María Victoria Barahona

Noradrenaline modulates smooth muscle activity of the isolated intravesical ureter of the pig through different types of adrenoceptors

1 We have studied the effects of α‐ and β‐adrenoceptor agonists and antagonists on both phasic peristaltic activity and basal tone of the isolated intravesical ureter of the pig by means of isometric techniques in vitro. 2 Spontaneous phasic activity was exhibited by 21% of pig intravesical ureter preparations manifested as rhythmic contractions with average frequency and amplitude of 2.54 ± 0.18 min−1 and 1.48 ± 0.16 g (n = 31), respectively. 3 Adrenaline, noradrenaline and phenylephrine induced concentration‐dependent increases in both phasic activity and basal tone of ureteral preparations, all three agonists being more potent in modifying ureteral phasic activity than baseline tone. B‐HT 920, B‐HT 933 and clonidine had no significant effect. 4 Phentolamine (10−9 − 10−7 m) and prazosin (3 × 10−11 − 3 × 10−8 m) significantly inhibited increases in both frequency of phasic activity and baseline tone induced by a submaximal dose of noradrenaline. Rauwolscine (10−9 − 10−7 m) affected only the tone evoked by noradrenaline and higher concentrations of this antagonist were needed to block phasic activity. 5 Pretreatment of ureteral strips with the β‐adrenoceptor antagonist, propranolol (10−6 m), significantly increased the maximum contraction evoked by noradrenaline. After incubation with phentolamine (10−6 m), noradrenaline (10−7 − 10−6 m) decreased phasic activity induced by prostaglandin F2α (10−5 m). Isoprenaline and salbutamol also abolished PGF2α‐induced phasic activity. Pafenolol (10−6 m) and butoxamine (10−6 m) blocked the inhibitory effect of noradrenaline, isoprenaline, and salbutamol on PGF2α‐induced phasic activity. 6 These results suggest that noradrenaline may modulate both phasic peristaltic activity and basal tone of pig intravesical ureter through both α‐ and β‐adrenoceptors.

Neuronal and smooth muscle receptors involved in the PACAP- and VIP-induced relaxations of the pig urinary bladder neck

As pituitary adenylate cyclase‐activating polypeptide 38 (PACAP 38)‐ and vasoactive intestinal peptide (VIP) are widely distributed in the urinary tract, the current study investigated the receptors and mechanisms involved in relaxations induced by these peptides in the pig bladder neck.

Nitric oxide is involved in the non-adrenergic, non-cholinergic inhibitory neurotransmission of the pig intravesical ureter

NADPH-diaphorase histochemical staining and electrical field stimulation (EFS) were performed in vitro to investigate whether nitric oxide (NO) is involved in non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission of pig intravesical ureter. NADPH-diaphorase activity was expressed in nerve trunks and thin nerve fibres around arteries and muscular bundles in the intravesical ureter. Relaxations to EFS were tetrodotoxin (10(-6) M)-sensitive which indicates their neurogenic origin. Addition of the NO-synthase inhibitor, L-NG-nitroarginine (L-NOARG, 3 x 10(-5) M), abolished the electrically induced relaxations, which were significantly reversed by L-arginine (3 x 10(-3) M). Addition of acidified sodium nitrite (NaNO2, 10(-5)-10(-3) M) evoked concentration-dependent relaxations of ureteral strips which were unaffected by L-NOARG. It is concluded that NO synthase is present in nerve fibres and NO seems to mediate the inhibitory neurotransmission of the porcine intravesical ureter.

Role of neuronal voltage-gated K+ channels in the modulation of the nitrergic neurotransmission of the pig urinary bladder neck

As nitric oxide (NO) plays an essential role in the inhibitory neurotransmission of the bladder neck of several species, the current study investigates the mechanisms underlying the NO‐induced relaxations in the pig urinary bladder neck.

5-hydroxytryptamine induced relaxation in the pig urinary bladder neck

Background and purpose:  5‐Hydroxytryptamine (5‐HT) is one of the inhibitory mediators in the urinary bladder outlet region. Here we investigated mechanisms involved in 5‐HT‐induced relaxations of the pig bladder neck.

Pre-junctional α2-adrenoceptors modulation of the nitrergic transmission in the pig urinary bladder neck†

AIMS To investigate the nitric oxide (NO)-mediated nerve relaxation and its possible modulation by pre-junctional alpha2-adrenoceptors in the pig urinary bladder neck. METHODS Urothelium-denuded bladder neck strips were dissected, and mounted in isolated organ baths containing a physiological saline solution (PSS) at 37 degrees C and continuously gassed with 5% CO2 and 95% O2, for isometric force recording. The relaxations to transmural nerve stimulation (electrical field stimulation [EFS]) or exogenously applied NO were carried out on strips pre-contracted with 1 microM phenylephrine (PhE) and treated with guanethidine (10 microM) and atropine (0.1 microM), to block noradrenergic neurotransmission and muscarinic receptors, respectively. RESULTS EFS (0.2-1 Hz, 1 msec duration, 20 sec trains, current output adjusted to 75 mA) evoked frequency-dependent relaxations which were abolished by the neuronal voltage-activated Na+ channel blocker tetrodotoxin (TTX, 1 microM). These responses were potently reduced by the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NOARG, 30 microM) and further reversed by the NO synthesis substrate L-arginine (L-ARG, 3 mM). The alpha2-adrenoceptor agonist BHT-920 (2 microM) reduced the electrically evoked relaxations, its effectiveness being higher on the responses induced by low frequency stimulation. BHT-920-elicited reductions were fully reversed by the alpha2-adrenoceptor antagonist rauwolscine (RAW, 1 microM). Exogenous NO (1 microM-1 mM) induced concentration-dependent relaxations which were not modified by BHT-920, thus eliminating a possible post-junctional modulation. CONCLUSIONS These results indicate that NO is involved in the non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission in the pig urinary bladder neck, the release of NO from intramural nerves being modulated by pre-junctional alpha2-adrenoceptor stimulation.

Involvement of a glibenclamide-sensitive mechanism in the nitrergic neurotransmission of the pig intravesical ureter

1 The present study was designed to investigate whether potassium (K+) channels are involved in the relaxations to nitric oxide (NO) of pig intravesical ureteral preparations suspended in organ baths for isometric tension recordings. In ureteral strips treated with guanethidine (10−5 m) and atropine (10−7 m) to block adrenergic neurotransmission and muscarinic receptors, respectively, NO was either released from nitrergic nerves by electrical field stimulation (EFS, 0.5–10 Hz, 1 ms duration, 20 s trains), or exogenously‐applied as an acidified solution of sodium nitrite (NaNO2, 10−6‐10−3 m). 2 Incubation with an inhibitor of guanylate cyclase activation by NO, methylene blue (10−5 m) did not change the basal tension of intravesical ureteral strips but inhibited the relaxation induced by EFS or exogenous NO on ureteral preparations contracted with the thromboxane analogue U46619 (10−7 m). 3 Incubation with charybdotoxin (3 × 10−8 m) and apamin (5 × 10−7 m), which are inhibitors of large and small conductance calcium (Ca2+)‐activated K+ channels, respectively, did not modify basal tension or the relaxations induced by EFS and exogenous NO. Treatment with charybdotoxin or apamin plus methylene blue (10−5 m) significantly reduced the relaxations to EFS and exogenous NO. However, in both cases the reductions were similar to the inhibition evoked by methylene blue alone. The combined addition of charybdotoxin plus apamin did not change the relaxations to EFS or exogenously added NO of the porcine intravesical ureter. 4 Cromakalim (10−8‐3 × 10−6 m), an opener of ATP‐sensitive K+ channels, evoked a dose‐dependent relaxation with a pD2of 7.3 + 0.2 and maximum relaxant effect of a 71.8 + 4.2% of the contraction induced by U46619 in the pig intravesical ureter. The blocker of ATP‐sensitive K+ channels, glibenclamide (10−6 m), inhibited markedly the relaxations to cromakalim. 5 Glibenclamide (10−6 m) had no effect on the basal tone of ureteral preparations but significantly reduced the relaxations induced by both EFS and exogenous NO. Combined treatment with methylene blue (10−5 m) and glibenclamide (10−6 m) did not exert an effect greater than that of methylene blue alone on either EFS‐or NO‐evoked relaxations of the pig ureter. 6 The present results suggest that NO acts as an inhibitory neurotransmitter in the pig intravesical ureter and relaxes smooth muscle through a guanylate cyclase‐dependent mechanism which seems to favour the opening of glibenclamide‐sensitive K+ channels.

A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission

The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. In U46619 (10−7  M)‐contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10−6  M), adenosine and related analogues induced relaxations with the following potency order: 5′‐N‐ethylcarboxamidoadenosine (NECA)=5′‐(N‐cyclopropyl)‐carboxamidoadenosine (CPCA)=2‐chloroadenosine (2‐CA)>adenosine>cyclopentyladenosine (CPA)=N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methylcarboxamide (IB‐MECA)=2‐[p‐(carboxyethyl)‐phenylethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS21680). Epithelium removal or incubation with indomethacin (3×10−6  M) and L‐NG‐nitroarginine (L‐NOARG, 3×10−5  M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 10−8 M) and 4‐(2‐[7‐amino‐2‐(2‐furyl) [1,2,4]‐triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol (ZM 241385, 3×10−8  M and 10−7  M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8‐phenyltheophylline (8‐PT, 10−5  M) and DPCPX (10−6  M), which block A1/A2‐receptors, reduced such relaxations. In strips treated with guanethidine (10−5  M), atropine (10−7  M), L‐NOARG (3×10−5  M) and indomethacin (3×10−6  M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10−4  M) induced contractions of preparations. 8‐PT (10−5  M) increased both contractions. DPCPX (10−8  M), NECA (10−4  M), CPCA, (10−4  M) and 2‐CA (10−4  M) did not alter the contractions to EFS. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids or NO, through activation of A2B‐receptors located in the smooth muscle. This relaxation may modulate the ureteral NANC excitatory neurotransmission through a postsynaptic mechanism.

Heterogeneity of neuronal and smooth muscle receptors involved in the VIP- and PACAP-induced relaxations of the pig intravesical ureter

The mechanisms and receptors involved in the vasoactive intestinal peptide (VIP)‐ and pituitary adenylate cyclase‐activating polypeptide (PACAP)‐induced relaxations of the pig intravesical ureter were investigated. VIP, PACAP 38 and PACAP 27 concentration‐dependently relaxed U46619‐contracted ureteral strips with a similar potency. [Ala11,22,28]‐VIP, a VPAC1 agonist, showed inconsistent relaxations. The neuronal voltage‐gated Ca2+ channel inhibitor, ω‐conotoxin GVIA (ω‐CgTX, 1 μM), reduced the VIP relaxations. Urothelium removal or blockade of capsaicin‐sensitive primary afferents, nitric oxide (NO) synthase and guanylate cyclase with capsaicin (10 μM), NG‐nitro‐L‐arginine (L‐NOARG, 100 μM) and 1H‐[1,2,4]‐oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 5 μM), respectively, did not change the VIP relaxations. However, the PACAP 38 relaxations were reduced by ω‐CgTX, capsaicin, L‐NOARG and ODQ. The VIP and VIP/PACAP receptor antagonists, [Lys1, Pro2,5, Arg3,4, Tyr6]‐VIP (1 μM) and PACAP (6–38) (0.4 μM), inhibited VIP and VIP and PACAP 38, respectively, relaxations. The nonselective and large‐conductance Ca2‐activated K+ channel blockers, tetraethylammonium (3 mM) and charybdotoxin (0.1 μM), respectively, and neuropeptide Y (0.1 μM) did not modify the VIP relaxations. The small‐conductance Ca2‐activated K+ channel blocker apamin (1 μM) did not change the PACAP 27 relaxations. The cAMP‐dependent protein kinase A (PKA) blocker, 8‐(4‐chlorophenylthio)adenosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐CPT‐cAMPS, 100 μM), reduced VIP relaxations. The phosphodiesterase 4 inhibitor rolipram and the adenylate cyclase activator forskolin relaxed ureteral preparations. The rolipram relaxations were reduced by Rp‐8‐CPT‐cAMPS. Forskolin (30 nM) evoked a potentiation of VIP relaxations. These results suggest that VIP and PACAP relax the pig ureter through smooth muscle receptors, probably of the VPAC2 subtype, linked to a cAMP‐PKA pathway. Neuronal VPAC receptors localized at motor nerves and PAC1 receptors placed at sensory nerves and coupled to NO release, seem also to be involved in the VIP and PACAP 38 relaxations.

Hydrogen Sulfide Plays a Key Role in the Inhibitory Neurotransmission to the Pig Intravesical Ureter

According to previous observations nitric oxide (NO), as well as an unknown nature mediator are involved in the inhibitory neurotransmission to the intravesical ureter. This study investigates the hydrogen sulfide (H2S) role in the neurogenic relaxation of the pig intravesical ureter. We have performed western blot and immunohistochemistry to study the expression of the H2S synthesis enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), measurement of enzymatic production of H2S and myographic studies for isometric force recording. Immunohistochemical assays showed a high CSE expression in the intravesical ureter muscular layer, as well as a strong CSE-immunoreactivity within nerve fibres distributed along smooth muscle bundles. CBS expression, however, was not consistently observed. On ureteral strips precontracted with thromboxane A2 analogue U46619, electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked frequency- and concentration-dependent relaxations. CSE inhibition with DL-propargylglycine (PPG) reduced EFS-elicited responses and a combined blockade of both CSE and NO synthase (NOS) with, respectively, PPG and NG-nitro-L-arginine (L-NOARG), greatly reduced such relaxations. Endogenous H2S production rate was reduced by PPG, rescued by addition of GYY4137 and was not changed by L-NOARG. EFS and GYY4137 relaxations were also reduced by capsaicin-sensitive primary afferents (CSPA) desensitization with capsaicin and blockade of ATP-dependent K+ (KATP) channels, transient receptor potential A1 (TRPA1), transient receptor potential vanilloid 1 (TRPV1), vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide (VIP/PACAP) and calcitonin gene-related peptide (CGRP) receptors with glibenclamide, HC030031, AMG9810, PACAP6–38 and CGRP8–37, respectively. These results suggest that H2S, synthesized by CSE, is involved in the inhibitory neurotransmission to the pig intravesical ureter, through an NO-independent pathway, producing smooth muscle relaxation via KATP channel activation. H2S also promotes the release of inhibitory neuropeptides, as PACAP 38 and/or CGRP from CSPA through TRPA1, TRPV1 and related ion channel activation.