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Aphallia, or penile agenesis, is rare (1:30 million) 1 and associated with other urogenital anomalies. 2 Under 100 cases of aphallia have been reported worldwide. 1 Pathogenesis is uncertain but thought to be failure of formation of the genital tubercle. 2 We present a case of complete penile agenesis with anorectal and urological malformations., (Copyright © 2020. Published by Elsevier Inc.)

Background and Purpose: Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders., Experimental Approach: Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders., Key Results: Hyoscine (3 micromol x L(-1)), but not tetrodotoxin (TTX, 1 micromol x L(-1)), reduced basal tension, distension-evoked contractile activity and physostigmine (1 micromol x L(-1))-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. omega-Conotoxin GVIA (0.3 micromol x L(-1)) did not affect physostigmine-induced contractions when given either alone or in combination with omega-agatoxin IVA (0.1 micromol x L(-1)) and SNX 482 (0.3 micromol x L(-1)). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5-500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 micromol x kg(-1) i.v.)., Conclusions and Implications: The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca(2+) channels.

We have investigated the effect of intravaginal application of capsaicin on micturition reflex in female rats. Urinary bladder contractility was measured by transurethral pressure recording at isovolumetric and subthreshold conditions in anaesthetized rats. The intravaginal application of capsaicin (15 microg/50 microl rat) induced reproducible bladder phasic contractions, without desensitization upon repeated applications, that were blocked by intravenous atropine (1 mg/kg) or hexamethonium (5 mg/kg) and prevented by removal of paracervical ganglia or systemic capsaicin pretreatment (125 mg/kg, s.c.). The inhibition of sympathetic transmission by guanethidine (30 mg/kg, s.c.) produced significant increase of the bladder reflex contractions activated by intravaginal capsaicin. Intravenous administration of the TRPV1 antagonist, capsazepine (3 mg/kg), significantly reduced the excitatory reflex response to capsaicin. Intravaginal administration of capsaicin (15 microg/50 microl), during distension-induced reflex bladder contractions, produced a transient block of reflexes, unaffected by guanethidine pretreatment. In conclusion, the stimulation of capsaicin-sensitive sensory nerve endings in the rat cervix-vagina induced a dual excitatory or inhibitory bladder response in anaesthetized female rats depending on the degree of bladder distension.

We have investigated the mechanism through which hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat isolated urinary bladder. Sodium hydrogen sulfide (NaHS), a donor of H2S, produced concentration-dependent contractile responses (pEC50=3.5+/-0.1) that were unaffected by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine (30 microM) and SB 366791 (10 microM) and by the N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CTX; 100 nM). In contrast, the unselective transient receptor potential (TRP) cation channels blocker ruthenium red (30 microM) almost abolished NaHS-induced contractions. Ruthenium red (30 microM) greatly reduced capsaicin-induced contractions, whereas it did not attenuate the contractile response to neurokinin A. The putative TRPV1 receptor antagonist iodo-resiniferatoxin, from 100 nM upward, produced agonist responses per se, and could not be tested against NaHS. We conclude that H2S either acts at TRPV1 receptorial sites unblocked by capsazepine or SB 366791, or stimulates a still unidentified transient receptor potential-like channel co-expressed with TRPV1 on sensory neurons.

In the rat isolated urinary bladder, NaHS (30 microm-3 mm) and capsaicin (10 nm-3 microm) produced concentration-dependent contractile responses (pEC(50)=3.5+/-0.02 and 7.1+/-0.02, respectively) undergoing dramatic tachyphylaxis. In preparations in which sensory nerves were rendered desensitized (defunctionalized) by high-capsaicin (10 microm for 15 min) pretreatment, neither capsaicin itself nor NaHS produced any motor effect. NaHS-induced contractile effects were totally prevented by the simultaneous incubation with tachykinin NK(1) (GR 82334; 10 microm) and NK(2) (nepadutant; 0.3 microm) receptor-selective antagonists. Tetrodotoxin (1 microm) only partially reduced the response to NaHS. These results provide pharmacological evidence that H(2)S stimulates capsaicin-sensitive primary afferent nerve terminals, from which tachykinins are released to produce the observed contraction by activating NK(1) and NK(2) receptors. While the molecular site of action of H(2)S remains to be investigated, our discovery may have important physiological significance since H(2)S concentrations capable of stimulating sensory nerves overlap those occurring in mammalian tissues under normal conditions.

The i.v. administration of nociceptin (10-100 nmol/kg) inhibits the micturition reflex in a naloxone-resistant manner. The effects induced by i.v. nociceptin were not observed in capsaicin-pretreated animals indicating that i.v. nociceptin inhibits the micturition reflex by inhibiting afferent discharge from capsaicin-sensitive nerves. Supporting this interpretation, nociceptin also inhibited the reflex but not the local bladder contraction induced by topical capsaicin and protects this reflex (but not the local contraction) by desensitization. Intrathecal nociceptin (10 nmol/rat) produces urodynamic modifications similar to those induced by the i.v. administration. Intracerebroventricular (i.c.v.) administration of nociceptin (0.3-1 nmol/rat) also inhibited the micturition reflex in a naloxone-resistant manner suggesting a direct effect on supraspinal sites controlling the micturition. Beyond the inhibitory effects exerted by nociceptin on the micturition reflex, a peripheral excitatory effect mediated by capsaicin-sensitive fibers was also detected. The application of nociceptin (5-50 nmol/rat) onto the bladder serosa when the intravesical volume was subthreshold for the triggering of the micturition reflex, activated the reflex in a dose-dependent manner; the same treatment produced a biphasic effect on the ongoing reflex. In addition to the triggering of micturition reflex, topical nociceptin evokes a local tonic-type contraction that was abolished by the coadministration of tachykinin NK(1) and NK(2) receptor antagonists. Altogether these results indicate that ORL(1) receptors are present at several sites for the integration of the micturition reflex, and that their activation may produce both excitatory or inhibitory effects, depending on the route of administration and the experimental conditions.

We have characterized the action of the tachykinin NK(2) receptor antagonist nepadutant (c¿[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2 beta-5 beta)¿) in the human isolated ileum, colon and urinary bladder. Nepadutant (30-1000 nM) competitively antagonized neurokinin A- or [beta Ala(8)]neurokinin A-(4-10)-induced contractions in all tissues, with pK(B)=8.3 (ileum and colon) and pK(B)=8.5 (bladder). In contrast, the nonpeptide tachykinin NK(2) receptor antagonist SR 48968 (or (S)-N-methyl-N [4-acetylamino-4-phenylpiperidino)-2-(3, 4-dichlorophenyl) butyl] benzamide) (30-1000 nM) produced insurmountable antagonism in all preparations. The tachykinin NK(2) receptor blockade produced by nepadutant in the colon was fully reversed by washout, whereas that produced by SR 48968 was not. Nepadutant (1 microM) greatly reduced (by 70-80%) the nonadrenergic noncholinergic (NANC) contractile off-response evoked by electrical field stimulation in the human ileum, and almost abolished it in the presence of the tachykinin NK(1) receptor antagonist GR 82334 (or: [[(S,S) Pro-Leu (spiro-gamma-lactam)](9,10),Trp(11)]Physalaemin (1-11)) (1 microM). The present results show that nepadutant is a potent, competitive and reversible antagonist at human tachykinin NK(2) receptors and provide further evidence that tachykinins act as excitatory NANC neurotransmitters in the human small intestine.

The application of nociceptin (5-50 nmol/rat) onto the serosa in the urinary bladder of urethane-anaesthetized rats, with the intravesical volume kept below threshold for activation of the micturition reflex, induced a low amplitude tonic contraction (local, i.e., resistant to ganglionectomy) with high amplitude phasic contractions (reflex, i.e., abolished by ganglionectomy) superimposed. The pharmacology of the local contraction was studied in animals with acute bilateral ablation in the pelvic ganglia: the combined administration of tachykinin NK(1) (S)1-¿2-[3-(3, 4-dichlorophenyl)-1-(3-isopropoxyphenyl-acetyl)-piperidin-3-yl]eth yl¿-4-phenyl-1-azoniabicyclo[2.2.2.]octane chloride (SR 140333) and NK(2) c¿[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta++ +)¿ (MEN 11420) receptor antagonists (given at doses of 1+0.1 micromol/kg, intravenous (i.v.), respectively) abolished the local bladder contraction induced by topical nociceptin (50 nmol/rat). These results indicate that the topical application of nociceptin onto the bladder evokes a tachykinin-mediated contraction.

Purpose: Nociceptin, the endogenous peptide ligand for the opioid receptor-like1 (ORL1) receptors, exerts a naloxone-resistant suppressant effect on micturition reflex after intravenous administration. This work aims to elucidate the mechanism and the site of action of the inhibitory effect of nociceptin on the micturition reflex., Materials and Methods: The bladder of urethane-anesthetized rats was cannulated through the dome (cystometries) or the urethra in isovolumetric conditions (distension-induced reflex contractions, DIRCs). In this latter model, the effect of the application of nociceptin onto the serosal surface of the urinary bladder was determined. The effect of intravenous, intrathecal and intracerebroventricular administration of nociceptin on ongoing cystometries at two different infusion rates (50 and 250 microL/min.) was assessed. The effect of the intravenous administration of nociceptin on cystometries was also studied in capsaicin-pretreated animals., Results: When cystometric recordings were obtained at a low infusion-rate (50 microL/min.), the intravenous administration of nociceptin (10 to 100 nmol./kg.) induced a dose-dependent reduction in the micturition frequency associated to an increase of the pressure threshold for activating the micturition reflex, whereas the amplitude of micturition contractions was unaffected. These effects faded within 60 minutes. The intracerebroventricular administration of nociceptin (0.3 nmol./rat) produced urodynamic changes similar to those observed after the intravenous route and, in addition, also reduced the amplitude of micturition contractions. The intrathecal administration of nociceptin up to 1 nmol./rat was ineffective. Capsaicin pretreatment (164 micromol./kg., s.c. 5 to 6 days before) significantly reduced the micturition frequency as compared with controls. In capsaicin pretreated animals intravenous nociceptin was ineffective. When cystometries were recorded at a high infusion-rate (250 microL/min.) either intravenous (100 nmol./kg.), i.t. (1 nmol./rat) nociceptin or capsaicin pretreatment had no effect. In contrast, intracerebroventricular nociceptin (0.3 and 1 nmol./rat) inhibited the micturition reflex by reducing both the frequency and the amplitude of micturition contractions: these effect were not modified by naloxone (0.5 micromol./kg., i.v.). The topical application of nociceptin (5 and 50 nmol./rat) caused a dose-dependent inhibition of DIRCs., Conclusion: Nociceptin inhibits the micturition reflex at a peripheral and at a supraspinal site. The effects observed after the intravenous administration of nociceptin indicate that the functional integrity of capsaicin-sensitive bladder afferents is required for exerting its inhibitory activity at the peripheral level. In contrast, the supraspinal effect of nociceptin involves both the afferent and the efferent pathways of the micturition reflex, possibly through a direct effect on ORL1 receptors located in the pontine micturition center.

The effect of three selective bradykinin B(2) receptor antagonists, MEN11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-1 0alpha)), Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH), and FR173567 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2, 4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylaminocarbonylmethyl]acrylamide) was evaluated in the human and rat urinary bladder in vitro and in vivo in anaesthetized rats. Bradykinin evoked a concentration-dependent contraction of human (pD(2)=7.2) and rat (pD(2)=7.7) detrusor muscle strips. In human preparations, all the antagonists tested produced a rightward-shift in the concentration-response curve for bradykinin. Schild plot analysis yielded pK(B) values of 8.4, 8.4 and 8.6 for MEN11270, Icatibant, and FR173567, respectively. In the rat preparations the three antagonists (at 100 nM concentration), produced a shift to the right which gave apparent pA(2) values of 8. 2, 8.0 and 8.1 for MEN11270, Icatibant, and FR173567, respectively. In anaesthetized rats, both MEN11270 and Icatibant (1-10 nmol/kg i.v. ) dose dependently reduced the bradykinin (100 nmol/kg i.v.)-induced urinary bladder contraction, their effect being prompt and long-lasting. In contrast, FR173567 (100 nmol/kg i.v.) produced a partial and short-lasting inhibition of bradykinin-induced bladder contractions. The present findings indicate that all the antagonists tested recognize with similar potencies the bradykinin B(2) receptors expressed in the detrusor muscle of both humans and rats. MEN11270 and Icatibant possess a higher potency and longer duration of action in vivo than FR173657, suggesting that the activity of this non-peptide antagonist in vivo is hampered by factors unrelated to its affinity for bradykinin B(2) receptors.

Chemical stimulation of primary afferent nerves in the rat urinary bladder in vivo with topical capsaicin (1 microg in 50 microl saline) determines a dual motor response, consisting of a contractile effect mediated by tachykinins released from sensory nerves in the bladder wall and a transient activation of a bladder-to-bladder micturition reflex organized at the supraspinal level (chemoceptive micturition reflex). Both responses undergo complete desensitization upon repeated applications of capsaicin. The i.v. administration of the novel neuropeptide nociceptin (100 nmol/kg) produced a long-lasting protection from capsaicin desensitization of afferent nerves which mediate the chemoceptive micturition reflex. In fact a chemoceptive micturition reflex could be repeatedly evoked by topical capsaicin in nociceptin-pretreated rats. In sharp contrast, nociceptin did not influence the development of desensitization of the local response to capsaicin, corresponding to the 'efferent' function of capsaicin-sensitive afferent neurons. These results suggest that the afferent and 'efferent' function of capsaicin-sensitive primary afferent neurons (CSPANs) in the rat bladder are differentiated by nociceptin. Alternative mechanisms underlying this phenomenon are discussed.

The rat urinary bladder is one of the few in vivo preparations in which kinin B1 receptor-mediated contractile responses have been described, but the nature (local or reflex) of these responses has not been characterized. We have investigated the motor effects of i.v. or topical (onto the bladder serosa) administration of the selective kinin B1 receptor agonist [des-Arg9]-bradykinin ([des-Arg9]-BK) in the normal or inflamed (cyclophosphamide-induced) urinary bladder in urethane-anaesthetized rats. In both normal and inflamed bladders [des-Arg9]-BK produced a tonic contraction of low amplitude (< 15 mmHg) with phasic contractions of high amplitude (> or = 15 mmHg) superimposed (micturition reflex contractions). In inflamed bladders, the response to [des-Arg9]-BK was more prominent than in controls. Similar observations were made after the topical administration of [des-Arg9]-BK. In order to evaluate any time-dependency in the expression of B1 receptor-mediated bladder responses, [des-Arg9]-BK was administered in separate groups of control animals at 30 and 240 min after the completion of surgical procedures required for set-up of the preparation: no bladder contraction was detected at 30 min whereas both local and reflex contractions could be elicited by [des-Arg9]-BK at 240 min after the set up. In ganglionectomized rats, the response to [des-Arg9]-BK or the selective tachykinin NK2 receptor agonist [betaAla8]NKA(4-10) was evaluated at 30 and 240 min after the set up in inflamed or in control animals. The response to [des-Arg9]-BK was greater after inflammation although a time-dependent increase was evident in both groups; in contrast, the response to [betaAla8]NKA(4-10) was similar in both groups and remained constant over the observation period. After induction of inflammation, the tonic contraction induced by [des-Arg9]-BK in ganglionectomized rats was dose-dependently reduced by the kinin B1 receptor antagonist [desArg10]Hoe 140. The contractile response (number of micturition reflex contractions) induced by [des-Arg9]-BK in normal rats with intact pelvic nerves at 240 min from the set up was not changed after the administration of the selective B2 receptor antagonist Hoe 140. These results indicate that stimulation of bladder kinin B1 receptors evokes a local, tonic-type contraction with reflex contractions superimposed in both normal and inflamed bladders, but in the latter situation the motor responses are magnified.

The kinin B1 receptor is generally expressed after inflammation or tissue injury. Kinin B1 receptor stimulation induces excitatory motor responses in the urinary bladder and, in this preparation, the effect of many excitatory transmitters involves the stimulation of capsaicin-sensitive afferent nerves. In this study we have investigated the effect of capsaicin pretreatment on the bladder contractions induced by [Sar0, D-Phe8, des-Arg9]bradykinin (SDABK), a kinin B1 receptor agonist, by inducing the expression of B1 receptors via the intravesical administration of a bacterial endotoxin (LPS, 1 mg/ml) in urethane-anaesthetized rats. Three and half hours after LPS, the bladder was filled with saline until the micturition reflex was evoked, then 0.15 ml of saline was withdrawn, in order to avoid spontaneous reflex contractions. In LPS-pretreated rats the threshold volume for micturition was lower than in the control group (248 +/- 44 vs. 534 +/- 112 microl). After capsaicin pretreatment the bladder capacity was increased in both control and LPS-treated groups and the LPS-induced hyperreflexia was abolished (threshold volumes: 901 +/- 96 vs. 837 +/- 120 microl, respectively). The administration of SDABK (30 nmol/kg i.v., 4 h after LPS or saline application) produced a local, low amplitude tonic contraction (< 15 mmHg) or a tonic contraction with superimposed high amplitude (> or = 15 mmHg) reflex contractions but no effect of LPS or capsaicin pretreatment was observed in the incidence of these responses. The amplitude of the local response was increased by LPS treatment (1.4 +/- 0.3 vs. 4.0 +/- 0.7 mmHg) but capsaicin pretreatment did not modify this effect (2.3 +/- 0.4 vs. 4.3 +/- 0.6 mmHg). Likewise, the number of reflex contractions induced by SDABK was increased after LPS treatment (1.1 +/- 0.4 vs. 2.7 +/- 0.5) irrespective of capsaicin pretreatment (1.3 +/- 0.4 vs. 2.8 +/- 0.6). These results indicate that: (1) topical application of LPS induces a bladder hyperreflexia that is sensitive to capsaicin pretreatment; (2) B1 receptor-mediated motor responses (either reflex or local) are enhanced after LPS treatment; (3) capsaicin pretreatment does not modify B1 receptor-mediated motor response (either reflex or local).

Purpose: To see whether a recently characterized model of bacterial toxin-induced urinary bladder inflammation (Stein et al., J. Urol. 155, 1133-1138, 1996) is associated with detrusor hyperreflexia, and whether endogenous tachykinins acting through NK2 or NK1 receptors were involved in this model., Materials and Methods: The bladder of urethane-anesthetized male Wistar rats was cannulated through the dome. Intravesical administration of protamine sulfate (PS, 10 mg./ml./rat) or vehicle for 1 hour was followed by the intravesical administration of E. coli lipopolysaccharide (LPS, 1 mg./ml./rat) or vehicle for 1 hour. Cystometries (50 microl./min.) were performed 3.5 hours after the exposure to LPS. MEN 11,420, a peptide tachykinin NK2 receptor antagonist, was administered before cystometries or, in a separate group of animals, during cystometries. The effect of SR 140,333, a non-peptide NK1 receptor antagonist, was also assessed in the presence or absence of MEN 11,420. The urodynamic effects of PS + LPS were also tested in capsaicin-pretreated rats., Results: Unlike PS or LPS alone, the intravesical administration of PS + LPS induced detrusor hyperreflexia. In PS + LPS treated animals during nonstop cystometries, the intermicturition interval was decreased by about 50% as compared to vehicle-pretreated rats. A quantitatively similar reduction in the bladder capacity was also observed. MEN 11,420 (100 nmol./kg., i.v.) restored the intermicturition interval in PS + LPS-pretreated rats at the level of controls by increasing the bladder capacity, whereas it had no effect in vehicle-pretreated rats. SR 140,333 (1 micromol./kg., i.v.) neither modified urodynamic parameters in controls and in PS + LPS-treated rats nor altered the effect of MEN 11,420 in these groups. Capsaicin pretreatment (164 micromol./kg., s.c., 4-5 days before) induced a two-fold increase of the bladder capacity in control rats and prevented PS + LPS-induced bladder hyperreflexia., Conclusions: The intravesical administration of PS + LPS produces the activation of capsaicin-sensitive afferents. Endogenous tachykinins released from these fibers act through NK2 receptors to induce detrusor hyperreflexia.

1. The effects of the tachykinin NK2 receptor antagonist MEN 11420 (100 nmol kg(-1), i.v.) and isoprenaline (400 nmol kg(-1), i.v.) were compared in a model of distension-induced bladder activity in isovolumetric conditions. MEN 11420 induced a relaxation of the basal tone of the urinary bladder that was dependent on the volume of the viscus: the effect was absent at low volumes (0.2 and 0.5 ml) and it was maximal at high volumes of distension (1 and 2 ml), approaching about 60% of the isoprenaline-induced relaxation. The relaxant effect of isoprenaline was always evident at all volumes of distension. 2. Tetrodotoxin (1-100 microM, intravesically applied) abolished distension-evoked micturition contractions, but did not prevent the relaxant effect of MEN 11420- or isoprenaline on the bladder tone. 3. The cyclo-oxygenase inhibitor S-ketoprofen (0.5 micromol kg(-1), i.v.) produced a marked decrease of the bladder tone and a concomitant reduction of bladder motility at 1 ml volume of distension. At 2 ml of distension, S-ketoprofen still decreased the minimal pressure but had no significant effect on other parameters of vesical motility. In S-ketoprofen-pretreated rats, the relaxant effect of MEN 11420 was significant at 2 but not at 1 ml of distension, and that of isoprenaline was reduced by 50% at both 1 and 2 ml. 4. Ruthenium red (10 micromol kg(-1), i.v.) had no effect at a low volume of distension (0.2 ml) or at highest volume (2 ml) but decreased the basal tone and the frequency of bladder contractions at 1 ml of distension. In ruthenium red-pretreated rats, MEN 11420 failed to decrease bladder tone at 1 ml, whereas at 2 ml the effect of MEN 11420 was not different from that observed in controls (43 vs 60% of isoprenaline-induced relaxation, respectively). 5. At both 1 and 2 ml of distension, capsaicin pretreatment (164 micromol kg(-1), s.c. 5 days before) reduced the frequency of micturition contractions but had no effect on the bladder tone. Capsaicin pretreatment prevented the relaxant effect of MEN 11420 on the bladder tone both at 1 and at 2 ml of distension. 6. It is concluded that the release of tachykinins from capsaicin-sensitive afferent nerves induced by bladder distension is resistant to tetrodotoxin and to prostaglandin synthesis inhibition. Tachykinins modulate the vesical tone by acting through NK2 receptors.

This study investigates the role of tachykinin NK1 and NK2 receptors in motor responses induced by the intravesical instillation of capsaicin in urethane-anaesthetized rats. SR 140,333 (1 micromol/kg, i.v.), a nonpeptide NK1 receptor antagonist, abolished urinary bladder contractions induced by the selective NK1 receptor agonist [Sar9]SP-sulfone (0.1-100 nmol/kg, i.v.) without affecting those induced by the NK2 receptor agonist [betaAla8]NKA(4-10). MEN 11,420 (100 nmol/kg, i.v.), a cyclic peptide NK2 receptor antagonist, abolished bladder contractions induced by [betaAla8]NKA(4-10) (0.3-300 nmol/kg, i.v.) without modifying those induced by [Sar9]SP-sulfone. Intravesical instillation of capsaicin (6 nmol/0.6 ml/rat) produced a motor response consisting in a primary contraction followed by a series of high amplitude phasic contractions. The intravesical instillation of saline (0.6 ml/rat) produced a primary contraction of lower amplitude with respect to that induced by capsaicin and the total area under the curve was also lower in saline-instilled rats, however the number and the amplitude of phasic contractions was similar to that induced by capsaicin. MEN 11,420 (100 nmol/kg, i.v.) did not modify motor responses induced by the intravesical administration of saline. In contrast, in capsaicin-instilled rats, MEN 11,420 (100 nmol/kg, i.v.) reduced the primary contraction, the area under the curve and also the number of phasic contractions. SR 140,333 (1 micromol/kg, i.v.) reduced the primary contraction but not other parameters. The combination of SR 140,333 (1 micromol/kg, i.v.) and MEN 11,420 (100 nmol/kg, i.v.) produced an additive inhibitory effect on the primary contraction but not a further inhibition on other parameters with respect to that observed with MEN 11,420 alone. In hexamethonium (110 micromol/kg, i.v.)-pretreated animals the intravesical instillation of capsaicin produced a tonic contraction having greater amplitude and area than that induced by saline. MEN 11,420, but not SR 140,333, significantly reduced the bladder response to capsaicin in hexamethonium-pretreated rats. Again, the combined administration of MEN 11,420 and SR 140,333 did not produce further inhibitory effect in comparison to MEN 11,420 alone. It is concluded that the motor responses induced by the intravesical instillation of capsaicin are mediated by the activation of peripheral tachykinin NK2 receptors.

1. We have studied several tachykinin NK2 receptor antagonists, bearing a monocyclic pseudopeptide (MEN 10,508, MEN 10,573, MEN 10,581, MEN 10,612, MEN 10,619 and MEN 10,677), or bicyclic peptide (MEN 10,627, MEN 10,692, MEN 10,771, MEN 10,882 and MEN 10,993) structure, on the human isolated urinary bladder detrusor muscle against neurokinin A as an agonist, and compared their affinities in this preparation with those for NK2 receptors expressed in the rabbit isolated pulmonary artery and hamster isolated trachea. 2. In the human bladder, all the antagonists tested produced a concentration-dependent and competitive antagonism of neurokinin A-mediated contractions: among the cyclic pseudopeptides MEN 10,677 (pKB = 8.0) was the most potent antagonist, while among the bicyclic analogues it was MEN 10,993 (pKB = 8.8). 3. In general, the bicyclic peptide antagonists tested were more potent than the monocyclic pseudopeptide compounds, either in the human urinary bladder or in the rabbit pulmonary artery or hamster trachea, showing a nanomolar affinity for the human NK2 receptor. 4. A highly significant correlation was found between the estimated pKB values of all the antagonists tested in the human urinary bladder and rabbit pulmonary artery (r2 = 0.94, n = 12, P < 0.01), whereas no linear correlation was found between pKB values measured in the human urinary bladder and hamster trachea (r2 = 0.52, n = 12, P > 0.05): these observations provide further pharmacological evidence for receptor homology between the human and rabbit NK2 receptor. 5. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN 10,627, as candidates for testing in pathological conditions, such as bladder hyperactivity, for which preclinical evidence indicates that a therapeutic effect could result from the block of the tachykinin NK2 receptor.

The aim of this study was to assess the effect of blocking the axonal transport of sensory neuropeptides, by local injection of colchicine at pelvic ganglia level, on the sensory and efferent functions mediated by capsaicin-sensitive primary afferent neurons innervating the rat urinary bladder. Bilateral injection of colchicine in the prostatic tissue underneath the pelvic ganglia of male rats induced a time-dependent reduction (maximal at 72 h, 100% reduction) of the in vitro contraction of the bladder strips induced by capsaicin (1 microM). The response to electrical field stimulation was also reduced, although to a lesser extent. The direct contractions induced by substance P (100 nM) or KCl (80 mM) were not affected by colchicine pretreatment. In vivo, perigangliar injection of colchicine (72 h before) greatly increased bladder capacity, and reduced the amplitude of micturition contractions and micturition frequency. Capsaicin-induced plasma protein extravasation was abolished in the urinary bladder and reduced in the distal, but not the proximal ureter of colchicine-treated rats. Topical application of capsaicin onto the urinary bladder or onto the stomach induced a cardiovascular pressor reflex in urethane-anaesthetized, spinalized rats. Colchicine pretreatment reduced (by about 50%) the pressor response elicited by chemonociceptive stimulation of the bladder but not that arising from the stomach. Colchicine pretreatment did not produce overt changes of nerve profiles immunoreactive for calcitonin gene-related peptide- or tachykinin-like material in the rat urinary bladder. A more intense staining of nerve fibres positive for calcitonin-gene related peptide-like immunoreactivity and tachykinin-like immunoreactivity was observed in pelvic ganglia of colchicine-pretreated rats. No changes were detected in the dorsal horns of spinal cord segments where pelvic bladder afferents project (L6-S1). Colchicine pretreatment reduced, but did not abolish, bladder levels of substance P-, neurokinin A-, calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity. However, vasoactive intestinal peptide-like immunoreactivity levels were not changed. The capsaicin-evoked (1 microM) release of calcitonin gene-related peptide was abolished in capsaicin as well as in colchicine-pretreated animals. The present findings demonstrate that local treatment of pelvic ganglia with colchicine totally eliminates the "efferent" functions of capsaicin-sensitive afferent nerves in the urinary bladder. Although reduced, tissue levels of sensory neuropeptides are not completely depleted, thus indicating the existence of a releasable versus non-releasable pool. The chemically induced blockade of axoplasmic transport also induces a limited impairment of the sensory function of capsaicin-sensitive afferents, and of the parasympathetic efferent system.

1. The topical application of bradykinin (BK) (0.05-5000 pmol/rat) onto the serosal surface of the urinary bladder in urethane-anaesthetized rats, evoked low amplitude tonic contractions (not exceeding 25 mmHg) or high amplitude (about 50 mmHg), phasic reflex contractions (chemoceptive micturition reflex) which were abolished by bilateral ablation of the pelvic ganglia. In ganglionectomized rats, BK induced only a local, tonic-type contraction. 2. Systemic capsaicin pretreatment (164 mumol kg-1, 4 days before) reduced the incidence of chemoceptive reflex induced by BK (500 pmol/rat) but had no effect on the magnitude of the tonic-type contraction elicited by BK in ganglionectomized rats. Indomethacin (11 mumol kg-1, 20 min before) reduced the incidence but not the amplitude of the reflex contractions induced by topical application of BK (500 pmol/rat). In ganglionectomized rats, indomethacin (11 mumol kg-1, 20 min before) decreased the amplitude of the tonic contraction evoked by BK. Indomethacin did not affect the chemoceptive reflex induced by topical application of capsaicin (15 nmol/rat) onto the bladder. 3. Intrathecal administration of the tachykinin NK1 receptor antagonists, RP 67,580 (10 nmol/rat) or SR 140,333 (10 nmol/rat), abolished the chemoceptive reflex induced by BK without modifying the magnitude of the tonic contraction. SR 140,333 (10 nmol/rat) also abolished the occurrence of the chemoceptive reflex induced by capsaicin. 4. Intravenous administration of the B2 receptor antagonist, Hoe 140 (35 nmol kg-1, 10 min before) abolished the reflex and local effects induced by BK on bladder motility but failed to modify the chemoceptive reflex induced by topical application of capsaicin (15 nmol/rat). 5. Intrathecal administration of Hoe 140 (10 nmol/rat) reduced the incidence of the chemoceptive reflex induced by BK but had no effect on the amplitude of the local motor response. Likewise, Hoe 140(10 nmol/rat, i.t.) reduced the incidence of reflex bladder contractions induced by topical application of capsaicin (15 nmol/rat) without affecting the magnitude of the tonic-type contraction.6. These findings indicate that BK stimulates motility through B2 receptors in the rat urinary bladder.BK activates the reflex response by stimulating capsaicin-sensitive afferent nerves with a contribution from prostanoids. At the spinal cord level, tachykinin NK1 and BK B2 receptors could also be involved in the chemoceptive reflex induced by BK or capsaicin.

The ability of the selective tachykinin NK1 and NK2 receptor agonists, [Sar9] substance P (SP) sulfone and [beta Ala8] neurokinin A (NKA), respectively, and neurokinin B (NKB) to stimulate urinary bladder contractions was determined in urethane-anaesthetized rats with intact bladder innervation and in animals with acute, bilateral ablation of pelvic ganglia. In addition, tachykinin receptors mediating the response to the agonists were characterized by means of the non-peptide NK1 and NK2 receptor selective antagonists, RP 67,580 and SR 48,968 respectively. In both experimental conditions (normal and ganglionectomized), the three tachykinin agonists induced a dose-dependent increase in intravesical pressure, however reflex bladder contractions were produced by the agonists only in animals with intact bladder innervation. RP 67,580 (10 mumol/kg, i.v.) reduced the response to [Sar9]SP sulfone (50 pmol/rat) in both preparations without modifying the effects induced by the NK2 receptor agonist. On the other hand, SR 48,968 (1 mumol/kg, i.v.) antagonized responses induced by [beta Ala8] NKA (50 pmol/rat) but not those evoked by [Sar9] SP sulfone. In animals with intact urinary bladder innervation, the effect of NKB (50 pmol/rat) was inhibited by SR 48,968 (1 mumol/kg, i.v.) but not by RP 67,580; on the contrary, in rats with ablation of pelvic ganglia, the direct bladder contraction induced by NKB was reduced by RP 67,580 (10 mumol/kg, i.v.) but not by SR 48,968. We conclude that NKB induces reflex and direct bladder muscle contractions by stimulating NK2 and NK1 receptors, respectively.

The behavioral response induced by the intravesical instillation of capsaicin has been investigated in catheter-implanted, freely moving, conscious male and female rats. Intravesical instillation of capsaicin (25 nmol/rat in 0.5 ml) evoked an intense licking directed toward the lower abdominal and perineal skin which lasted for 15 min. Capsaicin-induced licking was reduced by systemic capsaicin pretreatment at a dose of 150 mg/kg but not at 50 mg/kg, administered 4 days before the intravesical instillation. Both doses of capsaicin effectively abolished the eye-wiping response to local capsaicin application. The capsaicin-induced licking was preserved (and prolonged) in both male and female animals subjected to bilateral ablation of the pelvic ganglia, 48 h before the intravesical instillation of the irritant. The capsaicin-induced licking was almost abolished by placing a ligature around the proximal urethra (24 h before), in order to avoid the contact of the irritant with the urethra or by previous section of the pudendal nerves (48 h before). One hour intravesical infusion with tetrodotoxin (1 and 10 microM) reduced the amplitude of micturition contractions but did not prevent the licking response evoked by capsaicin. On the contrary, the intravesical infusion of lidocaine (100 mM) abolished both micturition contractions and the capsaicin-induced licking. We conclude that the behavioral response induced by the intravesical instillation of capsaicin is mainly due to the stimulation of urethral afferents travelling within pudendal nerves.

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

This study investigates the role of peripheral tachykinin receptors in the initiation of reflex urinary bladder contractions in urethane-anesthetized rats. Intravenous administration of the selective tachykinin neurokinin (NK)-1 receptor agonist [Sar9]substance P (SP) sulfone (0.3-30 nmol/kg) or of the selective tachykinin NK-2 receptor agonist [beta Ala8]NK-A(4-10) (0.3-100 nmol/kg) produced dose-related bladder contractions. Among NK-3 receptor agonists, senktide (1-100 nmol/kg) was not effective; [MePhe7] NK-B (3-100 nmol/kg) induced bladder contraction, but the magnitude of the response was only 20 to 25% of that produced by NK-1 or NK-2 agonists. The NK-1 receptor antagonist GR 82,334 (0.1 mumol/kg i.v.) blocked the urinary bladder contraction induced by [Sar9]SP sulfone (1 nmol/kg i.v.), but not that induced by [beta Ala8]NK-A(4-10). On the contrary, the NK-2 receptor antagonist L 659,877 (0.1-1 mumol/kg i.v.) abolished the effect of [beta Ala8] NK-A(4-10) (1 nmol/kg i.v.), but failed to affect the response to [Sar9]SP sulfone. The combined administration of GR 82,334 (0.1 mumol/kg i.v.) and L 659,877 (1 mumol/kg i.v.) blocked the tonic bladder contraction induced by topical application of capsaicin in pelvic ganglionectomized rats (efferent response of sensory nerves), but did not affect the hexamethonium-sensitive phasic reflex bladder contractions evoked by capsaicin in sham-operated rats (chemonociceptive micturition reflex). GR 82,334 (0.1 mumol/kg i.v.) or L 659,877 (1 mumol/kg i.v.), alone or in combination, did not modify cystometric parameters of the volume-evoked micturition reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. 2. Bombesin (0.1 nM-10 microM) produced a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCl (80 mM). The response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-13) OMe (0.3-10 microM). Degree of antagonism was concentration-dependent between 0.3 and 3 microM (dose ratios = 2.4, 9 and 39 in the presence of 0.3, 1, 3 microM of the antagonist). However, a larger concentration (10 microM) of the antagonist was not more effective (dose ratio = 36) than 3 microM. 3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3 microM each) or by atropine, indomethacin, chlorpheniramine and desensitization of P2x purinoceptors by alpha, beta methylene ATP. 4. The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder produced by the NK-1 tachykinin receptor-selective agonist, [Sar9] substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3 microM), a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5. The bombesin antagonist (1-10 microM) had no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravenous administration of the selective 5-hydroxytryptamine (5-HT)1A receptor agonist 8-hydroxy-2-(di-N-propylaminotetralin (8-OH-DPAT) and of a low doses of buspirone elicited the supraspinal micturition reflex (SMR) in urethane-anesthetized rats when the urinary bladder was filled with just a subthreshold volume of saline (threshold conditions). The effect of i.v. 8-OH-DPAT was abolished by hexamethonium or spiroxatrine. When SMR was elicited by bladder distension (suprathreshold conditions), i.v. 8-OH-DPAT increased the frequency of bladder contractions. In threshold conditions, stimulation of SMR was also induced by i.c.v. or by i.t. administration of 8-OH-DPAT and 5-HT but not by topical application of 8-OH-DPAT onto the bladder. Guanethidine pretreatment, which produced detrusor hyperreflexia, antagonized the effect of both i.c.v. and i.t. 8-OH-DPAT. In rats treated with capsaicin as adults, the response to 8-OH-DPAT was unchanged. In rats treated with capsaicin as newborns, instead, the response to i.t. 8-OH-DPAT was abolished and that to i.c.v. 8-OH-DPAT was shifted to higher doses. Pretreatment with 5,7-dihyroxytryptamine did not affect the response to i.t. 8-OH-DPAT but shifted to higher doses the response to i.c.v. 8-OH-DPAT. Intravenous administration of spiroxatrine, methysergide, NAN-190 [1-(2-methoxyphenyl)-4-[4-(2-phtalimido)butyl] piperazine] or high doses of buspirone but not of 1-sulpiride inhibited SMR in suprathreshold conditions. The inhibitory effect of spiroxatrine, NAN-190 and buspirone was not reduced by guanethidine pretreatment. In chronically spinalized animals, i.v. 8-OH-DPAT increased the amplitude of the reflex bladder contractions induced by bladder distension.(ABSTRACT TRUNCATED AT 250 WORDS)

The involvement of capsaicin-sensitive primary afferent neurons in cyclophosphamide (CYP)-induced cystitis has been investigated in rats. CYP (150 mg/kg) was administered 48 h before testing in both vehicle- and capsaicin- (50 mg/kg s.c., 4 days before) treated rats. Some experiments were also performed 96 h after bilateral removal of pelvic ganglia to produce bladder denervation. CYP administration produced a marked detrusor hyperreflexia which was abolished by capsaicin pretreatment, demonstrating that it is mediated through stimulation of capsaicin-sensitive afferent neurons. CYP administration was followed by a marked increase in bladder weight and plasma protein extravasation (measured by the Evans blue leakage technique). The latter effect was largely prevented by ganglionectomy but was aggravated by capsaicin pretreatment. The effect of capsaicin was suppressed by ganglionectomy. Isolated strips of detrusor muscle from CYP-treated animals developed less tension in response to various stimuli as compared to strips from vehicle-treated animals; however, when contractile responses were expressed as percentage of an internal standard (carbachol-induced contraction) no difference was evident between the two groups. The bladder content of calcitonin gene-related peptide, used as a marker of the bladder afferent fibres that are capsaicin-sensitive in adult rats, was slightly reduced as compared to controls, but the difference can be accounted for by the increased bladder weight. We conclude that CYP-induced cystitis is not accompanied by a toxic effect on bladder nerves and that the decrease in bladder capacity is entirely mediated through stimulation of capsaicin-sensitive afferent fibres, presumably linked to the formation of the irritant metabolite of CYP, acrolein.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of intrathecal or intracerebroventricular administration of the GABAB receptor agonist, baclofen, on rhythmic contractions induced by distension of the urinary bladder (micturition reflex) was evaluated in urethane-anesthetized rats. Baclofen inhibited bladder motility acting at central nervous system sites (spinal and supraspinal) with a comparable potency. The inhibitory effect of i.t. baclofen (0.1-10 nmol) was blocked by i.t. phaclofen (200 nmol) while i.c.v. phaclofen did not affect i.c.v. baclofen (0.1-1 nmol). The inhibition of the micturition reflex induced by bladder distension observed after i.t. administration of baclofen was unaffected by systemic capsaicin pretreatment (50 mg/kg s.c., four days before). On the other hand, i.t. baclofen suppressed, in a phaclofen-sensitive manner, the reflex bladder contraction evoked by chemical stimulation (topical capsaicin) of capsaicin-sensitive bladder afferents. Intrathecal baclofen did not affect the hexamethonium-resistant tonic contraction produced by topical application of capsaicin on to the urinary bladder, which is ascribable to local peptide release from sensory nerves. Bladder motility inhibition by i.t. or i.c.v. baclofen (1 nmol) was unchanged by previous administration of p-chlorophenylalanine, indicating that the serotonergic pathways do not play a role in its action. Baclofen (100 microM) suppressed the release of calcitonin gene-related peptide-like immunoreactivity evoked by electrical field stimulation from the dorsal half of the rat spinal cord. This response was also abolished by in vitro capsaicin desensitization or tetrodotoxin, indicating that baclofen suppresses transmitter release from central endings of capsaicin-sensitive primary afferents. The present findings indicate that baclofen acts at both spinal and supraspinal sites to inhibit, with different mechanisms, the micturition reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

We have assessed the ability of compound 48/80, a mast cell degranulating agent, to activate the sensory and efferent function of capsaicin-sensitive primary afferents in the rat urinary bladder. Compound 48/80 produced a calcium-dependent release of calcitonin gene-related peptide-like immunoreactivity from the superfused rat urinary bladder. This effect was prevented by in vitro capsaicin desensitization, but was not affected by indomethacin, methysergide, ondansetron, chlorpheniramine or cimetidine, nor by systemic pretreatment with compound 48/80 at a dose regimen which prevented lethality produced by intravenous administration of it in anesthetized rats. Compound 48/80 also produced a contraction of the rat isolated bladder which was not reduced by methysergide, indomethacin or in vitro capsaicin desensitization. In vivo, topical application of compound 48/80 on the serosal surface of the rat urinary bladder activated a series of high amplitude rhythmic bladder contractions which were hexamethonium-sensitive (micturition reflex). This effect was prevented by systemic capsaicin desensitization while it was unchanged by chlorpheniramine, methysergide, indomethacin or ondansetron. Administered intravenously, compound 48/80 produced a plasma protein extravasation (Evans blue leakage technique) in the rat urinary bladder which was abolished by systemic capsaicin pretreatment or chlorpheniramine while it was unaffected by methysergide or indomethacin. The present findings provide direct neurochemical evidence that compound 48/80 activates the peripheral endings of capsaicin-sensitive primary afferent neurons, leading to a stimulation of their sensory and efferent functions in the rat urinary bladder. The possibility of a direct action of compound 48/80 in producing excitation of capsaicin-sensitive sensory nerves should be considered.

Intracerebroventricular (i.c.v.) administration of endothelin-1 (ET-1, 10 and 100 pmol/rat) or endothelin-3 (ET-3, 100 pmol/rat) inhibited the supraspinal micturition reflex (SMR) in urethane-anesthetized rats. Guanethidine pretreatment, vagotomy or forced ventilation did not modify the effect of i.c.v. ET-1 on the SMR. Intravenous (i.v.) administration of ET-1 (350 pmol/rat) also blocked the SMR with a delay of about 8 min. Guanethidine pretreatment revealed a stimulatory effect of i.v. ET-1 on the frequency of the SMR and the late inhibition was also reduced. In the isolated rat urinary bladder ET-1, ET-3 and ET-(16-21) induced a dose-related contraction and a small enhancement of the neurogenic contraction caused by electrical field stimulation. Inhibition of bladder motility by i.c.v. administration of ET-1 or ET-3 was paralleled by an increase in blood pressure. Both ET-1 and ET-3, at the same doses which evoked these autonomic effects, also killed some animals. The ET-1-induced increase in blood pressure was not prevented by guanethidine pretreatment or by spinal cord transection. Guanethidine pretreatment also did not modify the pressor response induced by i.v. ET-1. Bilateral cervical vagotomy enhanced the pressor response to i.c.v. ET-1. Forced ventilation prevented the death of some animals following i.c.v. administration of ET-1.

1. We have investigated the ability of the N-formyl-methionyl-leucyl-phenylalanine (FMLP) a synthetic analogue of a chemotactic peptide derived from a variety of bacteria, to activate capsaicin-sensitive primary afferents in the guinea-pig atria and urinary bladder. 2. In the isolated, electrically-driven left atria from reserpine-pretreated guinea-pigs (atropine in the bath), FMLP (3 nM-1 microM) produced a biphasic positive inotropic response. The late component of this response was selectively abolished by in vitro capsaicin pretreatment while both the early and late responses were abolished by indomethacin. 3. The inotropic response to FMLP in the guinea-pig atria was unaffected by ruthenium red. The late but not the early response was strongly inhibited or abolished by tetrodotoxin (TTX), omega-conotoxin (CTX) or by the C-terminal fragment (8-37) of human alpha-calcitonin gene-related peptide (hCGRP). hCGRP-(8-37) acts as competitive antagonist at CGRP receptors. 4. In the guinea-pig isolated bladder, FMLP (10 nM-10 microM) produced a concentration-dependent contraction which was unchanged by previous in vitro capsaicin, TTX or CTX pretreatment. The response to low concentrations of FMLP was suppressed by indomethacin, irrespective of the capsaicin pretreatment. 5. FMLP (10 microM) produced a significant increase in the outflow of CGRP-like immunoreactivity (CGRP-LI) from superfused guinea-pig atria or urinary bladder. CGRP-LI outflow induced by FMLP was blocked by indomethacin or in vitro capsaicin pretreatment. 6. These findings indicate that FMLP activates the 'efferent' function of capsaicin-sensitive primary afferents via prostanoid generation. This action could provide a neurogenic contribution to the overall inflammatory response produced by bacteria-derived peptides in inflamed tissues. In addition the present data indicate that endogenous prostanoids generated during exposure to FMLP produce peptide secretion from sensory nerves via a TTX- and CTX-sensitive but ruthenium red-resistant mechanism.

The aim of this study was to assess whether omega conotoxin fraction GVIA, a potent blocker of N- and L-type voltage-sensitive calcium channels, might interfere with reflex responses (micturition, blood pressure rise in spinal rats) produced by activation of capsaicin-sensitive sensory nerves of the rat urinary bladder. The effect of conotoxin was also investigated on reflex micturition persisting after capsaicin pretreatment. Following topical application onto the bladder, conotoxin did not affect the volume threshold to elicit micturition although it reduced the amplitude of volume-evoked bladder contractions. Likewise, topical conotoxin did not prevent the reflex rise in blood pressure elicited by sudden bladder distension or topical application of capsaicin onto the bladder. In contrast, topical lidocaine strongly prevented both reflex responses. After intrathecal administration, conotoxin produced a dose-dependent inhibition of volume-evoked bladder contractions and the cardiovascular reflex produced by mechanical or chemical stimulation of bladder nerves. Intrathecal conotoxin inhibited micturition also in rats pretreated with capsaicin (50 mg/kg s.c., 4 days before). Depolarization by high potassium (80 mM) produced release of both substance P- and calcitonin gene-related peptide-like immunoreactivity from superfused slices of the dorsal half of rat spinal cord. Capsaicin (1 microM) produced a similar effect, and a previous exposure to capsaicin prevented the effect of potassium. Conotoxin (0.1 microM) significantly reduced (about 50%) the potassium-induced release of neuropeptides from the dorsal half of the rat spinal cord. These findings indicate that conotoxin-sensitive calcium channels in the rat spinal cord play a role in the neurotransmission along reflex pathways activated by stimulation of capsaicin-sensitive nerves in the urinary bladder.(ABSTRACT TRUNCATED AT 250 WORDS)

Capsaicin induced a contraction of isolated strips from the guinea-pig urinary bladder which was more evident in the dome than in the neck and inhibited contractions induced by field stimulation, particularly in the neck. Both responses exhibited prompt desensitization and were tetrodotoxin-resistant, suggesting a specific action on transmitter release from sensory nerve terminals. Indeed, the contractile response in the dome was prevented by a substance P antagonist while the inhibitory response in the neck was prevented by immunoblockade with anticalcitonin gene-related peptide (CGRP) serum. Substance P produced a contraction of the guinea-pig bladder, being about 5 times more potent in the dome than in the neck, while CGRP inhibited the evoked contractions, being about 8 times more potent in the neck than in the dome. Further, the maximal effect of CGRP in the neck was almost double that in the dome. Substance P- and CGRP-like immunoreactivity were detected in both the dome and the neck with no regional differences for each peptide. CGRP-like immunoreactivity was 6.3 and 7.9 times higher than substance P-like immunoreactivity in the dome and the neck, respectively. Exposure to capsaicin evoked release of both substance P- and CGRP-like immunoreactivity from the dome and the neck. Peak CGRP-like immunoreactivity released by capsaicin was 12.3 and 8 times greater than substance P-like immunoreactivity in the dome and the neck, respectively. For each peptide, no difference was found in peak release in the dome vs neck. Total substance P-like immunoreactivity released from the neck was 25% lower than that released from the dome. The ability of CGRP to stimulate accumulation of 3',5' cyclic adenosine monophosphate in membranes prepared from the bladder muscle was greater in preparations from the neck than from the dome. These findings indicate that postjunctional mechanisms (type and number of receptors for sensory neuropeptides, coupling with second messengers) are a major determinant of the type of motor responses consequent of the release of sensory neuropeptides from capsaicin-sensitive nerves.

The aim of this study was to determine the acute and delayed effect of topical application of high concentrations of capsaicin on the rat urinary bladder on micturition reflex and compare the effects of "topical" bladder desensitization with those produced by systemic (subcutaneous administration) capsaicin desensitization. On acute application, capsaicin (1-3%) produced a transient bladder contraction, not observed in capsaicin-pretreated rats. After a transient increase in excitability of the micturition reflex, topical capsaicin suppressed micturition and overflow incontinence ensued which was reverted by intravenous injection of 4-aminopyridine. Topical capsaicin also abolished reflex micturition in rats which had been systemically treated with capsaicin as adults (50 mg/kg, 7 days before) and reduced significantly the neurogenic bladder contractions produced by intravenous dimethylphenylpiperazinium or neurokinin A, while the direct (myogenic) response to neurokinin A was unaffected. In rats whose bladder was pre-exposed to 1-3% topical capsaicin (7 days before) the micturition reflex was affected in a manner which is qualitatively and quantitatively similar to that observed in rats treated with capsaicin as adults, e.g. increase in bladder capacity with no change in voiding efficiency. Topical capsaicin desensitization of the rat urinary bladder was shown to produce a selective impairment of bladder sensory nerves without any sign of desensitization in other areas of the body using both functional (hot plate, wiping, plasma extravasation) and neurochemical (determination of substance P-like immunoreactivity) assays. Systemically administered capsaicin (7 days before) had little effect on reflex micturition at 12.5 mg/kg but the change in bladder capacity produced at a dose of 25 mg/kg was comparable with that produced at 350 mg/kg. These findings provide evidence that selective desensitization of peripheral terminals of capsaicin-sensitive nerves of the rat urinary bladder inactivates their sensory and "efferent" function in a manner similar to that observed after systemic capsaicin desensitization in adult rats. The functional deficit of reflex micturition produced in this way can be overcome by increasing the stimulus to void. By contrast, neonatal capsaicin desensitization produced a long lasting abolition of reflex micturition. These data are in keeping with the hypothesis that adult versus neonatal capsaicin desensitization may be used as a tool to distinguish between two sets of sensory nerves in the rat urinary bladder.(ABSTRACT TRUNCATED AT 400 WORDS)

A new technique has been developed suitable for quantitative studies on physio-pharmacology of pain arising from the urinary bladder in conscious freely-moving rats. The method involves the intravesical instillation of xylene or its vehicle (0.3 cc of silicone oil) through a catheter chronically implanted into the rat bladder. The instillation of xylene (10 to 100%) produced behavioural effects (licking of lower abdomen or perineal region, hind paws hyperextension) suggestive of visceral pain. All the behavioural responses produced by xylene instillation were prevented by extrinsic bladder denervation (pelvic ganglionectomy). Morphine HCl (two to five mg./kg. s.c., 30 min. before) or (+/-)-baclofen (2.5-10 mg./kg. s.c., 60 min. before) reduced or abolished the response to xylene instillation, thus indicating that the action of analgesic drugs can be quantitated using the present model.

1. KCl, carbachol, neurokinin A and endothelin produced concentration-dependent contractions of mucosa-free muscle strips from the dome of the human urinary bladder. The maximal response to carbachol or neurokinin A exceeded that to KCl, while the maximal response to endothelin approached that to KCl. 2. Nifedipine (1 microM) abolished the response to KCl, reduced the response to carbachol or neurokinin A but had no effect on the response to endothelin. Bay K 8644 (1 microM) markedly potentiated the response to KCl but had little or no effect on the response produced by the other stimulants. 3. Superfusion of the strips with a nominally calcium (Ca)-free medium containing EDTA (1 mM) for 30 min markedly reduced the response to carbachol, neurokinin A and endothelin, although a small response was still evident at high concentrations. Likewise, after a prolonged (60 min) superfusion of the strips with a high K (80 mM) Ca-free medium plus EDTA (1 mM) these three agonists still produced a small contractile response. 4. The nifedipine (1 microM) resistant response to carbachol, neurokinin A or endothelin was markedly depressed by LaCl3 (1 mM). In contrast, the nifedipine-(1 microM) resistant response to carbachol was not modified by NiCl2 (0.1 mM) or omega-conotoxin (0.1 microM). 5. Caffeine produced divergent effects depending upon the temperature of incubation: a relaxation at 37 degrees C and a concentration-dependent (2.5-20 mM) contraction at 25 degrees C. The latter was markedly inhibited by procaine (3 mM) but unaffected by nifedipine (1 microM). 6. After a prolonged (60 min) superfusion with a high K, Ca-free medium containing EDTA the response to carbachol (100 microM) was abolished by previous exposure to procaine (3 mM). Conversely, the response to endothelin (1 microM) was unaffected by procaine. The response to endothelin in these experimental conditions was also resistant to LaCl3 (1 mM). 7. These findings indicate that multiple sources of Ca are mobilized for contraction of the human bladder muscle by different stimulants. Dihydropyridine- and voltage-sensitive Ca channels provide the major if not the sole source of Ca for the response to KCl, play some role in the response to muscarinic (carbachol) or NK-2 tachykinin receptor stimulation but are not involved in the response to endothelin. Carbachol, neurokinin A and endothelin all mobilize a Ca pool (either extracellular or located at membrane level) which is LaCl3-sensitive but nifedipine-resistant. Neither T- nor N-type channels appear to be involved in the response to carbachol. In addition, these agents mobilize a tightly bound Ca pool independently from membrane depolarization. This latter pool is probably a procaine-sensitive intracellular source of activator Ca mobilized by caffeine and carbachol. The failure of procaine to prevent the response to endothelin in high K, Ca-free medium raises the possibility that this peptide mobilizes an intracellular source of activator Ca, distinct from the caffeine- and carbachol-sensitive pool.

1. The effect of (+/-)-baclofen on micturition reflexes was investigated in urethane-anaesthetized rats. A 'low' dose of (+/-)-baclofen (0.5 mg/kg i.v.) barely affected the early phase of the transurethral cystometrogram (CMG) which involves activation of a spinal vesico-vesical excitatory reflex. 2. At a higher dose (2.5 mg/kg i.v.) (+/-)-baclofen suppressed both the spinal and supraspinal components of the bladder response to transurethral saline filling. 3. When the bladder was filled by the transvesical route a series of regular voiding cycles was obtained which are due to activation of a supraspinal vesico-vesical excitatory reflex. In this model, voiding efficiency of the rat bladder was markedly reduced even after a low dose of (+/-)-baclofen (0.5 mg/kg) and almost suppressed at 2.5 mg/kg. 4. (+/)-Baclofen reduction of voiding efficiency was mainly ascribable to an inhibitory effect on the expulsive phase of the voiding cycle which, in rats, depends critically upon the activation of a reflex which induces a twitch-like contraction of urethral/periurethral skeletal muscles. 5. (+/-)-Baclofen produced a small inhibition of the pinching-induced somatovesical excitatory reflex. (+/-)-Baclofen (2.5 mg/kg i.v.) produced also a marked but transient inhibition of bladder contractions induced by preganglionic nerve stimulation. However the time course of this effect was markedly shorter as compared to the long lasting suppression of voiding cycle observed with this same dose of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The effect of capsaicin on bladder motility in vivo (urethane anaesthesia) and in vitro, plasma extravasation (Evans blue leakage technique) and content of substance P-like immunoreactivity (SP-LI) of the urinary bladder was investigated in various mammalian species. 2. Systemic capsaicin desensitization (rat and hamster, 50 mg/kg s.c. 4 days before; guinea-pig 55 mg/kg s.c. 4-7 days before) increased bladder capacity in rats and guinea-pigs and reduced voiding efficiency in guinea-pigs. All other urodynamic parameters were unaffected in both rats, guinea-pigs and hamsters. 3. Reflex bladder voiding was abolished by spinal cord transection in anaesthetized rats and hamsters. On the other hand, hexamethonium-(20 mg/kg i.v.)sensitive voiding contractions were obtained in response to saline filling 45 min from cord transection in guinea-pigs, indicating a profound interspecies variation in the basic organization of micturition. 4. Exposure to capsaicin (1 microM) produced a contraction of the isolated bladder from rats, guinea-pigs (dome) and mice. Capsaicin produced only a slight contractile response in the guinea-pig bladder base. The motor response to capsaicin of the rat, guinea-pig and mouse bladder exhibited marked desensitization, suggesting a specific effect on sensory nerves. On the other hand, capsaicin (1 microM) produced a slight relaxation of the hamster isolated bladder but this effect was reproducible at 1-2 h intervals, suggesting an unspecific effect. Capsaicin (1-10 microM) did not affect motility of strips from the dome or the base of the rabbit bladder. 5. Intravenously administered capsaicin produced a marked plasma extravasation (Evans blue leakage) in the lower urinary tract of rats, mice and guinea pigs. In rats but not guinea-pigs the reaction in the bladder base was greater than in the dome. In hamsters intravenous capsaicin failed to induce any significant Evans blue leakage in the lower urinary tract. 6. SP-LI was detected in the lower urinary tract of rats, guinea-pigs, rabbits and mice but not hamsters. Bladder SP-LI was depleted by systemic capsaicin desensitization in rats, guinea-pigs and mice. Reverse phase HPLC indicated that all the immunoreactive material co-eluted with authentic substance P or its oxidized form. 7. These findings indicate that noticeable species-related differences exist with regard to the functions mediated by the capsaicin-sensitive neurons in the urinary bladder.

In urethane-anesthetized rats with an intact spinal cord, application of capsaicin on the outer surface of the urinary bladder produced a transient bradycardia, hypotension and negative cardiac inotropism which were neither prevented by i.v. atropine (0.5 mg/kg) nor by cervical vagotomy. In acute spinal rats (C2-C3) application of capsaicin (0.2 and 2 micrograms in 25 microliters) on the urinary bladder induced a transient hypertension, tachycardia and positive cardiac inotropism. A second application (30 min later) induced minor cardiovascular effects, expecially with the higher dose, indicating desensitization. All cardiovascular responses to topical capsaicin were abolished by systemic capsaicin desensitization (50 mg/kg s.c., 4 days before). The excitatory cardiovascular response to capsaicin in acute spinal rats was markedly reduced by bilateral section of pelvic but not hypogastric nerves. Further, it was abolished by pretreatment with hexamethonium (20 mg/kg i.v.) or reserpine (5 mg/kg i.p., 2 days before) and reduced, at various extent for the different components, by phentolamine (0.5 mg/kg i.v.) or propranolol (1 mg/kg). In rats with pelvic and hypogastric nerves intact, section of the cord at a level (T12-L1), just above the medullary segments which receive primary afferent input from the bladder (L6-S1), abolished the excitatory cardiovascular response to application of capsaicin on the bladder. In spinal rats (C2-C3) rapid distension of the urinary bladder with saline produced transient tachycardia, hypertension and positive cardiac inotropism similar to that evoked by capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)

The tachykinin-like immunoreactivity of the urinary bladder has been measured in various species by means of an antiserum (K12) having negligible cross-reactivity with substance P. The rank order for bladder content of tachykinin-like immunoreactivity was guinea-pig greater than mice greater than rat, similar to that found for substance P-like immunoreactivity. In all three species, both substance P- and tachykinin-like immunoreactivities were depleted by systemic capsaicin desensitization. The time course for depletion of substance P- and tachykinin-like immunoreactivities of the rat bladder following extrinsic denervation was almost superimposable. At reverse phase high pressure liquid chromatography, the major constituent of tachykinin-like immunoreactivity of the rat bladder co-eluted with neurokinin A. In vitro, the contractile response of the rat bladder to capsaicin (1 microM) was directly proportional to bladder tachykinin-like immunoreactivity while the response to field stimulation was not. In vivo, the volume threshold for reflex micturition was inversely proportional to bladder tachykinin-like immunoreactivity while amplitude of micturition contraction was not. Similar correlations were found in a previous study for substance P-like immunoreactivity. The contractile response to capsaicin or neurokinin A of the rat isolated bladder were significantly reduced by incubation with phenoxybenzamine at a concentration reported to produce a selective alkylation of neurokinin-2 receptors, while the response to substance P or KCl was unaffected. These findings indicate that multiple neurokinins co-exist in those bladder sensory nerves which are capsaicin-sensitive in adult rats. Both substance P- and tachykinin-like immunoreactivities in the rat bladder appear to be good functional markers of the sensory and "efferent" functions mediated by capsaicin-sensitive nerves, consistent with the hypothesis of a transmitter role for the corresponding peptides.

In acute spinal rats (C2-C3) the transvesical infusion of saline activates a vesico-vesical excitatory reflex (Brain Res., 380 (1986) 83-93). In bladders containing a subthreshold amount of fluid the topical application of capsaicin on the outer surface of the bladder dome activated this spinal reflex and also produced a transient rise in blood pressure and heart rate. The effects of systemic capsaicin desensitization (50 mg/kg s.c. 5 min, 60 days before) on the sensory (activation of the spinal vesico-vesical reflex) and 'efferent' (tetrodotoxin-insensitive capsaicin-induced contraction) functions mediated by the capsaicin-sensitive sensory fibers were correlated to changes in substance P-like immunoreactivity (SP-LI) content of the urinary bladder in adult rats. Blockade of both sensory and efferent functions was observed at a time (60 min from capsaicin administration) when the SP-LI content of the urinary bladder was unaffected. Four days after capsaicin desensitization the SP-LI levels of the bladder are almost depleted indicating that the neuropeptide(s) are entirely stored in sensory structures. At this time the sensory-efferent functions mediated by these fibers are still blocked. At 15-60 days from systemic capsaicin desensitization there was a progressive, time-related recovery of SP-LI levels in the bladder as well as of the sensory-efferent functions. These findings indicate a role of the capsaicin-sensitive innervation of the urinary bladder in activating the spinal vesico-vesical reflex. The present findings suggest that measurement of SP-LI levels in the rat bladder may be a useful biochemical index for monitoring the function(s) of the capsaicin-sensitive, peptidergic sensory neurons.

4-aminopyridine (4-AP) produced a dose-related (0.15-2 mg/kg i.v.) potentiation of the voiding cycle of the urinary bladder and increased frequency of micturition in urethane-anesthetized rats. In bladders containing a subthreshold amount of fluid for eliciting reflex micturition 4-AP (1-3 mg/kg i.v.) activated a series of high-amplitude, hexamethonium-sensitive rhythmic bladder contractions. In rats desensitized to capsaicin as newborns, reflex micturition was almost abolished: in these animals i.v. 4-AP did not affect bladder voiding unless at high doses (1-2 mg/kg), at which a reversal from anesthesia occurred. This was accompanied by a prompt micturition. In unanesthetized rats, neither the 4-AP-induced convulsions nor the behavioral response (assessed in an open field) to 4-AP were affected by neonatal capsaicin desensitization. Daily urine production of capsaicin-pretreated animals did not differ from that of controls. However, when measurements were made during daytime, almost no spontaneous urine emission was found in capsaicin-treated rats. On the rat isolated urinary bladder, 4-AP potentiated the response to field stimulation in preparations from both vehicle- and capsaicin-pretreated animals. These findings indicate that 4-AP has a potent excitatory action on bladder voiding in rats and support the hypothesis that in this species 'conscious' bladder voiding can be initiated through capsaicin-resistant mechanisms.

A new cystometric technique has been developed that is suitable for quantitative studies on physiopharmacology of micturition in conscious, freely moving rats. The method involves the transvesical infusion of warm saline at various filling rates through a catheter chronically implanted into the bladder dome. The various parameters of the collecting and voiding phase of the cystometrogram were recorded. Results obtained indicate that characteristics of the bladder voiding cycle of conscious, freely moving rats are qualitatively similar but quantitatively different from those described previously in anesthetized animals.

The mechanism(s) involved in determining the voiding cycle of the rat urinary bladder have been investigated in urethan-anesthetized animals. Fluid emission is almost confined to that phase of the voiding cycle which is characterized by the presence of a series of high-frequency oscillations in intraluminal pressure (IPHFO). During this phase the mean urethral flow rate reached a maximum and fluid was expelled in a stream-like fashion. The index obtained by multiplying the amplitude of IPHFO by their duration was significantly related to the maximal value of urethral flow rate. The IPHFO were selectively abolished by administration of d-tubocurarine at a dose that barely affects detrusor contractility. Moreover, d-tubocurarine reduced mean urethral flow rate and increased residual volume. The reflex (hexamethonium sensitive) mechanism(s) responsible for the generation of IPHFO is more developed in male than female rats. This mechanism, which involves activation of skeletal muscle, plays a significant role in determining bladder voiding in this species.

The ability of substance P (SP) and substance K (SK) to activate motility of the rat urinary bladder "in vivo" and "in vitro" have been compared. In urethane-anesthetized rats topical application of tachykinins on the serosal surface of the bladder elicited a series of neurogenic rhythmic contractions (micturition reflex). SK is about 13.5 times more potent than SP. The extent of the effects of both SP and SK depends on the bladder initial volume. SP and SK are equally effective in: contracting the tetrodotoxin-pretreated rat bladder in vivo; contracting the isolated rat bladder in vitro; enhancing the contractions induced by field stimulation; and delaying relaxation of bladder muscle in response to stretch. These findings suggest the existence of two different sites (receptors) for tachykinins in the rat bladder whose activation is responsible for initiation of the micturition reflex and direct smooth muscle contraction, respectively. The higher potency of SK, as compared to SP, in activating the micturition reflex suggests the involvement of a receptor of the SP-E type, possibly located on the afferent nerve endings, in the mediation of this type of response. The other receptor, located on the smooth muscle cell (and possibly on the efferent nerve endings) appears to be of the SP-P type, as it is equally sensitive to the two tachykinins.

1. The effect of nifedipine on spontaneous and stimulated motility of the rat urinary bladder has been investigated in vitro (isolated detrusor strips) and in vivo (micturition reflex). 2. Nifedipine inhibited tone and spontaneous activity of the isolated rat bladder, its effect being greater in indomethacin-treated preparations. Nifedipine suppressed the KCl induced phasic and tonic contraction and inhibited by 60-80% the carbachol- or ATP- induced contractions. Nifedipine reduced by about 70% amplitude of the nerve-mediated bladder contractions. 3. Exposure to Ca free medium containing EDTA suppressed tone and spontaneous activity of the rat bladder. In these conditions the response to KCl or ATP was rapidly abolished while a response to carbachol was still evident even after a long exposure to the Ca free medium. 4. In vivo, nifedipine affected reflex micturition e.g. increased volume threshold and slightly reduced amplitude of micturition contraction. In addition, nifedipine reduced voiding efficiency e.g. increased residual volume after micturition. These effects were evident following ligation of the ureters because in normal conditions nifedipine induced a marked diuresis which masked its effect on volume threshold. 5. These findings indicate that in the rat urinary bladder Ca from both intra- and extracellular pools is mobilized during spontaneous or stimulated contractions. Mobilization of an intracellular Ca pool by cholinomimetics or other neurotransmitter(s) may be responsible for the nifedipine-resistant component of the voiding contraction in vivo.

(1) Topical administration of Ruthenium Red (10-100 microM in saline) to the serosal surface of the urinary bladder in urethane-anesthetized rats prevented the motor response of the urinary bladder to topical administration of capsaicin and protected the sensory fibers from capsaicin desensitization, but had no effect on the volume-evoked contractions (micturition reflex). At 1 mM increased bladder capacity and decreased amplitude of micturition contraction were observed. (2) At 100 microM, topical Ruthenium Red prevented the blood pressure rise produced by topical administration of capsaicin onto the bladder but did not affect the blood pressure rise produced by sudden bladder distension in spinal rats. (3) After intrathecal administration, Ruthenium Red (80-800 ng/rat) produced a long lasting inhibition of the micturition reflex in urethane-anesthetized rats, this effect being evident in both vehicle- or capsaicin- (50 mg/kg s.c. 4 days before) pretreated rats. At 800 ng/rat, intrathecal Ruthenium Red did not affect the blood pressure rise produced by topical administration of capsaicin onto the rat bladder nor that produced by bladder distension. (4) These findings provide further evidence that Ruthenium Red acts quite selectively as a "capsaicin antagonist" preventing both reflex and "efferent" responses activated by peripherally administered capsaicin. By contrast, sensory impulse generation by a natural stimulus such as bladder distension is apparently unaffected by Ruthenium Red. The marked inhibition of the micturition reflex observed after intrathecal administration of Ruthenium Red does probably not involve an interaction with primary afferents in the spinal cord.

The relative ability of substance P, neurokinin A, neurokinin B and kassinin to activate the micturition reflex was investigated in urethane-anaesthetized rats. When administered topically neurokinin A, neurokinin B and kassinin were 14, 36 and 280 times, respectively, more potent than substance P to activate micturition. On the other hand substance P, neurokinin A and kassinin were practically equipotent (and neurokinin B was about 3-4 times less potent than substance P) to stimulate the contraction of the rat isolated bladder and to potentiate the contractions induced by electrical field stimulation. This indicates that neither a direct action on muscle cells nor a potentiating effect on efferent neurotransmission can account for the rank order of potency of tachykinins for activation of the micturition reflex. The ability of topical tachykinins to activate the micturition reflex was largely impaired in 2 months old rats pretreated with capsaicin (50 mg/kg s.c.) on their second day of life, indicating that integrity of the capsaicin-sensitive bladder mechanoreceptors is essential for the production of this effect. These findings indicate that an NK-B receptor, possibly located on sensory nerves in the bladder wall, participates in the tachykinin-induced activation of reflex micturition.

The effects of substance P and substance K on blood pressure, salivation and urinary bladder motility were investigated in urethane-anaesthetized rats. On intravenous administration, both peptides produced dose-related reductions in blood pressure, substance P being 25-30 times more potent than substance K. Substance P induced a dose-dependent increase of salivation, while substance K was half as active and induced only 40% of the maximum effect of substance P. The two tachykinins induced contractions of the urinary bladder, substance K being about twice as effective as substance P. Substance K was more effective than substance P in activating micturition. These findings provide further evidence that multiple receptor types are involved in mediating the effects of tachykinins in peripheral organs.