RESUMEN
Hydrogen sulfide (H2S) is a gasotransmitter employed for intra- and inter-cellular communication in almost all organ systems. This study investigates the role of endogenous H2S in nerve-evoked relaxation of pig terminal bronchioles with 260 µm medium internal lumen diameter. High expression of the H2S synthesis enzyme cystathionine γ-lyase (CSE) in the bronchiolar muscle layer and strong CSE-immunoreactivity within nerve fibers distributed along smooth muscle bundles were observed. Further, endogenous H2S generated in bronchiolar membranes was reduced by CSE inhibition. In contrast, cystathionine ß-synthase expression, another H2S synthesis enzyme, however was not consistently detected in the bronchiolar smooth muscle layer. Electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked smooth muscle relaxation. Inhibition of CSE, nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and of ATP-dependent K+, transient receptor potential A1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) channels reduced the EFS relaxation but failed to modify the GYY4137 response. Raising extracellular K+ concentration inhibited the GYY4137 relaxation. Large conductance Ca2+-activated K+ channel blockade reduced both EFS and GYY4137 responses. GYY4137 inhibited the contractions induced by histamine and reduced to a lesser extent the histamine-induced increases in intracellular [Ca2+]. These results suggest that relaxation induced by EFS in the pig terminal bronchioles partly involves the H2S/CSE pathway. H2S response is produced via NO/sGC-independent mechanisms involving K+ channels and intracellular Ca2+ desensitization-dependent pathways. Thus, based on our current results H2S donors might be useful as bronchodilator agents for the treatment of lung diseases with persistent airflow limitation, such as asthma and chronic obstructive lung disease.
Asunto(s)
Bronquiolos/metabolismo , Cistationina gamma-Liasa/metabolismo , Sulfuro de Hidrógeno/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Femenino , Histamina/metabolismo , Masculino , Morfolinas/farmacología , Relajación Muscular/fisiología , Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Compuestos Organotiofosforados/farmacología , Canales de Potasio/metabolismo , PorcinosRESUMEN
AIMS: Neuronal and non-neuronal bradykinin (BK) receptors regulate the contractility of the bladder urine outflow region. The current study investigates the role of BK receptors in the regulation of the smooth muscle contractility of the pig intravesical ureter. METHODS: Western blot and immunohistochemistry were used to show the expression of BK B1 and B2 receptors and myographs for isometric force recordings. RESULTS: B2 receptor expression was consistently detected in the intravesical ureter urothelium and smooth muscle layer, B1 expression was not detected where a strong B2 immunoreactivity was observed within nerve fibers among smooth muscle bundles. On ureteral strips basal tone, BK induced concentration-dependent contractions, were potently reduced by extracellular Ca(2+) removal and by B2 receptor and voltage-gated Ca(2+) (VOC) channel blockade. BK contraction did not change as a consequence of urothelium mechanical removal or cyclooxygenase and Rho-associated protein kinase inhibition. On 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α (U46619)-precontracted samples, under non-adrenergic non-cholinergic (NANC) and nitric oxide (NO)-independent NANC conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. Kallidin, a B1 receptor agonist, failed to increase preparation basal tension or to induce relaxation on U46619-induced tone. CONCLUSIONS: The present results suggest that BK produces contraction of pig intravesical ureter via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry mainly via VOC (L-type) channels. Facilitatory neuronal B2 receptors modulating NO-dependent or independent NANC inhibitory neurotransmission are also demonstrated.
Asunto(s)
Contracción Muscular/fisiología , Músculo Liso/metabolismo , Receptor de Bradiquinina B2/metabolismo , Uréter/metabolismo , Animales , Bradiquinina/farmacología , Femenino , Calidina/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Músculo Liso/efectos de los fármacos , Receptor de Bradiquinina B1/metabolismo , Porcinos , Uréter/efectos de los fármacos , Urotelio/efectos de los fármacos , Urotelio/metabolismo , Vasodilatadores/farmacologíaRESUMEN
INTRODUCTION: Endothelin 1 (ET-1) levels and receptors are up-regulated in the erectile tissue of diabetic patients and animal models of erectile dysfunction (ED). AIM: The present study assessed the role of ET-1 receptors in the impaired adrenergic vasoconstriction and nitrergic relaxation of penile arteries from a rat model of insulin resistance. METHODS: The effect of ET receptor antagonists was evaluated on the contractile responses to electrical field stimulation (EFS) of penile arteries from obese Zucker rats (OZRs) compared with lean Zucker rats (LZRs). ET receptor expression was determined by immunohistochemistry. MAIN OUTCOME MEASURES: Changes in neural nitrergic relaxation and adrenergic vasoconstriction and the expression of ET receptors in perivascular nerves were assessed. RESULTS: ET-1 (10(-10) M) enhanced EFS-induced vasoconstriction, and treatment with the adrenergic neurotoxin guanethidine reduced the contractions induced by ET-1 in penile arteries from both LZRs and OZRs, thus supporting the hypothesis that ET-1 releases noradrenaline from adrenergic nerves. ET-1 antagonized neural nitric oxide (NO)-mediated relaxant responses in LZR arteries, antagonizing relaxations induced by the NO donor S-nitroso-N-acetylpenicillamine to a larger extent in arteries from OZRs. ET(A) and ET(B) receptors were expressed in perivascular fibers colocalized with the neuronal marker protein gene product 9.5 in penile arteries from OZRs. The ET(A) receptor antagonist BQ-123 reversed the enhancing effect of ET-1 on the vasoconstriction elicited by EFS and the ET-1-induced inhibition of nitrergic relaxations in LZRs, restoring them to control levels in penile arteries of OZRs. CONCLUSIONS: ET-1 enhances adrenergic vasoconstriction through presynaptic ET(A) receptors and antagonizes neural NO-mediated relaxation through postsynaptic smooth muscle ET(A) receptors in penile arteries from OZRs, which likely contributes to the augmented vasoconstriction and blunted nitrergic relaxation of erectile tissue under conditions of insulin resistance.
Asunto(s)
Resistencia a la Insulina/fisiología , Pene/irrigación sanguínea , Receptor de Endotelina A/fisiología , Vasoconstricción/fisiología , Adrenérgicos/farmacología , Animales , Arterias/fisiología , Antagonistas de los Receptores de la Endotelina A , Endotelina-1/metabolismo , Endotelina-1/fisiología , Disfunción Eréctil/fisiopatología , Guanetidina/farmacología , Insulina/farmacología , Masculino , Relajación Muscular/fisiología , Músculo Liso Vascular/fisiología , Neurotoxinas/farmacología , Óxido Nítrico/fisiología , Obesidad/fisiopatología , Erección Peniana/efectos de los fármacos , Péptidos Cíclicos/farmacología , Ratas Zucker , Vasodilatación/fisiología , Vasodilatadores/farmacologíaRESUMEN
INTRODUCTION: Phosphodiesterase type 5 (PDE5) inhibitors act as effective drugs for the treatment of lower urinary tract symptom (LUTS). There is a poor information, however, about the role of the PDE4 inhibitors on the bladder outflow region contractility. AIM: To investigate PDE4 expression and the relaxation induced by the PDE4 inhibitor rolipram versus that induced by the PDE5 blockers sildenafil and vardenafil, in the pig and human bladder neck. METHODS: Immunohistochemistry for PDE4 expression, myographs for isometric force recordings and fura-2 fluorescence for simultaneous measurements of intracellular Ca2+ concentration ([Ca2+]i ) and tension for rolipram in bladder neck samples were used. MAIN OUTCOME MEASURES: PDE4 expression and relaxations to PDE4 and PDE5 inhibitors and simultaneous measurements of [Ca2+]i and tension. RESULTS: PDE4 expression was observed widely distributed in the smooth muscle layer of the pig and human bladder neck. On urothelium-denuded phenylephrine (PhE)-precontracted strips of pig and human, rolipram, sildenafil and vardenafil produced concentration-dependent relaxations with the following order of potency: rolipram> > sildenafil>vardenafil. In pig, the adenylyl cyclase activator forskolin potentiated rolipram-elicited relaxation, whereas protein kinase A (PKA) blockade reduced such effect. On potassium-enriched physiological saline solution (KPSS)-precontracted strips, rolipram evoked a lower relaxation than that obtained on PhE-stimulated preparations. Inhibition of large (BKCa ) and intermediate (IKCa ) conductance Ca2+ -activated K+ channels, neuronal voltage-gated Ca2+ channels, nitric oxide (NO) and hydrogen sulfide (H2 S) synthases reduced rolipram responses. Rolipram inhibited the contractions induced by PhE without reducing the PhE-evoked [Ca2+]i increase. CONCLUSIONS: PDE4 is present in the pig and human bladder neck smooth muscle, where rolipram exerts a much more potent relaxation than that elicited by PDE5 inhibitors. In pig, rolipram-induced response is produced through the PKA pathway involving BKCa and IKCa channel activation and [Ca2+]i desensitization-dependent mechanisms, this relaxation also being due to neuronal NO and H2S release.
Asunto(s)
Inhibidores de Fosfodiesterasa 4/farmacología , Rolipram/farmacología , Vejiga Urinaria/efectos de los fármacos , Adulto , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Imidazoles/farmacología , Masculino , Persona de Mediana Edad , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Fenilefrina/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Purinas/farmacología , Transducción de Señal/fisiología , Citrato de Sildenafil , Sulfonas/farmacología , Sus scrofa , Triazinas/farmacología , Vejiga Urinaria/metabolismo , Urotelio/metabolismo , Diclorhidrato de VardenafilRESUMEN
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. Neurourol. Urodynam. 33:558-565, 2014. © 2013 Wiley Periodicals, Inc.
Asunto(s)
Calcio/metabolismo , Contracción Muscular/fisiología , Relajación Muscular/fisiología , Músculo Liso/metabolismo , Receptor de Bradiquinina B1/metabolismo , Receptor de Bradiquinina B2/metabolismo , Vejiga Urinaria/metabolismo , Urotelio/metabolismo , Animales , Bradiquinina/farmacología , Antagonistas de los Receptores de Bradiquinina/farmacología , Canales de Calcio/metabolismo , Ciclooxigenasa 1/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptor de Bradiquinina B1/fisiología , Receptor de Bradiquinina B2/fisiología , Transducción de Señal , Porcinos , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/fisiología , Urotelio/efectos de los fármacosRESUMEN
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 ß-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 ß-synthase expression was observed in nerve fibers in the smooth muscle layer. Cystathionine γ-lyase and cystathionine ß-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 ß-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 ß-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.
Asunto(s)
Sulfuro de Hidrógeno , Transmisión Sináptica/fisiología , Vejiga Urinaria/fisiología , Animales , Femenino , Sulfuro de Hidrógeno/metabolismo , Masculino , PorcinosRESUMEN
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.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Canales KATP/metabolismo , Músculo Liso/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Transmisión Sináptica/efectos de los fármacos , Vejiga Urinaria/inervación , Vejiga Urinaria/metabolismo , Acetanilidas/farmacología , Acrilamidas/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Estimulación Eléctrica , Gliburida/farmacología , Guanilato Ciclasa/farmacología , Indometacina/farmacología , Morfolinas/farmacología , Compuestos Organotiofosforados/farmacología , Fenilefrina/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Purinas/farmacología , Pirazoles/farmacología , PorcinosRESUMEN
INTRODUCTION: Erectile dysfunction is considered as an early sign of subclinical vascular disease and endothelial dysfunction and a highly prevalent condition in diabetic patients. AIM: The current study assessed whether impaired vascular effects of endothelin (ET)-1 may contribute to the vascular dysfunction of penile arteries from a rat model of insulin resistance. METHODS: The effect of ETA and ETB receptor antagonists was assessed on the intracellular Ca(2+) [Ca(2+) ]i and contractile responses to ET-1 in penile arteries from obese Zucker rats (OZR) and lean Zucker rats (LZR), and ET receptor expression in the arterial wall was assessed by immunohistochemistry. MAIN OUTCOME MEASURE: Changes in ET-1 [Ca(2+) ]i and vasoconstriction and ET receptor expression were evaluated in penile arteries from insulin-resistant rats. RESULTS: ET-1-induced vasoconstriction was associated with a higher increase in smooth muscle [Ca(2+) ]i in penile arteries from OZR compared with LZR. Removal of the endothelium inhibited and enhanced contractions to the lowest and highest doses of ET-1, respectively, mainly in OZR. The selective ETA receptor antagonist BQ-123 inhibited ET-1 vasoconstriction and [Ca(2+) ]i response in both LZR and OZR. The ETB receptor antagonist BQ-788 had little effect in healthy arteries but markedly inhibited ET-1-induced increases in [Ca(2+) ]i and vasoconstriction in arteries from OZR. ETA receptors were located on the smooth muscle and endothelium of penile arteries, whereas ETB receptors were found on the arterial endothelium in LZR and OZR, and also on the smooth muscle in OZR, immunostaining for both receptors being higher in OZR. CONCLUSION: Penile arteries from OZR exhibit an impaired ET-1 Ca(2+) signaling along with changes in the ET receptor profile. Thus, whereas ET-1 contraction and the associated [Ca(2+) ]i increase are mediated by smooth muscle ETA receptors in healthy arteries, ETB receptors contribute to contraction and are coupled to the augmented ET-1 [Ca(2+) ]i response under conditions of insulin resistance.
Asunto(s)
Señalización del Calcio , Endotelina-1/fisiología , Endotelio Vascular/metabolismo , Impotencia Vasculogénica/etiología , Resistencia a la Insulina , Músculo Liso Vascular/metabolismo , Pene/irrigación sanguínea , Receptor de Endotelina B/metabolismo , Vasoconstricción , Animales , Arterias/metabolismo , Arterias/fisiopatología , Señalización del Calcio/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Disfunción Eréctil/metabolismo , Disfunción Eréctil/fisiopatología , Humanos , Impotencia Vasculogénica/metabolismo , Impotencia Vasculogénica/fisiopatología , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Ratas , Ratas Zucker , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/efectos de los fármacos , Vasoconstricción/efectos de los fármacosRESUMEN
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 ß-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.
Asunto(s)
Cloroquina , Gusto , Masculino , Animales , Ratas , Isoproterenol , GMP CíclicoRESUMEN
AIMS: There 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. METHODS: Bladder neck strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O(2) and 5% CO(2) , for isometric force recording to endothelin receptor agonists, noradrenaline (NA), and electrical field stimulation. Endothelin ET(A) receptor expression was also determined, by both immunohistochemistry and Western blot. RESULTS: ET(A) receptor expression (Western blot) was observed in the muscular layer and urothelium. A strong ET(A) -immunoreactivity (ET(A) -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 ET(B) receptor agonist BQ3020 failed to promote contraction. BMS182874, an ET(A) receptor antagonist, reduced ET-1-induced contraction whereas BQ788, an ET(B) antagonist, did not change such responses. ET-1 contractions were reduced by extracellular Ca(2+) removal and by inhibition of voltage-gated Ca(2+) (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. ET(A) receptor blockade enhanced nitric oxide-dependent nerve-mediated relaxations. CONCLUSIONS: These results suggest that ET-1 produces contraction via muscular ET(A) receptors coupled to extracellular Ca(2+) entry via VOC (L-type) and non-VOC channels. Intracellular Ca(2+) mobilization and a Rho/Rho-kinase pathway could also be involved in these responses. ET-1-evoked potentiation on noradrenergic contraction, and neuronal ET(A) receptors modulating nitrergic inhibitory neurotransmission, are also demonstrated.
Asunto(s)
Endotelina-1/fisiología , Contracción Muscular/fisiología , Transducción de Señal/fisiología , Vejiga Urinaria/fisiología , Animales , Calcio/fisiología , Canales de Calcio/fisiología , Estimulación Eléctrica , Endotelina-1/farmacología , Femenino , Masculino , Modelos Animales , Contracción Muscular/efectos de los fármacos , Receptor de Endotelina A/fisiología , Porcinos , Transmisión Sináptica/fisiologíaRESUMEN
AIMS: The involvement of endothelin receptors in the contraction of the lower urinary tract smooth muscle is well established. There is scarce information, however, about endothelin receptors mediating relaxation of the bladder outlet region. The current study investigates the possible existence of endothelin ET(B) receptors involved in the relaxation of pig bladder neck. METHODS: ET(B) receptor expression was determined by immunohistochemistry and urothelium-denuded bladder neck strips were mounted in organ baths for isometric force recording. RESULTS: ET(B) -immunoreactivity (ET(B) -IR) was observed within nerve fibers among smooth muscle bundles and urothelium. BQ3020 (0.01-300 nM), an ET(B) receptor agonist, produced concentration-dependent relaxations which were reduced by BQ788, an ET(B) receptor antagonist, and by inhibitors of protein kinase A (PKA) and large (BK(Ca) )- or small (SK(Ca) )-conductance Ca(2+) -activated K(+) channels. Pretreatment with BK(Ca) or SK(Ca) channel inhibitors plus PKA blocking did not cause further inhibition compared with that exerted by inhibiting BK(Ca) or SK(Ca) channels only. BQ3020-induced relaxation was not modified by blockade of either nitric oxide (NO) synthase, guanylyl cyclase, cyclooxygenase (COX) or of intermediate-conductance Ca(2+) -activated-(IK(Ca) ), ATP-dependent-(K(ATP) ), or voltage-gated-(K(v) ) K(+) channels. Under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation (0.5-16 Hz) evoked frequency-dependent relaxations, which were reduced by BQ788 and potentiated by threshold concentrations of BQ3020. CONCLUSIONS: These results suggest that BQ3020 produces relaxation of the pig bladder neck via activation of muscle endothelin ET(B) receptors, NO/cGMP- and COX-independent-, cAMP-PKA pathway-dependent-mechanisms, and involving BK(Ca) and SK(Ca) channel activation. ET(B) receptors are also involved in the NANC inhibitory neurotransmission.
Asunto(s)
Relajación Muscular , Músculo Liso/metabolismo , Receptor de Endotelina B/metabolismo , Vejiga Urinaria/metabolismo , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Endotelinas/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Fibras Nerviosas/metabolismo , Neurotransmisores/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Piperidinas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptor de Endotelina B/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Guanilil Ciclasa Soluble , Porcinos , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/inervación , Urotelio/metabolismoRESUMEN
AIMS: Nitric oxide (NO) and hydrogen sulfide (H2S) are involved in nerve-mediated corpus cavernosum (CC) relaxation. Expression of phosphodiesterase type 5 (PDE5) and type 4 (PDE4), cyclic guanosine monophosphate (cGMP)- and cyclic adenosine monophosphate (cAMP)-specific, respectively, has been described and PDE5- and PDE4-inhibitors induce cavernous smooth muscle relaxation. Whereas the NO/cGMP signaling pathway is well established in penile erection, the cAMP-mediated mechanism is not fully elucidated. The aim of this study is to investigate the localization and the functional significance of PDE4 in rat CC tone regulation. MAIN METHODS: We performed immunohistochemistry for the detection of the PDE4A isoenzyme. Isometric tension recordings for roflumilast and tadalafil, PDE4 and PDE5 inhibitors, respectively, electrical field stimulation (EFS) and ß-adrenoceptor agonist isoproterenol and endogenous H2S production measurement. KEY FINDINGS: A marked PDE4A expression was detected mainly localized in the nerve cells of the cavernous smooth muscle. Furthermore, roflumilast and tadalafil exhibited strong corpus cavernous relaxations. Endogenous H2S production was decreased by NO and H2S synthase inhibitors and increased by roflumilast. Isoproterenol- and EFS-induced relaxations were increased by roflumilast. SIGNIFICANCE: These results indicate that PDE4A is mainly expressed within the nerves cells of the rat CC, where roflumilast induces a potent corpus cavernous relaxation per se and potentiates the response induced by ß-adrenoceptor activation. The fact that roflumilast enhances H2S production, as well as EFS-elicited responses suggests that PDE4 inhibitors modulate, in a positive feedback fashion, nerve-mediated relaxation induced by gasotransmitters, thus indicating a key role for neuronal PDE4 in penile erection.
Asunto(s)
Aminopiridinas/farmacología , Benzamidas/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Gasotransmisores/metabolismo , Pene/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Aminopiridinas/administración & dosificación , Animales , Benzamidas/administración & dosificación , Ciclopropanos/administración & dosificación , Ciclopropanos/farmacología , Relación Dosis-Respuesta a Droga , Sulfuro de Hidrógeno/metabolismo , Masculino , Relajación Muscular/efectos de los fármacos , Nitroarginina/farmacología , Pene/efectos de los fármacos , Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/fisiología , Ratas Wistar , Tadalafilo/farmacologíaRESUMEN
Diabetes is associated with an increased vascular tone usually involved in the pathogenesis of diabetic cardiovascular complications such as hypertension, stroke, coronary artery disease, or erectile dysfunction (ED). Enhanced contractility of penile erectile tissue has been associated with augmented activity of the RhoA/Rho kinase (RhoK) pathway in models of diabetes-associated ED. The present study assessed whether abnormal vasoconstriction in penile arteries from prediabetic obese Zucker rats (OZRs) is due to changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and/or in myofilament Ca(2+) sensitivity. Penile arteries from OZRs and lean Zucker rats (LZRs) were mounted on microvascular myographs for simultaneous measurements of [Ca(2+)](i) and tension. The relationships between [Ca(2+)](i) and contraction for the α(1)-adrenergic vasoconstrictor phenylephrine (PE) were left shifted and steeper in OZRs compared with LZRs, although the magnitude of the contraction was similar in both groups. In contrast, the vasoconstriction induced by the thromboxane A(2) receptor agonist U-46619 was augmented in arteries from OZRs, and this increase was associated with an increase in both the sensitivity and maximum responses to Ca(2+). The RhoK inhibitor Y-27632 (10 µM) reduced the vasoconstriction induced by PE to a greater extent in OZRs than in LZRs, without altering Ca(2+). Y-27632 inhibited with a greater potency the contraction elicited by high KCl in arteries from OZRs compared with LZRs without changing [Ca(2+)](i). RhoK-II expression was augmented in arteries from OZRs. These results suggest receptor-specific changes in the Ca(2+) handling of penile arteries under conditions of metabolic syndrome. Whereas augmented vasoconstriction upon activation of the thromboxane A(2) receptor is coupled to enhanced Ca(2+) entry, a RhoK-mediated enhancement of myofilament Ca(2+) sensitivity is coupled with the α(1)-adrenergic vasoconstriction in penile arteries from OZRs.
Asunto(s)
Arterias/metabolismo , Calcio/metabolismo , Pene/irrigación sanguínea , Estado Prediabético/metabolismo , Quinasas Asociadas a rho/fisiología , Amidas/farmacología , Animales , Inhibidores Enzimáticos/farmacología , Masculino , Modelos Animales , Fenilefrina/farmacología , Piridinas/farmacología , Ratas , Ratas Zucker , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasoconstrictores/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/efectos de los fármacosRESUMEN
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.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/fisiología , Transmisión Sináptica , Vejiga Urinaria/inervación , Vejiga Urinaria/fisiología , Animales , Femenino , Masculino , PorcinosRESUMEN
Benign prostatic hypertrophy has been related with glandular ischemia processes and adenosine is a potent vasodilator agent. This study investigates the mechanisms underlying the adenosine-induced vasorelaxation in pig prostatic small arteries. Adenosine receptors expression was determined by Western blot and immunohistochemistry, and rings were mounted in myographs for isometric force recording. A(2A) and A(3) receptor expression was observed in the arterial wall and A(2A)-immunoreactivity was identified in the adventitia-media junction and endothelium. A(1) and A(2B) receptor expression was not obtained. On noradrenaline-precontracted rings, P1 receptor agonists produced concentration-dependent relaxations with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA) = CGS21680 > 2-Cl-IB-MECA = 2-Cl-cyclopentyladenosine = adenosine. Adenosine reuptake inhibition potentiated both NECA and adenosine relaxations. Endothelium removal and ZM241385, an A(2A) antagonist, reduced NECA relaxations that were not modified by A(1), A(2B), and A(3) receptor antagonists. Neuronal voltage-gated Ca(2+) channels and nitric oxide (NO) synthase blockade, and adenylyl cyclase activation enhanced these responses, which were reduced by protein kinase A inhibition and by blockade of the intermediate (IK(Ca))- and small (SK(Ca))-conductance Ca(2+)-activated K(+) channels. Inhibition of cyclooxygenase (COX), large-conductance Ca(2+)-activated-, ATP-dependent-, and voltage-gated-K(+) channel failed to modify these responses. These results suggest that adenosine induces endothelium-dependent relaxations in the pig prostatic arteries via A(2A) purinoceptors. The adenosine vasorelaxation, which is prejunctionally modulated, is produced via NO- and COX-independent mechanisms that involve activation of IK(Ca) and SK(Ca) channels and stimulation of adenylyl cyclase. Endothelium-derived NO playing a regulatory role under conditions in which EDHF is non-functional is also suggested. Adenosine-induced vasodilatation could be useful to prevent prostatic ischemia.
RESUMEN
AIMS: The current study investigates the mechanisms involved in nitric oxide (NO)-independent, nonadrenergic, noncholinergic (NANC) inhibitory neurotransmission to the pig urinary bladder neck. METHODS: Urothelium-denuded strips were mounted in organ baths containing physiological saline solution (PSS) at 37°C for isometric force recordings. The relaxations to electrical field stimulation (EFS) were carried out on strips treated with guanethidine, atropine and N(G) -nitro-L-arginine, to block noradrenergic neurotransmission, muscarinic receptors and NO synthase, respectively, and precontracted with phenylephrine. RESULTS: EFS (1-16 Hz) produced frequency-dependent relaxations which were abolished by the blockade of neuronal voltage-activated Na(+) channels. Nonselective and selective inhibition of COX and COX-1, respectively, and blockade of Na(+) -K(+) ATPase reduced the EFS-induced relaxations. However, blockade of COX-2, soluble guanylyl cyclase, large-, intermediate- and small-conductance Ca(2+) -activated K(+) channels, ATP-dependent K(+) channels, voltage-gated K(+) channels, cAMPc-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) failed to modify the nerve-mediated relaxations. CONCLUSIONS: The NO-independent inhibitory neurotransmission to the pig urinary bladder neck is mediated, in part, through prostanoids release from a COX-1 pathway, and through activation of the Na(+) -K(+) ATPase. PKA and PKG pathways and postjunctional K(+) channels do not appear to be involved in the NO-independent nerve-mediated relaxations.
Asunto(s)
Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Vejiga Urinaria/fisiología , Adrenérgicos/farmacología , Animales , Atropina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Ciclooxigenasa 1/metabolismo , Estimulación Eléctrica/métodos , Femenino , Guanetidina/farmacología , Técnicas In Vitro , Masculino , Antagonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos , Vejiga Urinaria/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Obesity is a risk factor for the development of chronic kidney disease independent of diabetes, hypertension and other co-morbidities. Obesity-associated nephropathy is linked to dysregulation of the cell energy sensor AMP-activated protein kinase (AMPK). We aimed here to assess whether impairment of AMPK activity may cause renal arterial dysfunction in obesity and to evaluate the therapeutic potential of activating renal AMPK. EXPERIMENTAL APPROACH: Effects of the AMPK activator A769662 were assessed on intrarenal arteries isolated from ob/ob mice and obese Zucker rats and then mounted in microvascular myographs. Superoxide and hydrogen peroxide production were measured by chemiluminescence and fluorescence, respectively, and protein expression was analysed by western blotting. KEY RESULTS: Endothelium-dependent vasodilation and PI3K/Akt/eNOS pathway were impaired in preglomerular arteries from genetically obese rats and mice, along with impaired arterial AMPK activity and blunted relaxations induced by the AMPK activator A769662. Acute ex vivo exposure to A769662 restored endothelial function and enhanced activity of PI3K/Akt/eNOS pathway in obese rats, whereas in vivo treatment with A769662 improved metabolic state and ameliorated endothelial dysfunction, reduced inflammatory markers and vascular oxidative stress in renal arteries and restored redox balance in renal cortex of obese mice. CONCLUSION AND IMPLICATIONS: These results demonstrate that AMPK dysregulation underlies obesity-associated kidney vascular dysfunction and activation of AMPK improves metabolic state, protects renal endothelial function and exerts potent vascular antioxidant and anti-inflammatory effects. The beneficial effects of vascular AMPK activation might represent a promising therapeutic approach to the treatment of obesity-related kidney injury.
Asunto(s)
Adenilato Quinasa , Endotelio Vascular , Proteínas Quinasas Activadas por AMP/metabolismo , Adenilato Quinasa/metabolismo , Animales , Endotelio Vascular/metabolismo , Inflamación/metabolismo , Riñón/metabolismo , Ratones , Obesidad/metabolismo , Estrés Oxidativo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Ratas Zucker , Arteria Renal , Roedores/metabolismo , VasodilataciónRESUMEN
Oxidative stress-associated endothelial dysfunction is a key pathogenic factor underlying the microvascular complications of metabolic disease. NADPH oxidase (Nox) is a major source of oxidative stress in diabetic nephropathy and chronic kidney disease, despite Nox4 and Nox2 have been identified as relevant sources of vasodilator endothelial H2O2.The present study was sought to investigate the role of Nox enzymes in renal vascular oxidative stress and endothelial dysfunction in a rat model of genetic obesity. Endothelial function was assessed in intrarenal arteries of obese Zucker rats (OZR) and their counterparts lean Zucker rats (LZR) mounted in microvascular myographs, and superoxide (O2.-) and H2O2 production were measured. Impaired endothelium-dependent relaxations to acetylcholine (ACh) were associated to augmented O2.- generation, but neither ROS scavengers nor the Nox inhibitor apocynin significantly improved these relaxant responses in renal arteries of OZR. Whereas NO contribution to endothelial relaxations was blunted, catalase-sensitive non-NO non-prostanoid relaxations were enhanced in obese rats. Interestingly, NADPH-dependent O2.- production was augmented while NADPH-dependent H2O2 generation was reduced, and cytosolic and mitochondrial SOD were up-regulated in kidney of obese rats. Nox4 was down-regulated in renal arteries and Nox4-dependent H2O2 generation and endothelial relaxation were reduced in OZR. Up-regulation of both Nox2 and Nox1 was associated with augmented O2.- production but reduced H2O2 generation and blunted endothelial Nox2-derived H2O2-mediated in obese rats. Moreover, increased Nox1-derived O2.- contributed to renal endothelial dysfunction in OZR. In summary, the current data support a main role for Nox1-derived O2.- in kidney vascular oxidative stress and renal endothelial dysfunction in obesity, while reduced endothelial Nox4 expression associated to decreased H2O2 generation and H2O2-mediated vasodilatation might hinder Nox4 protective renal effects thus contributing to kidney injury. This suggests that effective therapies to counteract oxidative stress and prevent microvascular complications must identify the specific Nox subunits involved in metabolic disease.
Asunto(s)
Endotelio Vascular/metabolismo , NADPH Oxidasa 1/genética , NADPH Oxidasa 2/genética , NADPH Oxidasa 4/genética , Obesidad/etiología , Obesidad/metabolismo , Estrés Oxidativo , Animales , Antioxidantes/metabolismo , Susceptibilidad a Enfermedades , Peróxido de Hidrógeno/metabolismo , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Masculino , Metabolómica , Modelos Biológicos , NADPH Oxidasa 1/metabolismo , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 4/metabolismo , Obesidad/patología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Arteria Renal/metabolismo , Arteria Renal/fisiopatología , Superóxidos/metabolismoRESUMEN
AMP-activated protein kinase (AMPK) is a cellular energy sensor activated during energy stress to stimulate ATP production pathways and restore homeostasis. AMPK is widely expressed in the kidney and involved in mitochondrial protection and biogenesis upon acute renal ischemia, AMPK activity being blunted in metabolic disease-associated kidney disease. Since little is known about AMPK in the regulation of renal blood flow, the present study aimed to assess the role of AMPK in renal vascular function. Functional responses to the selective AMPK activator A769662 were assessed in intrarenal small arteries isolated from the kidney of renal tumour patients and Wistar rats and mounted in microvascular myographs to perform simultaneous measurements of intracellular calcium [Ca2+]i and tension. Superoxide (O2.-) and hydrogen peroxide (H2O2) production were measured by chemiluminescence and fluorescence and protein expression by Western blot. Activation of AMPK with A769662 increased AMPKα phosphorylation at Thr-172 and induced potent relaxations compared to AICAR in isolated human and rat intrarenal arteries, through both endothelium-dependent mechanisms involving nitric oxide (NO) and intermediate-conductance calcium-activated potassium (IKCa) channels, as well as activation of ATP-sensitive (KATP) channels and sarcoplasmic reticulum Ca2+-ATPase (SERCA) in vascular smooth muscle (VSM). Furthermore, AMPK activator reduced NADPH oxidase 4 (Nox4) and Nox2-derived reactive oxygen species (ROS) production. These results demonstrate that A769662 has potent vasodilator and antioxidant effects in intrarenal arteries. The benefits of AMPK activation in rat kidney are reproduced in human arteries and therefore vascular AMPK activation might be a therapeutic target in the treatment of metabolic disease-associated kidney injury.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Vasodilatación , Proteínas Quinasas Activadas por AMP/genética , Adenosina Monofosfato , Adenilato Quinasa , Animales , Humanos , Peróxido de Hidrógeno , Riñón , Ratas , Ratas Wistar , Especies Reactivas de OxígenoRESUMEN
Erectile dysfunction frequently coexists with coronary artery disease and has been proposed as a potential marker for silent coronary artery disease in type 2 diabetes. In the present study, we comparatively assessed the structural and functional changes of both penile arteries (PAs) and coronary arteries (CAs) from a prediabetic animal model. PAs and CAs from 17- to 18-wk-old obese Zucker rats (OZRs) and from their control counterparts [lean Zucker rats (LZRs)] were mounted in microvascular myographs to evaluate vascular function, and stained arteries were subjected to morphometric analysis. Endothelial nitric oxide (NO) synthase (eNOS) protein expression was also assessed. The internal diameter was reduced and the wall-to-lumen ratio was increased in PAs from OZRs, but structure was preserved in CAs. ACh-elicited relaxations were severely impaired in PAs but not in CAs from OZRs, although eNOS expression was unaltered. Contractions to norepinephrine and 5-HT were significantly enhanced in both PAs and CAs, respectively, from OZRs. Blockade of NOS abolished endothelium-dependent relaxations in PAs and CAs and potentiated norepinephrine and 5-HT contractions in arteries from LZRs but not from OZRs. The vasodilator response to the phosphodiesterase 5 inhibitor sildenafil was reduced in both PAs and CAs from OZRs. Pretreatment with SOD reduced the enhanced vasoconstriction in both PAs and CAs from OZRs but did not restore ACh-induced relaxations in PAs. In conclusion, the present results demonstrate vascular inward remodeling in PAs and a differential impairment of endothelial relaxant responses in PAs and CAs from insulin-resistant OZRs. Enhanced superoxide production and reduced basal NO activity seem to underlie the augmented vasoconstriction in both PAs and CAs. The severity of the structural and functional abnormalities in PAs might anticipate the vascular dysfunction of the more preserved coronary vascular bed.