Novel mechanism of hydrogen sulfide-induced guinea pig urinary bladder smooth muscle contraction: role of BK channels and cholinergic neurotransmission

Hydrogen sulfide (H2S) is a key signaling molecule regulating important physiological processes, including smooth muscle function. However, the mechanisms underlying H2S-induced detrusor smooth muscle (DSM) contractions are not well understood. This study investigates the cellular and tissue mechanisms by which H2S regulates DSM contractility, excitatory neurotransmission, and large-conductance voltage- and Ca2+-activated K+ (BK) channels in freshly isolated guinea pig DSM. We used a multidisciplinary experimental approach including isometric DSM tension recordings, colorimetric ACh measurement, Ca2+ imaging, and patch-clamp electrophysiology. In isolated DSM strips, the novel slow release H2S donor, P-(4-methoxyphenyl)-p-4-morpholinylphosphinodithioic acid morpholine salt (GYY4137), significantly increased the spontaneous phasic and nerve-evoked DSM contractions. The blockade of neuronal voltage-gated Na+ channels or muscarinic ACh receptors with tetrodotoxin or atropine, respectively, reduced the stimulatory effect of GYY4137 on DSM contractility. GYY4137 increased ACh release from bladder nerves, which was inhibited upon blockade of L-type voltage-gated Ca2+ channels with nifedipine. Furthermore, GYY4137 increased the amplitude of the Ca2+ transients and basal Ca2+ levels in isolated DSM strips. GYY4137 reduced the DSM relaxation induced by the BK channel opener, NS11021. In freshly isolated DSM cells, GYY4137 decreased the amplitude and frequency of transient BK currents recorded in a perforated whole cell configuration and reduced the single BK channel open probability measured in excised inside-out patches. GYY4137 inhibited spontaneous transient hyperpolarizations and depolarized the DSM cell membrane potential. Our results reveal the novel findings that H2S increases spontaneous phasic and nerve-evoked DSM contractions by activating ACh release from bladder nerves in combination with a direct inhibition of DSM BK channels.National Institute of Diabetes and Digestive and Kidney DiseasesDepto. de FisiologíaFac. de FarmaciaTRUEpu

Cardiac System during the Aging Process

The aging process is accompanied by a continuous decline of the cardiac system, disrupting the homeostatic regulation of cells, organs, and systems. Aging increases the prevalence of cardiovascular diseases, thus heart failure and mortality. Understanding the cardiac aging process is of pivotal importance once it allows us to design strategies to prevent age-related cardiac events and increasing the quality of live in the elderly. In this review we provide an overview of the cardiac aging process focus on the following topics: cardiac structural and functional modifications; cellular mechanisms of cardiac dysfunction in the aging; genetics and epigenetics in the development of cardiac diseases; and aging heart and response to the exercise.Sección Deptal. de Fisiología (Farmacia)Depto. de EnfermeríaFac. de FarmaciaFac. de Enfermería, Fisioterapia y PodologíaTRUEpu

Role of Calcitonin Gene-Related Peptide in Inhibitory Neurotransmission to the Pig Bladder Neck

Purpose: We studied the role of calcitonin gene-related peptide in nonadrenergic, noncholinergic neurotransmission to the pig bladder neck. Materials and Methods: We used immunohistochemical techniques to determine the distribution of calcitonin gene-related peptide immunoreactive fibers as well as organ baths for isometric force recording. We investigated relaxation due to endogenously released or exogenously applied calcitonin gene-related peptide in urothelium denuded phenylephrine precontracted strips treated with guanethidine, atropine and NG-nitro-L-arginine to block noradrenergic neurotransmission, muscarinic receptors and nitric oxide synthase, respectively. Results: Rich calcitonin gene-related peptide immunoreactive innervation was found penetrating through the adventitia and distributed in the suburothelial and muscle layers. Numerous, variable size, varicose calcitonin gene-related peptide immunopositive terminals were seen close below the urothelium. In the muscle layer calcitonin gene-related peptide immunopositive nerves usually appeared as varicose terminals running along muscle fibers. Electrical field stimulation (2 to 16 Hz) and exogenous calcitonin gene-related peptide (0.1 nM to 0.3 μM) evoked frequency and concentration dependent relaxation, respectively. Nerve responses were potentiated by capsaicin, decreased by calcitonin gene-related peptide (8–37) and abolished by tetrodotoxin, capsaicin sensitive primary afferent blockers, calcitonin gene-related peptide receptors and neuronal voltage gated Na+ channels. Calcitonin gene-related peptide-induced relaxation was potentiated by the neuronal voltage gated Ca2+ channels blocker ω-conotoxin-GVIA and decreased by calcitonin gene-related peptide (8–37). Calcitonin gene-related peptide relaxation was not modified by blockade of endopeptidases, nitric oxide synthase, guanylyl cyclase and cyclooxygenase. Conclusions: Results suggest that calcitonin gene-related peptide is involved in the nonadrenergic, noncholinergic inhibitory neurotransmission of the pig bladder neck, producing relaxation through neuronal and muscle calcitonin gene-related peptide receptors. Nitric oxide/cyclic guanosine monophosphate and cyclooxygenase pathways do not seem to be involved in such responses.Ministerio de Ciencia e Innovación, SpainSección Deptal. de Fisiología (Farmacia)Fac. de FarmaciaTRUEpu

Hydrogen sulfide mediated inhibitory neurotransmission to the pig bladder neck: role of KATP channels, sensory nerves and calcium signaling.

Purpose: Because neuronal released endogenous H2S has a key role in relaxation of the bladder outflow region, we investigated the mechanisms involved in H2S dependent inhibitory neurotransmission to the pig bladder neck. Materials and methods: Bladder neck strips were mounted in myographs for isometric force recording and simultaneous measurement of intracellular Ca(2+) and tension. Results: On phenylephrine contracted preparations electrical field stimulation and the H2S donor GYY4137 evoked frequency and concentration dependent relaxation, which was reduced by desensitizing capsaicin sensitive primary afferents with capsaicin, and the blockade of adenosine 5'-triphosphate dependent K(+) channels, cyclooxygenase and cyclooxygenase-1 with glibenclamide, indomethacin and SC560, respectively. Inhibition of vanilloid, transient receptor potential A1, transient receptor potential vanilloid 1, vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide and calcitonin gene-related peptide receptors with capsazepine, HC030031, AMG9810, PACAP6-38 and CGRP8-37, respectively, also decreased electrical field stimulation and GYY4137 responses. H2S relaxation was not changed by guanylyl cyclase, protein kinase A, or Ca(2+) activated or voltage gated K(+) channel inhibitors. GYY4137 inhibited the contractions induced by phenylephrine and by K(+) enriched (80 mM) physiological saline solution. To a lesser extent it decreased the phenylephrine and K(+) induced increases in intracellular Ca(2+). Conclusions: H2S produces pig bladder neck relaxation via activation of adenosine 5'-triphosphate dependent K(+) channel and by smooth muscle intracellular Ca(2+) desensitization dependent mechanisms. H2S also promotes the release of sensory neuropeptides and cyclooxygenase-1 pathway derived prostanoids from capsaicin sensitive primary afferents via transient receptor potential A1, transient receptor potential vanilloid 1 and/or related ion channel activation. Keywords: 4-AP; 4-aminopyridine; AM; ATP; ATP dependent K(+); CGRP; COX; CSE; CSPA; Ca(2+) activated K(+); Emax; K(ATP); K(Ca); K(V); KPSS; L-NOARG; MLCP; N(G)-nitro-L-arginine; NO; PACAP; PKA; PSS; TRPA(1); TRPV(1); VOC; VPAC; [Ca(2+)](i); acetoxymethyl ester; adenosine 5′-triphosphate; calcitonin gene-related peptide; capsaicin sensitive primary afferent; cyclooxygenase; cystathionine γ-lyase; hydrogen sulfide; intracellular Ca(2+); maximum response; muscle, smooth; myosin light chain phosphatase; nitric oxide; physiological saline solution; pituitary adenylyl cyclase activating polypeptide; potassium channels; potassium rich PSS; protein kinase A; synaptic transmission; transient receptor potential A1; transient receptor potential vanilloid 1; urinary bladder; vasoactive intestinal peptide receptor; voltage gated Ca(2+); voltage gated K(+).Depto. de EnfermeríaFac. de Enfermería, Fisioterapia y PodologíaTRUEunpu

Mechanisms involved in endothelin‐1‐induced contraction of the pig urinary bladder neck

AbstractAimsThere is no information about the signaling pathways involved in the endothelin‐1 (ET‐1)‐induced contraction of bladder neck. The current study investigates the mechanisms involved in the ET‐1‐elicited contraction in the pig bladder neck.MethodsBladder neck strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O2 and 5% CO2, for isometric force recording to endothelin receptor agonists, noradrenaline (NA), and electrical field stimulation. Endothelin ETA receptor expression was also determined, by both immunohistochemistry and Western blot.ResultsETA receptor expression (Western blot) was observed in the muscular layer and urothelium. A strong ETA‐immunoreactivity (ETA‐IR) was identified within nerve fibers among smooth muscle bundles. ET‐1 and ET‐2 evoked similar concentration‐dependent contractions of urothelium‐denuded preparations. ET‐3 produced a slight response, whereas the ETB receptor agonist BQ3020 failed to promote contraction. BMS182874, an ETA receptor antagonist, reduced ET‐1‐induced contraction whereas BQ788, an ETB antagonist, did not change such responses. ET‐1 contractions were reduced by extracellular Ca2+ removal and by inhibition of voltage‐gated Ca2+ (VOC) (L‐type) and non‐VOC channels, Rho/Rho‐kinase pathway, and neuronal VOC channels. NA produced contractions which were enhanced by ET‐1 threshold concentrations. ETA receptor blockade enhanced nitric oxide‐dependent nerve‐mediated relaxations.ConclusionsThese results suggest that ET‐1 produces contraction via muscular ETA receptors coupled to extracellular Ca2+ entry via VOC (L‐type) and non‐VOC channels. Intracellular Ca2+ mobilization and a Rho/Rho‐kinase pathway could also be involved in these responses. ET‐1‐evoked potentiation on noradrenergic contraction, and neuronal ETA receptors modulating nitrergic inhibitory neurotransmission, are also demonstrated. Neurourol. Urodynam. 31:156–161, 2012. © 2011 Wiley Periodicals, Inc.Depto. de FisiologíaDepto. de EnfermeríaFac. de Enfermería, Fisioterapia y PodologíaFac. de FarmaciaTRUEpu

Neuronal and non-neuronal bradykinin receptors are involved in the contraction and/or relaxation to the pig bladder neck smooth muscle

Aims: The current study investigates the role played by bradykinin (BK) receptors in the contractility to the pig bladder neck smooth muscle. Methods: Bladder neck strips were mounted in myographs for isometric force recordings and BK receptors expression was also determined by immunohistochemistry. Results: B2 receptor expression was observed in the muscular layer and urothelium whereas B1 expression was consistent detected in urothelium. A strong B2 immunoreactivity was also observed within nerve fibers among smooth muscle bundles. On urothelium-denuded preparations basal tone, BK induced concentration-dependent contractions which were reduced in urothelium-intact samples, by extracellular Ca(2+) removal and by blockade of B2 receptors and voltage-gated Ca(2+) (VOC) and non-VOC channels, and increased by cyclooxygenase (COX) inhibition. On phenylephrine-precontracted denuded strips, under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. In urothelium-intact samples, the B1 receptor agonist kallidin promoted concentration-dependent relaxations which were reduced by blockade of B1 receptors, COX, COX-1 and large-conductance Ca(2+) -activated K(+) (BKCa ) channels and abolished in urothelium-denuded samples and in K(+) -enriched physiological saline solution-precontracted strips. Conclusions: These results suggest that BK produces contraction of pig bladder neck via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry via VOC and non-VOC channels with a minor role for intracellular Ca(2+) mobilization. Facilitatory neuronal B2 receptors modulating NANC inhibitory neurotransmission and urothelial B1 receptors producing relaxation via the COX-1 pathway and BKCa channel opening are also demonstrated.Ministerio de Ciencia e Innovación (España)Fundação para a Ciência e Tecnologia, Ministério da Educação e Ciência (Portugal)Depto. de FisiologíaFac. de FarmaciaTRUEpu

Bladder Dysfunction in an Obese Zucker Rat: The Role of TRPA1 Channels, Oxidative Stress, and Hydrogen Sulfide

Purpose: This study investigates whether functionality and/or expression changes of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels, oxidative stress, and hydrogen sulfide (H2S) are involved in the bladder dysfunction from an insulin-resistant obese Zucker rat (OZR). Materials and methods: Detrusor smooth muscle (DSM) samples from the OZR and their respective controls, a lean Zucker rat (LZR), were processed for immunohistochemistry for studying the expression of TRPA1 and TRPV1 and the H2S synthase cystathionine beta-synthase (CBS) and cysthathionine-γ-lyase (CSE). Isometric force recordings to assess the effects of TRPA1 agonists and antagonists on DSM contractility and measurement of oxidative stress and H2S production were also performed. Results: Neuronal TRPA1 expression was increased in the OZR bladder. Electrical field stimulation- (EFS-) elicited contraction was reduced in the OZR bladder. In both LZR and OZR, TRPA1 activation failed to modify DSM basal tension but enhanced EFS contraction; this response is inhibited by the TRPA1 blockade. In the OZR bladder, reactive oxygen species, malondialdehyde, and protein carbonyl contents were increased and antioxidant enzyme activities (superoxide dismutase, catalase, GR, and GPx) were diminished. CSE expression and CSE-generated H2S production were also reduced in the OZR. Both TRPV1 and CBS expressions were not changed in the OZR. Conclusions: These results suggest that an increased expression and functionality of TRPA1, an augmented oxidative stress, and a downregulation of the CSE/H2S pathway are involved in the impairment of nerve-evoked DSM contraction from the OZR.Universidad Complutense de MadridBanco SantanderSección Deptal. de Fisiología (Farmacia)Fac. de FarmaciaTRUEpu

The bitter taste receptor (TAS2R) agonist denatonium promotes a strong relaxation of rat corpus cavernosum

Bitter taste receptors (TAS2R) are found in numerous extra-oral tissues, including smooth muscle (SM) cells in both vascular and visceral tissues. Upon activation, TAS2R stimulate the relaxation of the SM. Nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway is involved in penile erection, and type 5 phosphodiesterase (PDE5) inhibitors, a cGMP-specific hydrolase are used as first-line treatments for erectile dysfunction (ED). Nevertheless, PDE5 inhibitors are ineffective in a considerable number of patients, prompting research into alternative pharmacological targets for ED. Since TAS2R agonists regulate SM contractility, this study investigates the role of TAS2Rs in rat corpus cavernosum (CC). We performed immunohistochemistry to detect TAS2R10, isometric force recordings for TAS2R agonists denatonium and chloroquine, the slow-release H2S donor GYY 4137, the NO donor SNAP, the β-adrenoceptor agonist isoproterenol and electrical field stimulation (EFS), as well as measurement of endogenous hydrogen sulfide (H2S) production. The immunofluorescence staining indicated that TAS2R10 was broadly expressed in the CC SM and to some extent in the nerve fibers. Denatonium, chloroquine, SNAP, and isoproterenol cause potent dose-dependent SM relaxations. H2S production was decreased by NO and H2S synthase inhibitors, while it was enhanced by denatonium. In addition, denatonium increased the relaxations induced by GYY 4137 and SNAP but failed to modify EFS- and isoproterenol-induced responses. These results suggest neuronal and SM TAS2R10 expression in the rat CC, where denatonium induces a strong SM relaxation per se and promotes the H2S- and NO-mediated inhibitory gaseous neurotransmission. Thus, TAS2R10 might represent a valuable therapeutic target in ED.Universidad Complutense de Madrid (Santander-UCM)Depto. de Farmacología y ToxicologíaFac. de VeterinariaTRUEpu

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.Ministerio de Ciencia e Innovación (España)Fundação para a Ciência e Tecnologia, Ministério da Educação e Ciência (Portugal)Depto. de FisiologíaFac. de FarmaciaTRUEpu

Phosphodiesterase type 4 inhibition enhances nitric oxide- and hydrogen sulfide-mediated bladder neck inhibitory neurotransmission

Nitric oxide (NO) and hydrogen sulfide (H2S) play a pivotal role in nerve-mediated relaxation of the bladder outflow region. In the bladder neck, a marked phosphodiesterase type 4 (PDE4) expression has also been described and PDE4 inhibitors, as rolipram, produce smooth muscle relaxation. This study investigates the role of PDE4 isoenzyme in bladder neck gaseous inhibitory neurotransmission. We used Western blot and double immunohistochemical staining for the detection of NPP4 (PDE4) and PDE4A and organ baths for isometric force recording to roflumilast and tadalafil, PDE4 and PDE5, respectively, inhibitors in pig and human samples. Endogenous H2S production measurement and electrical field stimulation (EFS) were also performed. A rich PDE4 and PDE4A expression was observed mainly limited to nerve fibers of the smooth muscle layer of both species. Moreover, roflumilast produced a much more potent smooth muscle relaxation than that induced by tadalafil. In porcine samples, H2S generation was diminished by H2S and NO synthase inhibition and augmented by roflumilast. Relaxations elicited by EFS were potentiated by roflumilast. These results suggest that PDE4, mainly PDE4A, is mostly located within nerve fibers of the pig and human bladder neck, where roflumilast produces a powerful smooth muscle relaxation. In pig, the fact that roflumilast increases endogenous H2S production and EFS-induced relaxations suggests a modulation of PDE4 on NO- and H2S-mediated inhibitory neurotransmission.Depto. de Farmacología y ToxicologíaFac. de VeterinariaTRUEpu