C Pharmacology & Toxicology 2002, 91, 179–184. Copyright CPrinted in Denmark . All rights reserved

ISSN 0901-9928

Nitric Oxide Synthase/Guanylate Cyclase PathwayModulates the Rat Vas Deferens Contractility Induced by

PhenylephrineRui Pinto, Helder Mota-Filipe and Beatriz Silva Lima

Unit of Pharmacology and Pharmacotoxicology, Faculty of Pharmacy, University of Lisbon, Portugal

(Received February 12, 2002; Accepted May 7, 2002)

Abstract: The involvement of the nitric oxide synthase/soluble guanylate cyclase pathway on the modulation of phenyleph-rine-induced contractility in the rat vas deferens was investigated. Phenlylephrine-concentration response curves wereobtained in absence and in presence of inhibitors, NG-Nitro-L-arginine (L-NOARG), NG-Nitro-L-arginine methyl esther(L-NAME) or NG-monomethyl-L-arginine (L-NMMA) or GC inhibitior, 1H-(1,2,4)-oxadiaziol-(4,3-a)quinoxalin-1-one(ODQ) or nitric oxide donor, 3-morpholinosydnonimine hydrochloride (SIN-1) alone or together with L-NMMA orODQ. Both nitric oxide synthase and GC inhibitors reduced the Phe-Emax. SIN-1 alone did not change phenylephrine-induced responses and it could reverse the L-NMMA effect but not ODQ effect. The reduction of the phenylephrine-induced contractility obtained in consequence of the inhibition of the nitric oxide/GC pathway suggest that, in the rat vasdeferens, despite its well identified relaxant properties, nitric oxide potentiates the contractility induced by adrenergicstimulation.

The first biological action identified for nitric oxide was itsrole as a second messenger involved in relaxation of bloodvessels (Ignarro et al. 1987).

It is now recognised that nitric oxide can act as a neur-onal messenger in the central and peripheral nervous sys-tems (Hoyle & Burnstock 1995) and also that it can modu-late the cellular responses induced by several neurotrans-mitters such as acetylcholine and norepinephrine (Rattan &Thatikunta 1993; Bartho & Lefebvre 1994a).

The release of nitric oxide-derived substances has beendemonstrated in several preparations with the use of mol-ecules which inhibit the enzyme responsible for its synthesis,the nitric oxide synthase. The effect of L-arginine analogueslike NG-Nitro-L-arginine (L-NOARG), NG-Nitro-L-argi-nine methyl esther (L-NAME) NG-monomethyl-L-arginine(L-NMMA) on cellular responses is therefore widelystudied in order to evaluate the role of the nitric oxide/GCpathway on such responses (Vladimirova et al. 1994).

In smooth muscle preparations from several systems, themajority of the information available suggests a relaxanteffect for nitric oxide/cGMP (Bucher et al. 1992). A relax-ant effect of nitric oxide has therefore been described inseveral regions of the gastrointestinal tract (Lefebvre et al.1994), in the respiratory tract (Li & Rand 1991) and inthe urogenital tract where nitrergic mechanisms have beenshown to influence the micturation at either the urethral orthe urinary bladder level (Nishizawa et al. 1992; Persson &Andersson 1992; Persson et al. 1992; Bennet et al. 1995).

Author for correspondence: Beatriz Silva Lima, Unit of Pharma-cology and Pharmacotoxicology, Faculty of Pharmacy, Universityof Lisbon, Av. Forcas Armadas, 1600–083 Lisbon, Portugal (faxπ351 21 7957458, e-mail beatrizlima/net.sapo.pt).

Although the action of nitric oxide in smooth muscle prep-arations is mainly reported as relaxant, there is some evi-dence that it may also display pro-contractile effects atsome levels, like the small intestine, where an excitatory rolefor nitrergic stimulation has been proposed (Bartho et al.1992; Bartho & Lefebvre 1994; 1994a & b).

In the urogenital tract a modulatory role for nitric oxidehas also been identified. Nitric oxide relaxes the smoothmuscle from corpus cavernosum via cGMP increase, con-tributing to increased penile blood flux and subsequent pen-ile erection (Ignarro et al. 1990; Kim et al. 1991). Nitricoxide synthase has been localised in several structures ofthe male genital tract, namely the seminal vesicles and vasdeferens smooth muscle coat (Jen et al. 1996) but the roleof nitric oxide at this level is not clarified. However, someauthors have described nitric oxide as a neuromodulator ofsympathetic transmission in the rat vas deferens (Vladimi-rova et al. 1994; Postorino et al. 1998) and it increases theelectrical stimulation–induced contractility in the guinea pigvas deferens (Ventura et al. 1998).

In order to contribute to the clarification of the role ofthe nitric oxide/cGMP pathway in the contractility of thevas deferens, we have analysed the effect of nitric oxide syn-thase inhibition using several molecular tools, the effect ofguanylate cyclase inhibition and the effect of a nitric oxidedonor on the concentration-response curves of rat vas de-ferens for phenylephrine-induced contractions.

Materials and Methods

Animals and preparations. Male Wistar rats, weighing 220–250 g, were used in this study. The rats were anaesthetised

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with ether and then killed by exsanguination and both vasdeferens were removed, cleaned of surrounding tissue andplaced in Mg2π-free Krebs-Henseleit solution with the fol-lowing composition: NaCl 113 mM, NaHCO3 29 mM, KCl4.7 mM, KH2PO4 1.2 mM, CaCl2 2.5mM and glucose 11.5mM gassed with 95% O2 and 5% CO2 and kept at 37 æ.Mg2π was not included in the perfusion solution since ithas been described to reduce the magnitude of contractileresponses through an antagonism with Ca2π. (Rico & Costa1978; Macara & Rico 1989).

The whole vas deferens was mounted in 20 ml organbaths. The preparations were allowed to stabilize for 30min. under a resting tension of 500 mg and washed every15 min. before the addition of phenylephine for construc-tion of concentration-response curves. Isometric responseswere recorded using a force transducer (TRI 201 – LET-ICA) connected to a LETICA 2006 polygraph.

Contractile responses induced by phenylephrine: model vali-dation. To ensure that vas deferens preparations were stabil-ized at the beginning of each concentration-response curve,a submaximal concentration of phenylephrine (1.0 or 2.0mM) was used until a reproducible contractile response wasobtained. Each preparation was used for two successiveconcentration-response curves, with 30 min. of stabilizationbetween the two curves, the first in absence (control curve)the second in presence of the tested drug(s). The reprodu-cibility of this model was tested using this protocol in ab-sence of drugs in both curves.

Effect of nitric oxide synthase/guanylate cyclase inhibition onphenylephrine-induced contractility. Non-cumulative con-centration-response curves were obtained using isolated in-cremental concentrations of phenylephrine (1.0–32.0 mM),applied every 10 min., first in the absence (control curve)and subsequently in the presence of one of the nitric oxidesynthase inhibitors or of the guanylate cyclase inhibitortested. In subsequent experiments the effect of further ad-dition of a nitric oxide donor was studied, to confirm theinvolvement of nitric oxide synthase pathway in the modifi-cations observed.

Analysis of parameters. The concentration-response curveparameters Emax and EC50%, were calculated by a non-lin-ear regression analysis using a specific software (Enzfitter).Results are given as mean (m) ∫S.E.M., where n is the num-ber of preparations from different animals.

Drugs. The following drugs were used in the concentration-response curves: Phenylephrine hydrochloride, L-NOARG,D-NOARG, L-NMMA, L-NAME, D-NAME, 1H-(1,2,4)-oxadiaziol-(4,3-a)quinoxalin-1-one (ODQ) and 3-morpholi-nosydnonimine hydrochloride (SIN-1) (all purchased fromSIGMA-ALDRICH, Madrid, Spain). All drugs, exceptODQ, were dissolved in distilled water and the working solu-tions were made in Krebs-Henseleit solution. The ODQ wasdissolved in dimethylsulfoxide (DMSO) and diluted in

Fig. 1. Effect of NOS inhibition on the Phe-c.r.c. A – c.r.c. in ab-sence and in presence of L-NOARG (0.001 and 0.01 mM). B – c.r.c.in absence and in presence of L-NAME (0.01 and 0.1 mM). C –c.r.c. in absence and in presence of L-NMMA (0.1 mM). The NOSinhibition induced a concentration-dependent decrease of the vasdeferens contractility. No changes were observed in EC50% (nΩ6,* P versus control ∞0.05).Abbreviations for fig. 1–3: Phe: phenylephrine; COROC: concen-tration response curve, NOS: nitric oxide synthase; GC: guanylatecyclase, NO: nitric oxide.

Krebs-Henseleit solution ([DMSO]finalΩ0.25%v/v). Care wastaken to avoid light exposure of phenylephrine, ODQ andSIN-1.

181NITRIC OXIDE/GUANYLATE CYCLASE MODULATES VAS DEFERENS CONTRACTILITY

Statistical analysis. Comparisons were made by paired Stu-dent’s t-test and PÆ0.05 was considered significant.

Results

Contractile responses induced by phenylephrine: model vali-dation.

In the experiments for model validation as described pre-viously no changes were observed in the phenylephrine Emax

(1351 ∫ 108 and 1333 ∫ 101 mg, nΩ6) and EC50% (2.1 ∫0.3 and 2.7 ∫ 0.4 mM) from the two successive concen-tration-response curves. It can therefore be concluded that,in this model, all the differences observed between the first(control) and the second concentration-response curves inthe subsequent protocols used, might have been related tothe tested drug(s).

Contractile responses induced by phenylephrine in presenceof nitric oxide synthase inhibitors.

Phenylephrine (1.0–32.0 mM) induced concentration-de-pendent contractions of the rat isolated vas deferens. Fig. 1shows the concentration-response curves obtained in ab-sence (control curve) or in presence of the nitric oxide syn-thase inhibitors L-NOARG (0.001 and 0.01 mM) (fig. 1A)or L-NAME (0.01 and 0.1 mM) (fig. 1B) or L-NMMA (0.1mM) (fig. 1C). It can be seen that nitric oxide synthase inhi-bition induced a concentration-dependent decrease of thevas deferens contractility. No changes were observed inEC50%. In order to justify the stereospecificity of our resultswe have also studied the effect of D-NOARG (0.01 mM)and D-NAME (0.1mM) in the concentration-response curveparameters. No changes in Emax and EC50% were observedbetween the curves obtained in presence of D-NOARG(Emax 103.2∫11.2% of control, EC50% 1.5∫0.1 mM) or D-NAME (Emax 94∫4.3% of control, EC50% 1.6∫0.3 mM) and

Fig. 2. Effect of GC inhibition on the Phe-c.r.c. in absence and inpresence of ODQ (0.01, 1 and 100 mM). The GC inhibition by ODQinduced a concentration-dependent decrease of the vas deferenscontractility. No changes were observed in EC50% (nΩ5, * P versuscontrol ∞0.05).

Fig. 3. Effect of a NO donor on the Phe-c.r.c. A – c.r.c. in absenceand in presence of SIN-1 (10 mM). B – c.r.c. in absence and inpresence of L-NMMAπsin-1 (0.1π10 mM). C – c.r.c. in absenceand in presence of ODQπsin-1 (100π10 mM). SIN-1 could reversethe effect of the L-NMMA. This effect was not observed when theGC had been inhibited by ODQ. No changes were observed inEC50% (nΩ8, * P versus control ∞0.05).

the control curves (Emax 100%, EC50% 1.3∫0.2 mM and1.2∫0.1 mM).

Contractile responses induced by phenylephrine in presenceof a GC inhibitor.

To ensure that the ODQ vehicle ([DMSO]finalΩ0.25%v/v inKrebs-Henseleit solution) did not modify the stability of vas

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deferens preparations, we tested the reproducibility of twosuccessive concentration-response curves in absence (con-trol) and in presence of DMSO 0.25% v/v. No changes inconcentration-response curve parameters were observed (re-sults not shown).

Fig. 2 shows the concentration-response curves obtainedin absence (control curve) or in presence of ODQ (0.01, 1and 100 mM), a soluble guanylate cyclase inhibitor. It canbe concluded that guanylate cyclase inhibition induced aconcentration-dependent decrease of the vas deferens con-tractility. No changes were observed in EC50%.

Contractile responses induced by phenylephrine in presenceof the nitric oxide donor ªSIN-1.

Fig. 3 shows the concentration-response curves obtained inthe absence (control curve) or in the presence of SIN-1 (10mM) (fig. 3A) or L-NMMAπSIN-1 (0.1 mMπ10 mM) (fig.3B) or ODQπSIN-1 (100 mMπ10mM) (fig. 3C). The nitricoxide donor SIN-1 could reverse the effect of the L-NMMAon the phenylephrine-induced vas deferens contractility.This effect was not observed when the GC had been pre-viously inhibited by ODQ. No changes were observed inEC50%.

Discussion

In the present study the involvement of the nitric oxide syn-thase/guanylate cyclase pathway on the vas deferens con-tractility was studied in several series of pharmacodynamicexperimental protocols. Phenylephrine was shown to induceconcentration-dependent contractile responses which weredecreased in the presence of any of the nitric oxide synthaseinhibitors used, L-NOARG, L-NAME but not D-NOARGand D-NAME, L-NMMA and also in the presence of theGC inhibitor ODQ.

The stereospecificity of the L-NOARG and L-NAME ac-tion and the qualitative similarity of the effects of the nitricoxide synthase and the guanylate cyclase inhibitors stronglyargue in favour of an involvement, in the rat vas deferens,of the nitric oxide synthase/guanylate cyclase pathway andnitric oxide on the adrenergic-induced stimulation. How-ever, the nitric oxide effect as suggested by our data resultedin a potentiation rather than in a depression of the contrac-tility which should be expected in line with the relaxantproperties that are well described in the smooth muscle sys-tems. Indeed, the known relaxant effect at the vascular levelhas also been widely described at the respiratory level,where nitric oxide was shown to relax the tracheal and thebronchial smooth muscle (Li & Rand 1991; Bai & Bramley1993), at the urinary system where nitric oxide decreasedthe cholinergic-induced tonus of the detrusor (Persson &Andersson 1992; Persson et al. 1992) and at the gastrointes-tinal level, in the stomach and the intestine (Boeckxstaenset al. 1991; Lefebvre et al. 1994).

The effects of nitric oxide on vas deferens contractility isnot widely studied yet but some authors have also con-

cluded, using different approaches on a potentiation of con-traction at this level.

Ventura et al. (1998) have shown that, in the guinea-pigvas deferens, sodium nitroprusside could increase the con-tractions evoked by electric field stimulation but not by ATPor noradrenaline and concluded that sodium nitroprussideenhances electrically evoked contractions of the preparationby reducing the threshold voltage for action potential firingin smooth muscle cells. Although in our experiments thenitric oxide donor SIN-1 did not significantly increase thephenylephrine-induced contractions, it could clearly reversethe depressive effect of L-NMMA on the phenylephrine-concentration-response curves, leading to Emax values closeto the control ones. In the same direction, when ODQ wasapplied with SIN-1, the depression of phenylephrine-Emax

also observed with ODQ alone was not reversed, thus sug-gesting that guanylate cyclase is involved in the modulationby nitric oxide (SIN-1) of the phenylephrine-induced con-traction. Put together, the effects of either nitric oxide syn-thase inhibition or guanylate cyclase inhibition on the phen-ylephrine-induced concentration response curves clearlysuggest a stimulating effect for nitric oxide resulting in in-creased contractility of the rat vas deferens. The mechanismfor such effect needs to be clarified and several plausibleexplanations may be anticipated, which propose that nitricoxide may act as co-transmitter or modulate the levels ofneurotransmitter in the nerve terminals. Previously, Ventu-ra & Burnstock (1997) have described that high concen-trations of L-NAME (3 mM) could decrease the contractileresponses of rat vas deferens to electrical field stimulation.However, no effect was observed for 1 nM–1 mM of L-NAME which correspond to the concentrations range usedin our protocols (10 and 100 nM). No effect was also de-scribed for sodium nitroprusside or L-arginine on the sameset of experiments. In the same work, when the prepara-tions were stimulated with 10 mM noradrenaline or 1mMATP, no effects on the induced contractile responses wereobserved by the previous administration of L-NAME, L-arginine or sodium nitroprusside. These authors concludedthat, despite the well documented presence of nitric oxidesynthase-immunoreactive nerves in the vas deferens, theirroles does not appear to be a contractile one. These findingsand conclusions are in disagreement with our results. How-ever, important methodological differences can be pointedout between the protocols used, which might, at least par-tially, explain the lack of concordance. Indeed, while in ourexperiments we have studied the effects of L-NAME in thephenylephrine-dose-response curves, and the preparationswere allowed to equilibrate for 30 min. at least in the pres-ence of the nitric oxide synthase inhibitors, Ventura &Burnstock (1997) have only tested one concentration ofeach stimulant. More importantly, L-NAME was addedonly 1 min. before the stimuli, either electrical or chemical.The short time allowed for L-NAME to equilibrate withthe preparation and inhibit the nitric oxide synthase enzymemight even explain why a depressant effect on the electri-cally-stimulated preparation could be seen only with the

183NITRIC OXIDE/GUANYLATE CYCLASE MODULATES VAS DEFERENS CONTRACTILITY

very high concentration of 3 mM. In line with this, as aplausible explanation for the discrepancy between theirfindings and those reported by others like Vladimirova et al.(1994), the authors suggested that a poor access to nitrergicnerves which are located in the inner muscle layers mighthave occurred in the experiments with intact vas deferenspreparations. We tend to agree and further suggest that thismight also have been the case even when longitudinal stripswere used to mimic Vladimirova et al. (1994), who howeverhave allowed the preparations to stabilise for 10 min. in thepresence of L-NAME. At the intestine, Bartho & Lefebvre(1994a & b) have also described a pro-contractile effect fornitric oxide. They have shown that such an effect might bedue to the nitric oxide-induced release of acetylcholine fromcholinergic nerves. Rattan & Thatikunta (1993) showedthat nitric oxide may play a significant role in the facilitato-ry modulation of sympathetic responses in the internal analsphincter.

In the rat vas deferens, Postorino et al. (1998) suggestedthe existence of a facilitatory prejunctional modulatory rolefor nitric oxide in the sympathetic neurotransmission andpostulated that endogenous nitric oxide released in theextracellular space is presumed to potentiate neurotransmis-sion by acting at prejunctional level via cGMP. These pro-posals might fit with our results.

Also in the rat vas deferens Vladimirova et al. (1994) havedescribed a L-NAME-induced depression of both the fastand the slow components of contractile responses to intra-mural nerve stimulation. Provided that intramural nervestimulation induces release, Vladimirova et al. (1994) putforward several hypothesis to explain the nitric oxide-in-duced increased contractility as suggested by the conse-quences of nitric oxide synthase inhibition: nitric oxide hasan indirect action by a) increasing the release of neurotrans-mitters during nerve stimulation or b) helping to sustain anappropriate resting concentration of the neurotransmittersin nerve terminals. Nitric oxide would therefore act as a co-transmitter at the adrenergic terminals of the rat vas defer-ens. However, in the same set of experiments Vladimirovaet al. (1994) could not show a modulatory effect on theexogenously applied noradrenaline-induced contractility.This is not in agreement with our results where phenyleph-rine was applied and a reduction of the contractions by ni-tric oxide synthase or GC inhibition was clearly observed.Nevertheless, in our model, if a stimulation-induced releaseof endogenous neurotransmitter has occurred and has con-tributed to the final contractile response, this componentcould not be differentiated from the one induced by theexogenous phenylephrine.

The presence of immunoreactive nerves for nitric oxidesynthase and tyrosine hydroxylase in the human neonatalmale genitourinary organs, including the vas deferens, havebeen described (Jen et al. 1996) and can support the hypoth-esis that both NE and nitric oxide can be synthesized insame noradrenergic nerves. Jen et al. (1996) have thereforeconcluded that the ability to synthesise nitric oxide is notconfined to parasympathetic nerves but is also present in

sympathetic neurones supplying the human neonatal uro-genital tract. The existence of a co-transmission at suchlevel needs to be confirmed.

In conclusion, in the rat vas deferens the nitric oxide syn-thase/guanylate cyclase pathway is involved in the modu-lation of the contractility induced by adrenergic stimula-tion. An increase of the phenylephrine-induced contractilitywas observed. The mechanism for such an increase is notclarified but co-transmission at the adrenergic nerve ter-minals cannot be excluded.

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