limited to the initial rapid phase of the relaxation; the slow phase was not affected. The inhibitory effect of L-NOARG was attenuated by L-arginine. L-NOARG-resistant relaxation is specific to the dog urethra, because L-NOARG almost completely attenuated the relaxing responses, even at high stimulation frequencies, in rabbit, sheep and human urethras (Andersson et al., 1992, 1991; Dokita et al., 1991; Gracia-Pascual et al., 1991). These findings strongly suggest that the NANC relaxation evoked at high stimulation frequencies in the dog urethra is composed of an unknown component in addition to a NO-ergic component. VIP produced a slowly developing relaxation in the isolated urethra, and VIP-containing nerves have been demonstrated in the urethra of several species (Hashimoto et al., 1992; Mattiasson et al., 1990; Sj6gren et al., 1985; Larsen et al., 1981; Aim et al., 1980; Fahrenkrung, 1979). On the basis of this evidence, VIP is considered a neurotransmitter of the NANC relaxation in the urethra. This possibility was examined by using a competitive VIP antagonist, [4-C1-D-Phe 6, Leut7]vIP (Pandol et al., 1986), which has been shown to block the following responses: VIP-induced amylase release from the guinea-pig pancreas (Pandol et al., 1986), VIP-induced relaxation in the guinea-pig teania-coli (Grider et al., 1990), NANC relaxation in the guinea-pig gastric fundus (Grider and Rivier, 1990), and specific 125I-VIP binding in rat seminal vesicle membranes (Rodriguez-Pena et al., 1991). However, the antagonist is also reported to be ineffective on VIP- and NANC-induced relaxations of the guinea-pig trachea and the rat gastric fundus (Ellis and Farmer, 1989; De Beurme and Lefebvre, 1988). In the present study, the antagonist also failed to block the relaxation induced by transmural electrical stimulation in the presence of a NO synthase inhibitor and by exogenous VIP. We therefore could not determine whether VIP is involved in the neurogenic response. The VIP receptors of the dog urethra might be different from those in the tissues where the antagonist produces an effective blockade. The development of selective antagonists is necessary to clarify this point. However, even though VIP is released from VIP-containing nerves, the released VIP does not stimulate the generation of NO, because the NO synthase inhibitor did not affect the relaxation induced by VIP. In conclusion, the present study shows that the NANC relaxation is composed of two distinct components (NO-dependent and -independent) in the dog urethra. The different stimulation frequency depen-
dence of the two neurogenic responses suggests that the two components may be involved differently in the physiological functions of the urethra.
Acknowledgement We thank H. Takahashi for secretarial assistance.
References Aim, P., J. Alumets, R. H~kanson, Ch. Owman, N.-O. Sj6berg, F. Sundler and B. Walles, 1980, Origin and distribution of VIP (vasoactive intestinal polypeptide)-nerves in the genito-urinary tract, Cell Tiss. Res. 205, 337. Andersson, K.-E., A. Mattiasson and C. Sj6gren, 1983, Electrically induced relaxation of the noradrenaline contracted isolated urethra from rabbit and man, J. Urol. 129, 210. Andersson, K.-E., A. Garcia-Pascual, A. Forman and A. T0ttrup, 1991, Non-adrenergic, non-cholinergic nerve-mediated relaxation of rabbit urethra is caused by nitric oxide, Acta Physiol. Scand. 141, 133. Andersson, K.-E., A. Garcia-Pascual, K. Persson, A. Forman and A. TC~ttrup, 1992, Electrically-induced, nerve-mediated relaxation of rabbit urethra involves nitric oxide, J. Urol. 147, 253. Bult, H., G.E. Boeckxstaens, P.A. Pelckmans, F.H. Jordaens, Y.M. Van Maercke and A.G. Herman, 1990, Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature 345, 346. De Beurme, F.A. and R.A. Lefebvre, 1988, Vasoactive intestinal polypeptide as Possible mediator of relaxation in the rat gastric fundus, J. Pharmacol. 40, 711. Dokita, S., W.R. Morgan, M.A. Wheeler, M. Yoshida, J. Latifpour and R.M. Weiss, 1991, NG-Nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle, Life Sci. 48, 2429. Ellis, J.L. and S.G. Farmer, 1989, The effects of vasoactive intestinal peptide (VIP) antagonists, and VIP and peptide histidine isoleucine antisera on non-adrenergic, non-cholinergic relaxations of tracheal smooth muscle, Br. J. Pharmacol. 96, 513. Fahrenkrug, J., 1979, Vasoactive intestinal polypeptide; measurement, distribution and putative neurotransmitter function, Digestion 19, 149. Garcia-Pascual, A., G. Costa, A. Garcia-Sacristan and K.-E. Andersson, 1991, Relaxation of sheep urethral muscle induced by electrical stimulation of nerves: involvement of nitric oxide, Acta Physiol. Scand. 141,531. Gibson, A., S. Mirazadeh, A.J. Hobbs and P.K. Moore, 1990, L-N 6Monomethyl arginine and L-NG-nitro arginine inhibit non-adrenergic, non-cholinergic relaxation of the mouse anococcygeus muscle, Br. J. Pharmacol. 99, 602. Gillespie, J.S., X. Liu and W. Martin, 1989, The effect of L-arginine and N°-monomethyl L-arginine on the response of the rat anococcygenus muscle to NANC nerve stimulation, Br. J. Pharmacol. 98, 1080. Grider, J.R. and J.R. Rivier, 1990, Vasoactive intestinal peptide (V1P) as transmitter of inhibitory motor neurons of the gut: evidence from the use of selective VIP antagonists and VIP antiserum, J. Pharmacol. Exp. Ther. 253, 738. Hashimoto, S., S. Kigoshi and I. Muramatsu, 1992, Neurogenic responses of urethra isolated from the dog, Eur. J. Pharmacol. 213, 117. Ito, Y. and Y. Kimoto, 1985, The neural and non-neural mechanisms involved in urethral activity in rabbits, J. Physiol. (London) 367, 57.
214 Klarskov, P., T.C. Gerstenberg, D. Ramirez and T. Hald, 1983, Non-cholinergic, non-adrenergic nerve mediated relaxation of trigone, bladder neck and urethral smooth muscle in vitro, J. Urol. 129, 848. Larsen, J.-J., B. Ottesen, J. Fahrenkrug and L. Fahrenkrug, 1981, Vasoactive intestinal polypeptide (VIP) in the male genitourinary tract, Invest, Urol. 19, 211. Li, C.G. and M.J. Rand, 1990, Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus, Eur. J. Pharmacol. 191,303. Mattiasson, A., K.-E. Andersson and C. Sj6gren, 1985, Contractant and relaxant properties of the female rabbit urethral submucosa, J. Urol. 133, 304. Mattiasson, A., K.-E. Andersson, P.-O. Andersson, B. Larsson, C. Sj6gren and B. Uvelius, 1990, Nerve-mediated functions in the circular and longitudinal muscle layers of the proximal female rabbit urethra, J. Urol. 143, 155. Moncada, S., R.M.J. Palmer and E.A. Higgs, 1989, Biosynthesis of nitric oxide from L-arginine, Biochem. Pharmacol. 38, 1709.
Miilsch, A. and R. Busse, 1990, NG-Nitro-L-arginine (N 5[imino(nitroamino)methyl]-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine, Naunyn-Schmiedeb. Arch. Pharmacol. 341, 143. Pandol, S.J., K. Dharmsathaphoren, M.S. Schoeffield, W. Vale and J. Rivier, 1986, Vasoactive intestinal peptide receptor antagonist [4-CI-D-Phe6,LeulT]vIP, Am. J. Physiol. 250, G553. Rodriguez-Pena, M,S., L.G. Guijarro and J.C. Prieto, 1991, Vasoactive intestinal peptide receptor antagonists in rat seminal vesicle membranes, Eur. J. Pharmacol. 208, 207. SjSgren, C., K.-E. Andersson and A. Mattiasson, 1985, Effects of vasoactive intestinal polypeptide on isolated urethral and urinary bladder smooth muscle from rabbit and man, J. Urol. 133, 136. Toda, N. and T. Okamura, 1991, Role of nitric oxide in neurally induced cerebroarterial relaxation, J. Pharmacol. Exp. Ther. 258, 1027. Tucker, J.F., S.R., Brave, L. Charalambous, A.J. Hobbs and A. Gibson, 1990, L-NG-Nitro arginine inhibits non-adrenergic, noncholinergic relaxations of guinea-pig isolated tracheal smooth muscle, Br. J. Pharmacol. 100, 663.
209
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 52893
Nitric oxide-dependent and -independent neurogenic relaxation of isolated dog urethra Shigeki H a s h i m o t o , S h i g e r u Kigoshi a n d I k u n o b u M u r a m a t s u Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11, Japan Received 27 July 1992, revised MS received 28 October 1992, accepted 10 November 1992
In the presence of adrenergic and cholinergic blocking agents, transmural electrical stimulation evoked a relaxation in isolated dog urethra precontracted with histamine. The response was abolished by tetrodotoxin, indicating its neurogenic origin. The non-adrenergic and non-cholinergic relaxation developed rapidly and was transient at low stimulation frequencies (< 1 Hz). However, at higher frequencies (> 5 Hz) the recovery phase of the relaxation became slow and often showed a notch, suggesting the presence of transient and slow components. NO-Monomethyl-L-arginine, a nitric oxide synthase inhibitor, inhibited the transient relaxation but did not affect the relaxation evoked at high stimulation frequencies. N~-Nitro-L-arginine, a more potent nitric oxide synthase inhibitor, abolished the transient relaxation produced at low stimulation frequencies and markedly attenuated the transient component at high frequencies. However, NG-nitro-L-arginine did not affect the slow component. The inhibition by NG-monomethyl-L-arginine and NG-nitro-L-arginine was reversed by the addition of L- but not D-arginine. Exogenously applied vasoactive intestinal polypeptide (VIP) produced a slowly developing relaxation. The slow relaxation induced by transmural electrical stimulation and VIP was not affected by [4-C1-D-Phe6,Leu17]VIP, a reportedly competitive VIP antagonist. NG-Nitro-L-arginine did not affect the relaxation induced by VIP and sodium nitroprusside. These results suggest that the non-adrenergic and non-cholinergic relaxation induced by transmural electrical stimulation is composed of nitric oxide-dependent and -independent components in the isolated dog urethra. Urethra (dog); Non-adrenergic, non-cholinergic (NANC) relaxation; Nitric oxide (NO) synthase inhibitor; VIP receptor antagonists
1. Introduction
Relaxations mediated by non-adrenergic and noncholinergic (NANC) nerves have been demonstrated in the urethra isolated from rabbits (Andersson et al., 1983; Ito and Kimoto, 1985; Mattiasson et al., 1990, 1985), pigs (Klarskov et al., 1983) and humans (Andersson et al., 1983). In the isolated dog urethra, we have also demonstrated NANC relaxation in response to transmural electrical stimulation (Hashimoto et al., 1992). There are several reports indicating that vasoactive intestinal polypeptide (VIP) causes relaxation, and that VIP-containing nerves are distributed in the urethra of several species (Aim et al., 1980; Fahrenkrug, 1979; Hashimoto et al., 1992; Larsen et al., 1981; Mattiasson et al., 1990; Sj6gren et al., 1985). The peptide is therefore considered a candidate NANC transmitter, although there are no reports on the ef-
Correspondence to: I. Muramatsu, Department of Pharmacology, Fukui Medical School, Matsuoka, Fukui 910-11, Japan.
fects of VIP antagonists or VIP antiserum on the NANC relaxation in the urethra. Nitric oxide (NO) or a NO-like substance was recently suggested to be a mediator of NANC relaxation in several smooth muscles including mouse and rat anococcygeus muscle (Gibson et al., 1990; Gillespie et al., 1989), rat gastric fundus (Li and Rand, 1990), guinea-pig trachea (Tucker et al., 1990), dog ileocolonic junction (Bult et al., 1990) and dog cerebral artery (Toda and Okamura, 1991). This suggestion is based on the finding that NG-mono methyl-L-arginine (L-NMMA) and NC-nitro-L-arginine (L-NOARG) inhibit the synthesis of NO from Larginine in several types of cells (Moncada et al., 1989; Miilsch and Busse, 1990) and also block the NANC relaxation in the tissues mentioned above. Recently, Andersson et al. (1992, 1991), Dokita et al. (1991) and Garcia-Pascual et al. (1991) reported that the NANC relaxation in response to electrical stimulation was inhibited by L - N O A R G in isolated rabbit, sheep and human urethras. In the present study, we evaluated the NANC relaxation in response to transmural electrical stimulation by using L-NMMA, L - N O A R G and [4-C1-
210 D-Phe6,LeuI7]VIP, a competitive VIP antagonist (Pandol et al., 1986), in isolated circular muscle preparations of the dog urethra.
nitro-L-arginine (L-NOARG), prazosin hydrochloride, [4-CI-D-Phe6,Leu17]VIP (Sigma, MO, U.S.A.), D,Lpropranolol hydrochloride (Sumitomo, Osaka, Japan), tetrodotoxin (Sankyo, Tokyo, Japan) and VIP (Peptide Institute, Osaka, Japan).
2. Materials and methods Female mongrel dogs, weighing 8 to 12 kg, were anesthetized with sodium thiopental (20 m g / k g i.v.) and exanguinated from the common carotid arteries. The proximal and middle portions of the urethra were cut longitudinally, and the connective tissue and mucosal layer were removed under a dissecting microscope (Hashimoto et al., 1992). Transverse muscle strips (approximately 3 mm in width, 10 mm in length and 2 mm in thickness) were mounted vertically in an organ bath containing 10 ml (5 ml when the VIP antagonist was used) of Krebs-Henseleit solution of the following composition (mM): NaC1 112, KCI 5.9, MgCI 2 1.2, CaC12 2.0, N a H C O 3 25, N a H 2 P O 4 1.2 and glucose 11.5. The bathing medium was maintained at 37°C, pH 7.4, and was equilibrated with a gas mixture consisting of 95% 0 2 and 5% CO 2. An initial resting tension of 1.0 g was applied; responses were recorded isometrically with force-displacement transducers. Indomethacin (3 x 10 -6 M), prazosin (10 -7 M), propanolol (10 -6 M) and atropine (10 -6 M) were present in the bath solution to block spontaneous contractile activity, a 1and/3-adrenoceptors and muscarinic receptors, respectively. All preparations were equilibrated for at least 60 min before the experiments were started. The preparations were exposed to histamine (10 -5 M), which produced an active tension. Transmural electrical stimulation was applied through a pair of platinum-wire electrodes. Stimulus parameters were 0.3 ms in duration, single pulse or trains at a frequency of 1, 5 or 10 Hz for 5 s and supramaximal voltage (10 V). Drugs were added directly to the bath. The relaxation in response to transmural electrical stimulation and agonists was investigated before and after the tissue was exposed to VIP antagonist or NO synthase inhibitors, which were left in contact with the preparation for 30 or 15 min, respectively. Experimental values are expressed as means _ S.E. The relaxation responses were normalized against the maximal relaxation induced by papaverine ( 1 0 - 4 M ) . The statistical significance of differences between groups was determined by paired Student's t-test or an analysis of variance followed by ScheffCs multiple comparison test. Differences were considered to be significant when P < 0.05. The following drugs were used: L- and D-arginine, atropine sulfate (Wako, Tokyo, Japan), histamine dihydrochloride, sodium nitroprusside, papaverine hydrochloride (Nacalai, Kyoto, Japan), indomethacin, N Gmonomethyl-L-arginine acetate salt (L-NMMA), N °-
3. Results
3.1. Relaxing responses to transmural electrical stimulation Histamine (10 -5 M) produced a sustained contraction (2.5 + 0.2 g, n = 49), which corresponded to about 65% of the maximal contraction elicited by histamine (10 -4 M) in all urethra preparations. Transmural electrical stimulation with a single pulse or a train at 1, 5 or 10 Hz produced a rapid relaxation (figs. 1A and 2A). The amplitude of relaxation was frequency dependent and the maximal relaxation was reached at 5 Hz (table 1). The relaxations produced with a single pulse and at 1 Hz were transient: the tension recovered to the original level within 1 min (figs. 1A and 2A). However, the recovery phase of the relaxations elicited at 5 and 10 Hz was slow and often showed a notch (figs. 1A and 2A). These relaxations were reproducible upon repeated stimulation and were abolished by tetrodotoxin (3 X 10 -7 M).
3.2. Effects of L-NMMA and L-NOARG on the relaxing responses to transmural electrical stimulation In the preparations precontracted with histamine (10 -5 M), application of L-NMMA (10 -5 and 10 -4 M) further increased tension to 3.5 _ 1.5 (n = 7) and 16.4 _+ 2.9% (n = 18) of the contraction elicited by his-
Single
1Hz
5Hz
10Hz
A L-NMMA 10"4M
B lml
1.0g[
Fig. 1. Representative recordings of the effect of L-NMMA on the NANC relaxation induced by transmural electrical stimulation in isolated dog urethra. (A) Control; (B) in the presence of L-NMMA (10 -4 M). The preparation was contracted with histamine (10-5 M) and was electricallystimulated with a single pulse or at 1, 5 or 10 Hz for 5 s. Indomethacin (3 × 10 -6 M), atropine (10 -6 M), propranolol (10-6 M) and prazosin (10-7 M) were present throughout the experiment.
211 Single
1Hz
SHz
10Hz
L-NOARG
10-4M
L-NOARG
10"4M ÷ L-Arg 3x10-3M
I
B !
i
(3
,,j
"' ~
1rain 1.0g[-Fig. 2. Representative recordings of the effect of L-NOARG on the NANC relaxation induced by transmural electrical stimulation and of its reversal by L-arginine in isolated dog urethra. (A) Control; (B) in the presence of L - N O A R G (10 -4 M); (C) in the presence of LN O A R G (10 -4 M) and L-arginine (3 x 10 .3 M). Other experimental conditions were the same as in fig. 1.
tamine (10 -s M). The contraction induced by LNMMA was not affected by tetrodotoxin (3 x 10 -7 M, n = 3). L-NMMA (10 -5 and 10 -4 M) inhibited the relaxation induced by a single pulse of transmural electrical stimulation in a concentration-dependent manner (fig. 1B, table 1). The inhibitory effects of L-NMMA (1.0 -4 M) were reversed by L- but not Darginine (10 -3 M) (table 1). On the other hand, no
inhibitory effects of L-NMMA (10 -4 M) were observed at stimulation frequencies of 5 and 10 Hz (fig. 1B, table 1). In the absence of L-NMMA (10 -4 M), neither relaxations induced by transmural electrical stimulation nor contractions induced by histamine were affected by L- and D-arginine (10 -3 M). However, in the presence of L-NMMA (10 -4 M), L- but not D-arginine (10 -3 M) produced a decease in muscle tension (25.2 + 4.4% reduction, n = 7). L-NOARG (10 -5 M) also inhibited the relaxations induced by transmural electrical stimulation with a single pulse and at a frequency of 1 Hz. The inhibitory potency of L-NOARG (10 -5 M) was the same as that of L-NMMA (10 -4 M) (tables 1 and 2). At 10 -4 M the inhibitory effects of L-NOARG became more pronounced (table 2, fig. 2B). In addition, L-NOARG (10 -4 M but not 10 -5 M) partially inhibited the relaxations induced at 5 and 10 Hz (table 2, fig 2B). Close inspection revealed that the inhibitory effect of LN O A R G (10 -4 M) was evident only on the initial rapid phase of the relaxation and that the slow recovery phase was not affected (fig. 2B). The inhibitory effects of L-NOARG (10 -4 M) were reversed by Lbut not D-arginine (3 × 10 -3 M) at all stimulation frequencies tested (table 2, fig. 2C). In addition to these inhibitory effects, L-NOARG itself (10 -5 and 10 -4 M) increased muscle tension in preparations precontracted with histamine (data not shown).
TABLE 1
Effects of L-NMMA and L- and D-arginine on the NANC relaxation induced by transmural electrical stimulation of the dog urethra. Transmural electrical stimulation
Drugs
Control L-NMMA L-NMMA L-NMMA L-NMMA
(10 -5 (10 -4 (10- 4 (10 -4
M) M) M) + D-arginine (10- 3 M) M ) + L-arginine (10 -3 M)
Single pulse
1 Hz
5 Hz
10 Hz
53.1±3.6 37.9±3.7 14.7±2.0 a'b 14.6±3.6 a 51.4±4.6 c,d
75.1±3.7 74.0±7.0 55.9±7.3 _
91.4±2.2 86.4±2.5 _ _
90.4±1.4 92.0±2.4 _ _
Each value is the mean ± S.E. (n = 4-13) and is expressed as a percentage of the maximal relaxation induced by papaverine (10 -4 M). a p < 0.01 versus control, b p < 0.05 versus L-NMMA (10 -5 M). c p < 0.01 versus L-NMMA (10 -4 M). d p < 0.01 versus L-NMMA (10 -4 M ) + D-arginine (10 -3 M). ( - ) not tested. The experimental conditions are the same as in fig. 1.
TABLE 2
Effects of L-NOARG and L- and D-arginine on the NANC relaxation induced by transmural electrical stimulation of the dog urethra. Transmural electrical stimulation
Drugs
Control L-NOARG L-NOARG L-NOARG L-NOARG
(10 -5 (10 -4 (10 -4 (10 -4
M) M) M ) + D-arginine (3 × 10-3 M) M ) + L-arginine (3 X 10 -3 M)
Single pulse
1 Hz
44.0±2.3 15.4±1.8 0.9±0.5 a 0.7±0.7 a 20.1±2.8
79.5±1.6 47.1±1.9 4.8±2.5 3.1±1.3 66.3±3.6
a a'b mb a'b'c'd
5 Hz
10 Hz
88.3± 1.0 78.3± 5.3 50.2± 9.4 a 45.5±12.7 a 87.6± 2.4
89.2± 1.1 85.7± 5.7 70.9±12.9 69.5±14.2 ~ . 3 ± 2.2
Each value is the mean + S.E. (n = 4-12) and is expressed as a percentage of the maximal relaxation induced by papaverine (10 -4 M). a p < 0.01 versus control, b p <0.01 versus L - N O A R G (10 -5 M). c p <0.01 versus L - N O A R G (10 -4 M). d p <0.01 versus L - N O A R G (10 -4 M ) + D arginine (3 x 10 -3 M). The experimental conditions are the same as in fig. 2.
212 100-
DControl
o o0
50
1114-CI - D - Phe6, Leu '7] -VIP (10"%1)
i
i
ETS 10Hz
i
VIP 10"aM
10TM
Fig. 3. Effect of [4-CI-D-Phe6,Leu17]VIP (10 -5 M) on the relaxation induced by transmural electrical stimulation (ETS) and by exogenously applied VIP. L-NOARG (10 -5 M) was present throughout the experiment. Other experimental conditions were the same as in fig. I. The results are the means + S.E. of four to five experiments.
3.3. Effects of VIP antagonist on the relaxing responses to transmural electrical stimulation Application of [4-CI-D-Phe6,Leu17]VIP (10 -5 M) did not change the amplitude of the contraction elicited by histamine (10 -5 M). The agent also did not affect the relaxation induced by transmural electrical stimulation at any of the stimulation frequencies tested (data not shown). In the presence of L-NOARG (10 -4 M), [4-C1D-Phe6,LeulT]vIP (10 -5 M) did not affect the other components of the contraction induced by transmural electrical stimulation (10 Hz) (fig. 3).
3.4. Effects of L-NMMA, L-NOARG and VIP antagonist on the relaxing responses to nitroprusside and VIP Exogenously applied sodium nitroprusside (10 -810 -4 M) caused a concentration-dependent relaxation, with the maximum relaxation being reached at 10 -4 M (99.4 + 0.5%, n = 5). The ECs0 value of the agent was 2.3 + 0.3 × 10 -7 M (n = 5). L-NMMA and L-NOARG (10 -4 M) did not affect the relaxation induced by sodium nitroprusside (data not shown). VIP (10 -8 and 10-7 M) produced a slowly developing relaxation which was not affected by L-NOARG (10 -4 M). In the presence of L-NOARG (10 -4 M), the VIP-induced relaxation was not changed by [4-CI-D-Phe6,Leu 17]VIP (10-5 M) (fig. 3).
4. Discussion
Recently, we reported that, in isolated dog urethra precontracted with prostaglandin F2,~ transmural electrical stimulation produced relaxations which were sensitive to tetrodotoxin but which were not affected by
adrenergic and cholinergic blocking agents (Hashimoto et al., 1992). In the present study these NANC relaxations were observed in preparations contracted with histamine. The relaxations patterns were similar under the different conditions, indicating that NANC responses are not influenced by either contractile agent. The relaxation elicited at low stimulus frequencies ( < 1 Hz) was transient, but at higher frequencies a notch was seen in the slowly decaying recovery phase, suggesting the involvement of at least two distinct neurogenic components in the dog urethra (Hashimoto et al., 1992). In contrast, only a transient relaxation has been reported in rabbit, sheep and human urethras (Andersson et al., 1992, 1991, 1983; Dokita et al., 1991; Garcia-Pascual et al., 1991). L-NMMA and L-NOARG prevent the synthesis of NO from L-arginine in several cell types, including vascular endothelial cells, macrophages and brain cells (Moncada et al., 1989; Miilsch and Bussue, 1990), and also inhibit the NANC relaxation in various smooth muscles (see Introduction). These reports suggest a possible involvement of NO or a NO-like substance in NANC relaxation. In the present study, L-NMMA also inhibited the relaxation of dog urethra evoked by low stimulation frequencies (< 1 Hz). A similar but more potent inhibition was produced by L-NOARG. The inhibitory effects of L-NMMA and L-NOARG were reversed by L- but not D-arginine, and neither LNMMA nor L-NOARG inhibited the relaxation induced by sodium nitroprusside. These results strongly suggest that the NANC relaxation produced at low stimulation frequencies is predominantly mediated by NO derived from L-arginine. The fact that the inhibitory effects of L-NOARG were more potent than those of L-NMMA may reflect a difference in the potency of the two compounds to inhibit NO synthesis (Gibson et al., 1990). In addition, L-NMMA and LNOARG by themselves increased the tension of preparations precontracted with histamine, and this increase in muscle tension was reversed by L-arginine. In preliminary experiments we could not observe any effects of L-NMMA and L-NOARG on the resting tension of dog urethra preparations. These results suggest that NO (or a NO-like substance) may be released continuously from contracted, but not resting, urethral muscle. Since the increase in muscle tension produced by LNMMA and L-NOARG was not affected by tetrodotoxin, the enhanced basal release of NO (or a NO-like substance) from contracted muscle may not be associated with Na ÷ channel-dependent mechanisms. L-Arginine per se did not affect either the active tension induced by histamine or the relaxation induced by transmural electrical stimulation. In contrast to these results. Andersson et al. (1992) reported that, in isolated rabbit urethra, L-arginine enhanced the relaxation induced by electrical stimulation without chang-
213
ing the noradrenaline-induced contraction. The causes for this difference are unclear. The relaxation produced at high stimulation frequencies (5 and 10 Hz) was not inhibited by L-NMMA but was partially inhibited by L-NOARG. The LNOARG-induced inhibition was limited to the initial rapid phase of the relaxation; the slow phase was not affected. The inhibitory effect of L-NOARG was attenuated by L-arginine. L-NOARG-resistant relaxation is specific to the dog urethra, because L-NOARG almost completely attenuated the relaxing responses, even at high stimulation frequencies, in rabbit, sheep and human urethras (Andersson et al., 1992, 1991; Dokita et al., 1991; Gracia-Pascual et al., 1991). These findings strongly suggest that the NANC relaxation evoked at high stimulation frequencies in the dog urethra is composed of an unknown component in addition to a NO-ergic component. VIP produced a slowly developing relaxation in the isolated urethra, and VIP-containing nerves have been demonstrated in the urethra of several species (Hashimoto et al., 1992; Mattiasson et al., 1990; Sj6gren et al., 1985; Larsen et al., 1981; Aim et al., 1980; Fahrenkrung, 1979). On the basis of this evidence, VIP is considered a neurotransmitter of the NANC relaxation in the urethra. This possibility was examined by using a competitive VIP antagonist, [4-C1-D-Phe 6, Leut7]vIP (Pandol et al., 1986), which has been shown to block the following responses: VIP-induced amylase release from the guinea-pig pancreas (Pandol et al., 1986), VIP-induced relaxation in the guinea-pig teania-coli (Grider et al., 1990), NANC relaxation in the guinea-pig gastric fundus (Grider and Rivier, 1990), and specific 125I-VIP binding in rat seminal vesicle membranes (Rodriguez-Pena et al., 1991). However, the antagonist is also reported to be ineffective on VIP- and NANC-induced relaxations of the guinea-pig trachea and the rat gastric fundus (Ellis and Farmer, 1989; De Beurme and Lefebvre, 1988). In the present study, the antagonist also failed to block the relaxation induced by transmural electrical stimulation in the presence of a NO synthase inhibitor and by exogenous VIP. We therefore could not determine whether VIP is involved in the neurogenic response. The VIP receptors of the dog urethra might be different from those in the tissues where the antagonist produces an effective blockade. The development of selective antagonists is necessary to clarify this point. However, even though VIP is released from VIP-containing nerves, the released VIP does not stimulate the generation of NO, because the NO synthase inhibitor did not affect the relaxation induced by VIP. In conclusion, the present study shows that the NANC relaxation is composed of two distinct components (NO-dependent and -independent) in the dog urethra. The different stimulation frequency depen-
dence of the two neurogenic responses suggests that the two components may be involved differently in the physiological functions of the urethra.
Acknowledgement We thank H. Takahashi for secretarial assistance.
References Aim, P., J. Alumets, R. H~kanson, Ch. Owman, N.-O. Sj6berg, F. Sundler and B. Walles, 1980, Origin and distribution of VIP (vasoactive intestinal polypeptide)-nerves in the genito-urinary tract, Cell Tiss. Res. 205, 337. Andersson, K.-E., A. Mattiasson and C. Sj6gren, 1983, Electrically induced relaxation of the noradrenaline contracted isolated urethra from rabbit and man, J. Urol. 129, 210. Andersson, K.-E., A. Garcia-Pascual, A. Forman and A. T0ttrup, 1991, Non-adrenergic, non-cholinergic nerve-mediated relaxation of rabbit urethra is caused by nitric oxide, Acta Physiol. Scand. 141, 133. Andersson, K.-E., A. Garcia-Pascual, K. Persson, A. Forman and A. TC~ttrup, 1992, Electrically-induced, nerve-mediated relaxation of rabbit urethra involves nitric oxide, J. Urol. 147, 253. Bult, H., G.E. Boeckxstaens, P.A. Pelckmans, F.H. Jordaens, Y.M. Van Maercke and A.G. Herman, 1990, Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature 345, 346. De Beurme, F.A. and R.A. Lefebvre, 1988, Vasoactive intestinal polypeptide as Possible mediator of relaxation in the rat gastric fundus, J. Pharmacol. 40, 711. Dokita, S., W.R. Morgan, M.A. Wheeler, M. Yoshida, J. Latifpour and R.M. Weiss, 1991, NG-Nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle, Life Sci. 48, 2429. Ellis, J.L. and S.G. Farmer, 1989, The effects of vasoactive intestinal peptide (VIP) antagonists, and VIP and peptide histidine isoleucine antisera on non-adrenergic, non-cholinergic relaxations of tracheal smooth muscle, Br. J. Pharmacol. 96, 513. Fahrenkrug, J., 1979, Vasoactive intestinal polypeptide; measurement, distribution and putative neurotransmitter function, Digestion 19, 149. Garcia-Pascual, A., G. Costa, A. Garcia-Sacristan and K.-E. Andersson, 1991, Relaxation of sheep urethral muscle induced by electrical stimulation of nerves: involvement of nitric oxide, Acta Physiol. Scand. 141,531. Gibson, A., S. Mirazadeh, A.J. Hobbs and P.K. Moore, 1990, L-N 6Monomethyl arginine and L-NG-nitro arginine inhibit non-adrenergic, non-cholinergic relaxation of the mouse anococcygeus muscle, Br. J. Pharmacol. 99, 602. Gillespie, J.S., X. Liu and W. Martin, 1989, The effect of L-arginine and N°-monomethyl L-arginine on the response of the rat anococcygenus muscle to NANC nerve stimulation, Br. J. Pharmacol. 98, 1080. Grider, J.R. and J.R. Rivier, 1990, Vasoactive intestinal peptide (V1P) as transmitter of inhibitory motor neurons of the gut: evidence from the use of selective VIP antagonists and VIP antiserum, J. Pharmacol. Exp. Ther. 253, 738. Hashimoto, S., S. Kigoshi and I. Muramatsu, 1992, Neurogenic responses of urethra isolated from the dog, Eur. J. Pharmacol. 213, 117. Ito, Y. and Y. Kimoto, 1985, The neural and non-neural mechanisms involved in urethral activity in rabbits, J. Physiol. (London) 367, 57.
214 Klarskov, P., T.C. Gerstenberg, D. Ramirez and T. Hald, 1983, Non-cholinergic, non-adrenergic nerve mediated relaxation of trigone, bladder neck and urethral smooth muscle in vitro, J. Urol. 129, 848. Larsen, J.-J., B. Ottesen, J. Fahrenkrug and L. Fahrenkrug, 1981, Vasoactive intestinal polypeptide (VIP) in the male genitourinary tract, Invest, Urol. 19, 211. Li, C.G. and M.J. Rand, 1990, Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus, Eur. J. Pharmacol. 191,303. Mattiasson, A., K.-E. Andersson and C. Sj6gren, 1985, Contractant and relaxant properties of the female rabbit urethral submucosa, J. Urol. 133, 304. Mattiasson, A., K.-E. Andersson, P.-O. Andersson, B. Larsson, C. Sj6gren and B. Uvelius, 1990, Nerve-mediated functions in the circular and longitudinal muscle layers of the proximal female rabbit urethra, J. Urol. 143, 155. Moncada, S., R.M.J. Palmer and E.A. Higgs, 1989, Biosynthesis of nitric oxide from L-arginine, Biochem. Pharmacol. 38, 1709.
Miilsch, A. and R. Busse, 1990, NG-Nitro-L-arginine (N 5[imino(nitroamino)methyl]-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine, Naunyn-Schmiedeb. Arch. Pharmacol. 341, 143. Pandol, S.J., K. Dharmsathaphoren, M.S. Schoeffield, W. Vale and J. Rivier, 1986, Vasoactive intestinal peptide receptor antagonist [4-CI-D-Phe6,LeulT]vIP, Am. J. Physiol. 250, G553. Rodriguez-Pena, M,S., L.G. Guijarro and J.C. Prieto, 1991, Vasoactive intestinal peptide receptor antagonists in rat seminal vesicle membranes, Eur. J. Pharmacol. 208, 207. SjSgren, C., K.-E. Andersson and A. Mattiasson, 1985, Effects of vasoactive intestinal polypeptide on isolated urethral and urinary bladder smooth muscle from rabbit and man, J. Urol. 133, 136. Toda, N. and T. Okamura, 1991, Role of nitric oxide in neurally induced cerebroarterial relaxation, J. Pharmacol. Exp. Ther. 258, 1027. Tucker, J.F., S.R., Brave, L. Charalambous, A.J. Hobbs and A. Gibson, 1990, L-NG-Nitro arginine inhibits non-adrenergic, noncholinergic relaxations of guinea-pig isolated tracheal smooth muscle, Br. J. Pharmacol. 100, 663.