Paz Recio

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.

Mechanisms involved in testosterone-induced vasodilatation in pig prostatic small arteries

AIMS Testosterone is beneficial to the cardiovascular system due to its direct coronary vasodilatory action and its circulatory deficiency is associated with coronary artery disease (CAD), which has been proposed as an extrinsic risk factor for benign prostatic hyperplasia (BPH). Therefore, the current study investigated the mechanisms involved in the testosterone-induced vasodilatation in pig prostatic small arteries. MAIN METHODS The testosterone vasoactive effects were assessed in small arterial rings mounted in microvascular myographs for isometric force recordings. KEY FINDINGS Testosterone and the non-aromatizable metabolite 4, 5alpha-dihydrotestosterone (DHT) evoked a similar concentration-dependent relaxation on noradrenaline (NA)-precontracted rings. Similar responses were obtained in preparations contracted with 60 mM K(+)-enriched physiological saline solution. Endothelium mechanical removal or pre-treatment with blockers of nitric oxide (NO) synthase, guanylate cyclase, aromatase activity, intracellular androgenic receptor (AR), 5alpha-reductase, prostanoid synthesis and K(+) channels, failed to modify the responses to testosterone. In Ca(2+)-free 124 mM KPSS, testosterone markedly inhibited in a concentration-dependent manner the contraction curve t degrees CaCl(2). In arteries pretreated with an L-type voltage-activated Ca(2+) channels (VOCCs) inhibitor, nifedipine, testosterone still relaxed noradrenaline-precontracted arteries. SIGNIFICANCE These data suggest that testosterone induces a direct vasodilatory action in pig prostatic small arteries independent of either endothelium, NO, prostanoids, aromatase or 5alpha-reductase activities, AR or K(+) channels. Such an effect is suggested to be produced via blockade of extracellular Ca(2+) entry through L-type VOCCs and non-L-type Ca(2+) channels. Testosterone-induced vasodilatation could be useful to prevent prostatic ischemia.

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.

Nitrergic relaxation of the horse corpus cavernosum. Role of cGMP.

The involvement of nitric oxide (NO) and the mechanisms mediating neurogenic relaxation were investigated in the horse corpus cavernosum. NADPH-diaphorase activity was expressed in nerve fibres around arteries and muscular bundles in the horse trabecular tissue. Relaxations in response to electrical field stimulation were tetrodotoxin (10(-6) M)-sensitive, indicating their neurogenic origin. The NO synthase inhibitor, L-NO-arginine (L-NO-Arg, 3 x 10(-5) M), abolished the electrically induced relaxations, which were significantly reversed by L-arginine (3 x 10(-3) M). Exogenous NO (10(-6)-10(-3) M) evoked relaxations which were unaffected by L-NO-Arg. 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 5 x 10(-6) M), an inhibitor of guanylate cyclase activation by NO, reduced the relaxations in response to electrical stimulation and exogenous NO. Iberiotoxin (3 x 10(-8) M) or apamin (5 x 10(-7) M), inhibitors of large and small conductance Ca2+-activated K+ channels, respectively, and glibenclamide (3 x 10(-6) M), a blocker of ATP-sensitive K+ channels, failed to modify the relaxations with NO. It is suggested that NO is present in nerve fibres of the horse corpus cavernosum and relaxes smooth muscle through a guanylate cyclase-dependent mechanism. Neither Ca2+-activated nor ATP-sensitive K+ channels seem to be involved in these relaxations.

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.

Functional evidence of nitrergic neurotransmission in the human urinary bladder neck.

Nitric oxide (NO) is involved in the non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission of the lower urinary tract. However, functional evidence of this involvement in the human urinary bladder neck has not been consistently demonstrated. Therefore, the current study investigates the relaxations to endogenously released and/or exogenously added NO, in the human bladder neck. 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% CO(2) and 95% O(2), for isometric force recording. The relaxations to transmural nerve stimulation (EFS) or to exogenously applied NO, as an acidified solution of NaNO(2) were carried out on strips precontracted with phenylephrine, and treated with guanethidine and atropine, to block noradrenergic neurotransmission and muscarinic receptors, respectively. EFS (0.5-16Hz) and exogenous NaNO(2) (1muM to 1mM) evoked frequency- and concentration-dependent relaxations, respectively. The nerve responses were abolished by the blockade of neuronal voltage-activated Na(+) channels with tetrodotoxin, indicating their neurogenic character. N(G)-nitro-l-arginine (l-NOARG), a NO synthase inhibitor, abolished the relaxations to nerve stimulation, which were partially reversed by the substrate of NO synthesis l-arginine. l-NOARG failed to modify the relaxations to exogenous NaNO(2). These results suggest that NO is the major NANC inhibitory neurotransmitter in the human urinary bladder neck. Blockers of NO synthase could be useful in therapy for the urinary incontinence produced by intrinsic sphincteric deficiency.

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.

Characterization of the 5‐hydroxytryptamine receptors mediating contraction in the pig isolated intravesical ureter

This study was designed to investigate the effect of 5‐hydroxytryptamine (5‐HT) and to characterize the 5‐HT receptors involved in 5‐HT responses in the pig intravesical ureter. 5‐HT (0.01–10 μM) concentration‐dependently increased the tone of intravesical ureteral strips, whereas the increases in phasic contractions were concentration‐independent. The 5‐HT2 receptor agonist α‐methyl 5‐HT, mimicked the effect on tone whereas weak or no response was obtained with 5‐CT, 8‐OH‐DPAT, m‐chlorophenylbiguanide and RS 67333, 5‐HT1, 5‐HT1A, 5‐HT3 and 5‐HT4 receptor agonists, respectively. 5‐HT did not induce relaxation of U46619‐contracted ureteral preparations. Pargyline (100 μM), a monoaminooxidase A/B activity inhibitor, produced leftward displacements of the concentration‐response curves for 5‐HT. 5‐HT‐induced tone was reduced by the 5‐HT2 and 5‐HT2A receptor antagonists ritanserine (0.1 μM) and spiperone (0.2 μM), respectively. However, 5‐HT contraction was not antagonized by cyanopindolol (2 μM), SDZ–SER 082 (1 μM), Y‐25130 (1 μM) and GR 113808 (0.1 μM), which are respectively, 5‐HT1A/1B, 5‐HT2B/2C, 5‐HT3, and 5‐HT4 selective receptor antagonists. Removal of the urothelium did not modify 5‐HT‐induced contractions. Blockade of neuronal voltage‐activated sodium channels, α‐adrenergic receptors and adrenergic neurotransmission with tetrodotoxin (1 μM), phentolamine (0.3 μM) and guanethidine (10 μM), respectively, reduced the contractions to 5‐HT. However, physostigmine (1 μM), atropine (0.1 μM) and suramin (30 μM), inhibitors of cholinesterase activity, muscarinic‐ and purinergic P2‐receptors, respectively, failed to modify the contractions to 5‐HT. These results suggest that 5‐HT increases the tone of the pig intravesical ureter through 5‐HT2A receptors located at the smooth muscle. Part of the 5‐HT contraction is indirectly mediated via noradrenaline release from sympathetic nerves.