Salvador Bustamante

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.

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.

An approach to personalized cell therapy in chronic complete paraplegia: The Puerta de Hierro phase I/II clinical trial.

BACKGROUND AIMS Cell transplantation in patients suffering spinal cord injury (SCI) is in its initial stages, but currently there is confusion about the results because of the disparity in the techniques used, the route of administration, and the criteria for selecting patients. METHODS We conducted a clinical trial involving 12 patients with complete and chronic paraplegia (average time of chronicity, 13.86 years; SD, 9.36). The characteristics of SCI in magnetic resonance imaging (MRI) were evaluated for a personalized local administration of expanded autologous bone marrow mesenchymal stromal cells (MSCs) supported in autologous plasma, with the number of MSCs ranging from 100 × 10(6) to 230 × 10(6). An additional 30 × 10(6) MSCs were administered 3 months later by lumbar puncture into the subarachnoid space. Outcomes were evaluated at 3, 6, 9 and 12 months after surgery through clinical, urodynamic, neurophysiological and neuroimaging studies. RESULTS Cell transplantation is a safe procedure. All patients experienced improvement, primarily in sensitivity and sphincter control. Infralesional motor activity, according to clinical and neurophysiological studies, was obtained by more than 50% of the patients. Decreases in spasms and spasticity, and improved sexual function were also common findings. Clinical improvement seems to be dose-dependent but was not influenced by the chronicity of the SCI. CONCLUSION Personalized cell therapy with MSCs is safe and leads to clear improvements in clinical aspects and quality of life for patients with complete and chronically established paraplegia.

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.

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.

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.

Endogenous hydrogen sulfide has a powerful role in inhibitory neurotransmission to the pig bladder neck.

PURPOSE We investigated the possible involvement of H2S in nitric oxide independent inhibitory neurotransmission to the pig bladder neck. MATERIALS AND METHODS We used immunohistochemistry to determine the expression of the H2S synthesis enzymes cystathionine γ-lyase and cystathionine β-synthase. We also used electrical field stimulation and myographs for isometric force recordings to study relaxation in response to endogenously released or exogenously applied H2S in urothelium denuded, phenylephrine precontracted bladder neck strips under noradrenergic, noncholinergic, nonnitrergic conditions. RESULTS Cystathionine γ-lyase and cystathionine β-synthase expression was observed in nerve fibers in the smooth muscle layer. Cystathionine γ-lyase and cystathionine β-synthase immunoreactive fibers were also identified around the small arteries supplying the bladder neck. Electrical field stimulation (2 to 16 Hz) evoked frequency dependent relaxation, which was decreased by DL-propargylglycine and abolished by tetrodotoxin (blockers of cystathionine γ-lyase and neuronal voltage gated Na(+) channels, respectively). The cystathionine β-synthase inhibitor O-(carboxymethyl)hydroxylamine did not change nerve mediated responses. The H2S donor GYY4137 (0.1 nM to 10 μM) induced potent, concentration dependent relaxation, which was not modified by neuronal voltage gated Na(+) channels, or cystathionine γ-lyase or cystathionine β-synthase blockade. CONCLUSIONS Results suggest that endogenous H2S synthesized by cystathionine γ-lyase and released from intramural nerves acts as a powerful signaling molecule in nitric oxide independent inhibitory transmission to the pig bladder neck.

Mechanisms involved in testosterone-induced relaxation to the pig urinary bladder neck.

OBJECTIVES Testosterone replacement therapy improves bladder capacity in urinary tract dysfunction. There is no information, however, about the role of this steroid hormone on the muscle tension of the bladder outflow region. The current study investigated the mechanisms underlying the testosterone-induced action in the pig bladder neck. METHODS Urothelium-denuded bladder neck strips were mounted in myographs for isometric force recordings and for simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension. The relaxations to testosterone, the non-aromatizable metabolite 4,5α-dihydrotestosterone (DHT) and electrical field stimulation (EFS) were carried out on phenylephrine (PhE)-precontracted strips. RESULTS Testosterone and DHT evoked similar concentration-dependent relaxations only at very high pharmacological concentrations. The presence of the urothelium and the inhibition of intracellular androgenic receptor (AR), aromatase, 5α-reductase, nitric oxide (NO) synthase, guanylyl cyclase, cyclooxygenase (COX), large-, intermediate- and small-Ca(2+)-activated K(+) channels or ATP-dependent K(+) channels failed to modify the testosterone relaxations. Neuronal voltage-gated Ca(2+) (VOC) channels and voltage-gated K(+) (K(V)) channel blockers potentiated these responses. EFS evoked frequency-dependent relaxations, which were not changed by threshold concentrations of testosterone. In Ca(2+)-free potassium rich physiological saline solution, testosterone inhibited the contractions induced by CaCl(2) and the L-type VOC channel activator (±)-BAY K 8644. Relaxations elicited by testosterone were accompanied by simultaneous decreases in smooth muscle [Ca(2+)](i). CONCLUSIONS Testosterone produces relaxation of the pig urinary bladder neck through mechanisms independent of urothelium, AR, aromatase, 5α-reductase, NO synthase, guanylyl cyclase, COX and K(+) channels. Testosterone-induced relaxation is produced via the inhibition of the extracellular Ca(2+) entry through L-type VOC channels.