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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.

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 β-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.

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.Bladder neck strips were mounted in myographs for isometric force recording and simultaneous measurement of intracellular Ca(2+) and tension.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+).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.

Metabolic syndrome (MS) is a known risk factor for lower urinary tract symptoms. This study investigates whether functional and expression changes of cannabinoid CB1 and CB2 receptors are involved in the bladder dysfunction in an obese rat model with insulin resistance. Bladder samples from obese Zucker rat (OZR) and their respective controls lean Zucker rat (LZR) were processed for immunohistochemistry and western blot for studying the cannabinoid receptors expression. Detrusor smooth muscle (DSM) strips from LZR and OZR were also mounted in myographs for isometric force recordings. Neuronal and smooth muscle CB1 and CB2 receptor expression and the nerve fiber density was diminished in the OZR bladder. Electrical field stimulation (EFS) and acetylcholine (ACh) induced frequency- and concentration-dependent contractions of LZR and OZR DSM. ACh contractile responses were similar in LZR and OZR. EFS-elicited contractions, however, were reduced in OZR bladder. Cannabinoid receptor agonists and antagonists failed to modify the DSM basal tension in LZR and OZR In LZR bladder, EFS responses were inhibited by ACEA and SER-601, CB1 and CB2 receptor agonists, respectively, these effects being reversed by ACEA plus the CB1 antagonist, AM-251 or SER-601 plus the CB2 antagonist, AM-630. In OZR bladder, the inhibitory action of ACEA on nerve-evoked contractions was diminished, whereas that SER-601 did not change EFS responses. These results suggest that a diminished function and expression of neuronal cannabinoid CB1 and CB2 receptors, as well as a lower nerve fiber density is involved in the impaired excitatory neurotransmission of the urinary bladder from the OZR.

The role of endothelial and neural factors as modulators of neurogenic- and noradrenaline-induced vasoconstriction was examined in healthy pig internal mammary artery (IMA). Tetrodotoxin-, guanethidine-sensitive electrical field stimulation (EFS)-, and noradrenaline-elicited contractions were significantly diminished by prazosin (n=8, P0.001) and less so by rauwolscine, indicating functional α₁- and α₂-adrenoceptor-mediated noradrenergic innervation of the IMA. Endothelium removal reduced neurogenic (n=8, P0.01) but augmented noradrenaline responses (n=8, P0.01), suggesting the release of two endothelium-dependent factors with opposite effects. In the presence of endothelium, neurogenic and exogenous noradrenaline vasoconstrictions were enhanced by L-NOArg (n=7, P0.05 and P0.01 respectively) and ODQ (n=7, both P0.05); in denuded arteries, nNOS inhibition with N(ω)-propyl-L-arginine increased neurogenic contraction (n=7, P0.05). Western blotting indicated the presence of neural and endothelial origin NO (n=6, P0.001). Tetraethylammonium (n=9, P0.001), iberiotoxin (n=7, P0.001) and 4-aminopyridine (n=8, P0.01) enhanced vasoconstrictions revealing a modulatory role of big conductance Ca²⁺-activated K⁺ (BK(Ca)) and voltage-dependent K⁺ (K(v)) channels in noradrenergic responses. Bosentan pretreatment (n=8, P0.05) suggested endothelin-1 as the inferred contractile neurogenic endothelial-dependent factor. Indomethacin-induced inhibition involved a muscular prostanoid (n=9, P0.05), functionally and immunologically localized, and derived from cyclooxygenase (COX)-1 and COX-2, as revealed by Western blots (n=5, P=0.1267). Thus, noradrenergic IMA contractions are controlled by contractile prostanoid activation and endothelin-1 release, and offset by BK(Ca) and K(v) channels and neural and endothelial NO. These results help clarify the mechanisms of vasospasm in IMA, as the preferred vessel for coronary bypass.

Benign prostatic hypertrophy has been known to be related with glandular ischemia processes, and nitric oxide (NO) is a potent vasodilator agent. Therefore, the current study investigates the mechanisms underlying the NO-induced vasorelaxation in pig prostatic small arteries. In microvascular myographs, relaxation to electrical field stimulation (EFS), or to exogenous (S)-nitroso-N-acetylpenicillamine (SNAP) and acetylcholine (ACh), was observed on noradrenaline-precontracted prostatic small arterial rings under non-adrenergic and non-cholinergic (NANC) conditions. EFS (1-16 Hz) and exogenous SNAP (0.1-30 μM) evoked frequency- and concentration-dependent relaxation, respectively. Tetrodotoxin, a neuronal voltage-gated Na(+) channel blocker, abolished the EFS-evoked relaxation. ACh (1 nM-10 μM) induced concentration-dependent relaxation, which was reduced by the NO synthase inhibitor N(G)-nitro-L: -arginine (L: -NOARG). L: -NOARG also reduced the EFS-elicited relaxation but failed to modify the response to SNAP. 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and iberiotoxin (IbTX), blockers of soluble guanylyl cyclase and large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, respectively, reduced EFS-, SNAP-, and ACh-induced relaxation. The combination of ODQ with IbTX did not produce further inhibition of the responses to either SNAP or ACh, compared with ODQ alone. Blockade of cyclooxygenases and intermediate and small conductance Ca(2+)-activated, ATP-dependent, and voltage-gated K(+) channels did not change the EFS and SNAP responses. In conclusion, our results suggest that NO and non-NO non-prostanoid factor(s) derived from NANC nerves are involved in the vasodilatation of pig prostatic small arteries. NO produces relaxation through soluble guanylyl cyclase activation-dependent BK(Ca) channel opening and through guanylyl cyclase-independent mechanisms. The vasodilatation elicited by NO could be useful to prevent prostatic ischemia.

This study was designed to determine whether K+ channels play a role in nitric oxide (NO)-dependent acetylcholine relaxation in porcine internal mammary artery (IMA). IMA segments were isolated and mounted in organ baths to record isometric tension. Acetylcholine-elicited vasodilation was abolished by muscarinic receptor blockade with atropine (10(-6)M). Incubation with indomethacin (3 x 10(-6)M), superoxide dismutase (150 U/ml) and bosentan (10(-5)M) did not modify the acetylcholine response ruling out the participation of cyclooxygenase-derivates, reactive oxygen species or endothelin. The relaxation response to acetylcholine was strongly diminished by NO synthase- or soluble guanylyl cyclase-inhibition using L-NOArg (10(-4)M) or ODQ (3 x 10(-6)M), respectively. The vasodilation induced by acetylcholine and a NO donor (NaNO(2)) was reduced when rings were contracted with an enriched K+ solution (30 mM), by voltage-dependent K+ (K(v)) channel blockade with 4-amynopiridine (4-AP; 10(-4)M), by Ca(2+)-activated K+ (K(Ca)) channel blockade with tetraethylammonium (TEA; 10(-3)M), and by apamin (5 x 10(-7)M) plus charybdotoxin (ChTx; 10(-7)M) but not when these were added alone. In contrast, large conductance K(Ca) (BK(Ca)), ATP-sensitive K+ (K(ATP)) and inwardly rectifying K+ (K(ir)) channel blockade with iberiotoxin (IbTx; 10(-7)M), glibenclamide (10(-6)M) and BaCl(2) (3 x 10(-5)M), respectively, did not alter the concentration-response curves to acetylcholine and NaNO(2). Na+-K+ ATPase pump inhibition with ouabain (10(-5)M) practically abolished acetylcholine and NaNO(2) relaxations. Our findings suggest that acetylcholine-induced relaxation is largely mediated through the NO-cGMP pathway, involving apamin plus ChTx-sensitive K+ and K(v) channels, and Na+-K+-ATPase pump activation.

The localisation and function of noradrenergic perivascular innervation of the radial artery were examined in a porcine model. Through immunohistochemical techniques, we explored the possible existence of dopamine beta-hydroxylase and choline-acetyltransferase in the nerve fibres supplying the radial artery. Arterial rings suspended in organ baths were used to isometrically record tension in functional tests designed to determine the vasoconstriction response to electrical field stimulation (EFS) or exogenous noradrenaline. Morphological studies revealed the presence of noradrenergic, but not cholinergic, nerve fibres in the tunica adventitia and adventitia-media boundary of the artery wall. EFS-elicited frequency-dependent contractions (EF50 = 3.37 ± 0.19 Hz and Emax = 87.7 ± 3.8%; n = 47) were abolished by tetrodotoxin. The contractile effect was markedly reduced by guanethidine, phentolamine and prazosin and slightly inhibited by rauwolscine, but unaltered by propranolol, atropine, bosentan or capsaicine. Endothelium removal increased EFS-evoked contractions but the addition of L-NOArg, ODQ or indomethacin had no effect. Pre-incubation with tetraethylammonium and 4-aminopyridine, but not glibenclamide, enhanced these neurogenic responses. SOD and apocynin reduced EFS-elicited responses at low frequencies. Exposure of the arterial rings to the same agents did not affect the noradrenaline concentration–response curves except for the α-adrenoceptor antagonists. These results led to the conclusions that neurogenic contractions in the pig radial artery are predominantly mediated by noradrenaline released from periarterial adrenergic nerves. This neurogenic vasoconstriction is modulated by a non-NO, non-prostanoid endothelium-dependent relaxing factor and by Ca2+-activated and voltage-dependent K+ channels.

The present study was designed to characterize the neurogenic contraction of rat radial artery. Electrical field stimulation (EFS) evoked frequency-dependent contraction that was abolished by tetrodotoxin (neuronal Na+ channel blocker), guanethidine (sympathetic neuron blocker), or phentolamine (α-adrenoceptor blocker). The α1-adrenoceptor antagonist prazosin inhibited endothelium-independent contractions to EFS, noradrenaline (NA), and the α1-adrenoceptor agonist phenylephrine. Rauwolscine, an α2-adrenoceptor antagonist, augmented nerve-mediated contractions and reduced sensitivity to NA and the α2-adrenoceptor agonist BHT-920. The β-adrenoceptor antagonist propranolol diminished EFS-elicited contractions, while sensitivity to NA was enhanced by propranolol. Relaxations evoked by isoproterenol, a β-adrenoceptor agonist, were abolished by propranolol. NG-Nitro-L-arginine (L-NOARG), a nitric oxide (NO) synthase inhibitor, increased both nerve-mediated and NA-induced responses in endothelium-intact, but not in endothelium-denuded arteries. Moreover, endothelium-dependent responses to BHT-920 and isoproterenol were modified by L-NOARG. Tetraethylammonium (TEA) or 4-amynopyridine, the Ca2+-activated (KCa) or voltage-dependent K+ (KV) channel blockers, respectively, enhanced the neurogenic contractions observed. TEA but not 4-amynopyridine increased NA-induced contractions. The ATP-sensitive K+ (KATP)–channel blocker glibenclamide failed to modify adrenergic contractions. Blockade of capsaicin-sensitive primary afferents increased EFS-induced contractions. In conclusion, adrenergic contractions are predominantly mediated by muscular α1-adrenoceptors, while endothelial α2- and β-adrenoceptors play a minor role. Presynaptic α2- and β-adrenoceptors cannot be precluded. Noradrenergic neurotransmission in rat radial artery seems to be modulated by both stimulation of endothelial NO, KCa, and KV channels and sensory C-fiber activation. Keywords:: rat radial artery, electrical field stimulation, adrenergic receptor, nitric oxide, K+ channel

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, 5α-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), 5α-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 °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 5α-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.

Objectives To investigate the effect of acetylcholine (ACh) on horse deep dorsal penile vein and to characterize the muscarinic receptor subtypes involved in this response. Methods Vein rings were mounted in an organ bath chamber, and the isometric tension was recorded. Results In phenylephrine-contracted veins, ACh (1 nM to 1 μM) induced endothelium-dependent relaxation. The muscarinic receptor antagonist, atropine, produced parallel rightward shifts of the ACh response curves (pA 2 = 10.04; pK B = 9.98). Carbachol (10 nM to 100 μM) also evoked relaxation in the vein segments, but showed a lower potency and similar relaxation to that induced by ACh. Pirenzepine, the high, intermediate, and low-affinity antagonist for M 1 , M 3 , and M 2 receptors, respectively, inhibited ACh and carbachol-induced relaxation, yielding pA 2 values of 7.51 and 7.37, and pK B values of 7.38 and 7.28, respectively. Methoctramine, a high-affinity M 2 antagonist, showed no significant effect on the response to ACh. However, a high-affinity M 3 antagonist, pFHHSiD, potently blocked the relaxation induced by carbachol and ACh, yielding pA 2 and pK B values of 7.72 and 7.70 for pFHHSiD against ACh, respectively, and of 7.77 and 7.65 against carbachol, respectively. Conclusions These results indicate that ACh induces an endothelium-dependent relaxation in horse deep dorsal penile vein. The antagonist profile suggests that M 3 muscarinic receptors mediate ACh-induced relaxation in this tissue.

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.Western blot and immunohistochemistry were used to show the expression of BK B1 and B2 receptors and myographs for isometric force recordings.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.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.

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. Sanchez A, Contreras C, Martinez P, Munoz M, Martinez AC, Garcia-Sacristan A, Hernandez M, and Prieto D. Endothelin (ET A ) receptors are involved in augmented adrenergic vasoconstriction and blunted nitric oxide-mediated relaxation of penile arteries from insulin-resistant obese Zucker rats. J Sex Med 2014;11:1463–1474.

Objective: Peripheral arterial disease is one of the macrovascular complications of type 2 diabetes mellitus. This study addresses femoral artery regulation in a prediabetic model of obese Zucker rats (OZR) by examining cross-talk between endothelial and neural factors. Methods and Results: Arterial preparations from lean (LZR) and OZR were subjected to electrical field stimulation (EFS) on basal tone. Nitric oxide synthase (NOS) and cyclooxygenase (COX) isoform expression patterns were determined by immunohistochemical labelling and Western blotting. Results indicate significantly reduced noradrenergic contractions in preparations from OZR compared with those of LZR. Functional inhibition of endothelial NOS (eNOS) indicated a predominant role of this isoform in LZR and its modified activity in OZR. Neural (nNOS) and inducible NOS (iNOS) were activated and their expression was higher in femoral arteries from OZR. Neurotransmission modulated by largeconductance Ca 2+ -activated (BK Ca ) or voltage-dependent (K V ) K + channels did not seem compromised in the obese animals. Endothelial COX-1 and COX-2 were expressed in LZR and an additional adventitial location of COX-2 was also observed in OZR, explaining the higher COX-2 protein levels detected in this group. Prostanoids derived from both isoforms helped maintain vasoconstriction in LZR while in OZR only COX-2 was active. Superoxide anion inhibition reduced contractions in endothelium-intact arteries from OZR. Conclusions: Endothelial dysfunction led to reduced neurogenic vasoconstriction in femoral arteries from OZR. In a setting of obesity, NO-dependent nNOS and iNOS dilation activity could be an alternative mechanism to offset COX-2- And reactive oxygen species-mediated vasoconstriction, along with impaired endothelial NO relaxation.

The effect of histamine (10(-9)-10(-3) M) on horse penile dorsal artery was evaluated. Precontracted vessels showed a biphasic response (relaxation-contraction) to histamine, while at basal tone, histamine only induced a contractile effect. The H1 receptor agonist, 2-pyridylethylamine (PEA) (10(-9)-10(-3) M), induced concentration-dependent relaxation in precontracted rings and provoked vasoconstriction at basal tone. Mepyramine (10(-9)-10(8) M), an H1 receptor antagonist, competitively antagonized the relaxant response to histamine (pA2 = 9.7) and PEA (pA2 = 9.2). At basal tone, mepyramine (10(-10)-10(-8) M) also caused a rightward shift in the histamine contraction curve (pA2 = 10.1). Mepyramine (10(-9)-10(-8) M)/PEA Schild plots for resting vessels yielded a pA2 value of 9.4. A regulatory role for H2 and H3 receptors was precluded since there was no response to their agonists (dimaprit (10(-9)-10(-3) M), (R)-alpha-methylhistamine (10(-10)- 3 x 10(-4) M)), and antagonists (cimetidine (10(-5) M), thioperamide (10(-6) M)) did not affect control curves. Removal of the endothelium abolished the relaxant component causing a leftward shift in the contractile component in precontracted rings, with no effect on maximum contraction. Inhibitors of nitric oxide (NO) synthesis, L-NAME (3 x 10(-4) M) and L-NOARG (3 x 10(-4) M), modified the relaxant response while contraction was unaffected. L-Arginine (3 x 10(-4) M) potentiated maximum relaxation but did not affect contraction in precontracted rings. Effects of a prostanoid and K+ channels were ruled out. The biphasic response of precontracted vessels persisted in the presence of indomethacin (3 x 10(-6) M), tetraethylammonium (10(-3) M) and gliblenclamide (10(-5) M). L-NAME plus indomethacin, or this combination plus TEA or glibenclamide produced similar effects as isolated treatments. In resting vessels, histamine contraction was also unaffected by the lack of endothelium, or L-NAME, L-arginine or indomethacin pretreatment. The biphasic response to histamine is probably mediated by H1 receptors with a partial role for NO in the relaxant response in precontracted vessels. In the absence of tone, the contractile effect may be mediated by direct action on smooth muscle.

The present in vitro study was designed to evaluate the effect of histamine on isolated rings of bovine oviductal artery and to characterize the histamine receptors involved in the histamine-induced response. Endothelial dependence of the response was also investigated. Cumulative addition of histamine and 2-pyridylethylamine (histamine H receptor agonist) induced a concentration-dependent relaxation in intact arterial segments precontracted with noradrenaline. The histamine H1 receptor antagonist mepyramine showed non-competitive antagonism in the histamine-induced concentration-response curve. However, when the response to histamine was evaluated in the presence of mepyramine and histamine H1 and H3 receptors were blocked, Schild analysis yielded a line with a slope of 1.10 and a pA2 value of 8.91, indicating simple competitive antagonism of mepyramine at histamine H1 receptor sites. The histamine H2 receptor agonist, dimaprit, caused marked dilatation only at high doses. Cimetidine, propranolol and mepyramine failed to inhibit this relaxant effect. In precontracted oviductal arteries, cimetidine did not modify the histamine-induced concentration-response curves. Combined treatment with histamine H1 and H2 receptor antagonists did not induce an additional displacement with respect to the isolated effect of mepyramine thus excluding activation of histamine H2 receptors. Histamine and (R)-alpha-methylhistamine, a selective histamine H3 receptor agonist, produced a moderate contractile effect on the resting tone of preparations. Pretreatment with the selective histamine H1 receptor antagonist decreased the (R)-alpha-methylhistamine response but increased the maximal relaxant effect and abolished the contractile effect of histamine, suggesting the presence of a limited population of contractile histamine H3 receptors. Removal of the endothelium or pretreatment with methylene blue produced a significant inhibition of the relaxant response to histamine. Remaining dilatation was practically abolished by mepyramine and also by indomethacin. The L-arginine analogue, N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of histamine and basal production of nitric oxide. L-Arginine, which on its own induced significant endothelium-dependent vasodilatation, reversed the effect of L-NAME on histamine relaxation. Indomethacin only caused a slight modification of the sensitivity of the vessels to histamine, suggesting that prostacyclin or other cyclo-oxygenase products did not make a significant contribution to the model. The absence of the endothelium did not modify the contractile effect of histamine. The results suggest that the relaxant response of isolated oviductal arteries to histamine is dependent on the functional integrity of the endothelium and is mainly mediated by histamine H1 receptors. These receptors may mask a minority presence of histamine H3 contractile receptors located on smooth muscle. The main relaxing factor released from the endothelium by mediation of histamine is nitric oxide, which may also exert an effect on vascular tone.

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.

The aim of the present study was to investigate the mechanisms underlying the vasorelaxation induced by the selective phosphodiesterase 5 (PDE5) inhibitor vardenafil in rat penile small arteries. Segments of the rat dorsal penile artery were mounted in microvascular myographs for isometric tension recording. Concentration-response curves for vardenafil (1 nM-3 microM) and other PDE inhibitors (sildenafil, rolipram and milrinone) were constructed by adding cummulative concentrations of the drugs to arteries precontracted with phenylephrine. The effect of mechanical endothelial cell removal and of selective blockers of the nitric oxide (NO)/cGMP pathway and K+ channels were evaluated on the vardenafil relaxant responses. Vardenafil was the most potent of the four PDE inhibitors tested that maximally relaxed penile arteries, pD2 and maximum relaxation being 6.96+/-0.08 and 97+/-1% (n=48), respectively. Blockade of guanylate cyclase with ODQ (5 microM), mechanical removal of the endothelium or inhibition of NO synthase with l-NOARG (100 microM) markedly reduced vardenafil-induced relaxations, without altering maximum response. Inhibitors of both the cGMP-dependent (PKG) and the cAMP-dependent (PKA) protein kinases, Rp-8-Br-PET-cGMPS (5 microM) and Rp-8-CPT-cAMPS (50 microM), respectively, both reduced vardenafil relaxant responses and the later abolished that of rolipram. Vardenafil-elicited relaxation was reduced by the selective inhibitor of the large-conductance Ca2+-activated K+ channels (BK(Ca)), iberiotoxin (30 nM) and also by the ATP-sensitive K+ channel (K(ATP)) inhibitor, glibenclamide (1 microM). Vardenafil induces a potent vasodilatation in rat penile arteries that is partially dependent on the endothelium and the NO/cGMP pathway and involves activation of both BK(Ca) and K(ATP) channels.

To investigate the nitric oxide (NO)-mediated nerve relaxation and its possible modulation by pre-junctional alpha2-adrenoceptors in the pig urinary 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% 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.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.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.

The present study was designed to establish whether penile dorsal arteries isolated from rabbits fed a high cholesterol diet show an enhanced contractile and/or impaired vasodilator response to histamine, and to characterize the histamine receptor subtype involved through in vitro isometric techniques. New Zealand White rabbits were fed a normal diet or a 1% cholesterol diet for 16 weeks. Arteries from cholesterol-fed rabbits retained the ability to relax in response to acetylcholine, whereas histamine and noradrenaline induced a greater contraction response compared to that observed in controls. In both groups, histamine-induced contraction was unaffected by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), its precursor L-arginine or the cyclooxygenase inhibitor indomethacin. Treatment of arterial rings in the control and hypercholesterolemia groups with the H1 receptor antagonist, mepyramine, unmasked a vasodilation response to histamine. This was followed by contraction at higher concentrations showing a leftward displacement of the histamine curve compared to controls. The histamine receptor that induced contraction in preparations from the hypercholesterolemic animals was of the H1 subtype, whereas the receptor involved in histamine-induced relaxation was H2. The affinity of histamine receptor agonists was comparable to their effects in control animals, and receptor antagonists showed the same potency in both groups. Our findings indicate a preserved endothelial function and enhanced contraction in response to histamine in penile dorsal arteries, probably due to a change in the sensitivity of the contractile machinery of smooth muscle but not a mechanism mediated by a receptor.

This study evaluates the mechanisms underlying endothelium-dependent responses to acetylcholine in horse deep dorsal penile veins. Acetylcholine-induced relaxation was abolished by endothelium removal, the soluble guanylyl cyclase-inhibitor, and the nitric oxide (NO) synthase inhibitors. Acetylcholine-induced relaxation was inhibited by high K+ concentrations and blockade of large-conductance Ca(2+)-activated potassium (BK(Ca)) channels, and voltage-dependent potassium (K(v)) channels. Relaxations were unaffected by a small-conductance K(Ca) (SK(Ca)) channel blocker, or an ATP-sensitive potassium (K(ATP)) channel blocker. Relaxation in response to a NO donor was unaffected by K(Ca) channel blockers, but inhibited by high K+ concentrations and a K(v) channel blocker. In the presence of a NO synthase inhibitor, acetylcholine-induced contractions were inhibited by a cyclooxygenase blocker and abolished by endothelial removal. The contractile response was competitively inhibited by muscarinic receptor antagonists, high affinity M1 and M3 antagonists, while the M2 antagonist had no effect. The pharmacological profile suggests that acetylcholine contraction is mediated by muscarinic M1 receptors. Our findings indicate that acetylcholine-induced relaxation in the horse deep dorsal penile vein is essentially mediated by NO, acting via the cGMP-dependent pathway and opening of K+ channels. The contraction elicited by acetylcholine is prostanoid-mediated and induced by endothelial muscarinic M1 receptor activation.

We developed an in vitro method that allows us to study the physiopharmacological responses of penile resistance arteries under isobaric conditions.Second to third order penile resistance arteries (internal diameter 170 to 210 microm) were mounted in a pressure myograph and cannulated at each end with small glass cannulas (tip external diameter 150 to 180 microm). Internal diameter was continuously recorded and monitored under an intraluminal pressure of 60 mm Hg.Noradrenaline (0.1 to 0.3 microM) induced a decrease in the luminal diameter of the penile arteries, ie vasoconstriction. This effect was reversed by 1 microM acetylcholine, 1 microM prostaglandin E1 (PGE1) and 1 nM to 1 microM sildenafil citrate. Furthermore, the vasodilatation induced by sildenafil was compared by artery internal diameter values under isometric and isobaric conditions. Although the mean potency of this drug +/- SEM, expressed in pD2, was higher in 5 isometric (7.60 +/- 0.04) than in 4 isobaric (7.03 +/- 0.20) preparations (p0.05), the slope of the curve was lower in 4 isobaric (0.49 +/- 0.02) than in 5 isometric (1.34 +/- 0.11) studies (p0.01).Under isobaric conditions all vasoactive agents tested inhibited the noradrenaline induced vasoconstriction. Furthermore, the vasodilatory effect of PGE1 beyond baseline diameter could suggest an inhibitory effect of PGE1 on spontaneous myogenic tone. On the other hand, the effect of sildenafil was more potent under isometric than under isobaric conditions. However, the lower slope of the curve under isobaric conditions suggests that the pressure myograph could be a more suitable in vitro model for the study of the activity of penile resistance arteries, and so isobaric conditions correspond more closely to the in vivo situation.

Our aim was to study the presence of noradrenergic nerves and to characterize the alpha-adrenergic receptors involved in the contractions to electrical field stimulation and to alpha-adrenergic agonists of the horse penile deep dorsal vein. Noradrenergic fibres were visualized by immunohistochemistry using an antibody against dopamine-beta-hydroxylase (DBH). For functional studies, the responses of the venous rings to electrical field stimulation and to alpha-adrenergic agonists (noradrenaline, phenylephrine and BHT 920) were studied in the absence and the presence of noradrenergic transmission- and neuronal sodium channel-blockers (guanethidine and tetrodotoxin, respectively) and of alpha1- and alpha2-adrenergic antagonists (prazosin and rauwolscine, respectively). DBH-immunoreactive fibres were present in the adventitia and in the media layer of the venous rings. Electrical field stimulation (0.5-32 Hz) caused frequency-dependent contractions that were abolished by guanethidine (10(-6) M) and tetrodotoxin (10(-6) M) and reduced by prazosin (10(-9)-10(-7) M) and rauwolscine (3 x 10(-8)-3 x 10(-7) M). Noradrenaline, phenylephrine and BHT 920 induced equipotent contractions of the rings. Prazosin and rauwolscine competitively antagonized the contractions to phenylephrine and BHT 920, respectively. In conclusion, DBH-immunoreactive nerve fibres are present in the horse penile dorsal vein. Both transmural nerve stimulation and alpha-adrenergic agonists induce contraction of the venous rings through a heterogeneous population of alpha1- and alpha2-adrenoceptors.

The present investigation was designed to evaluate the effect of histamine on isolated rings of horse deep dorsal penile vein. Under precontracted or basal conditions, histamine evoked an endothelium-independent contraction. Preincubation of the vein rings with the selective H1 receptor antagonist, mepyramine, shifted the concentration-response curve for histamine and to the H1 receptor agonist 2-pyridylethylamine to the right in a competitive manner. Pretreatment with cimetidine, a specific H2 receptor antagonist, did not modify the pEC50 and maximal contraction of the histamine response. Cimetidine and propranolol failed to induce a change in the relaxation caused by dimaprit, the H2 receptor agonist. Histamine contraction was unaffected by thioperamide, the specific H3 receptor antagonist. (R)-alpha-methylhistamine, the H3 receptor agonist, also induced contractions which persisted in the presence of either thioperamide or tetrodotoxin. These data indicate that horse deep dorsal penile vein shows an endothelium-independent contraction response to histamine, mainly mediated by H1 receptors.

The effects of specific histamine agonists and antagonists on isolated rabbit penile dorsal artery segments were explored using in vitro isometric techniques. Histamine caused the constriction of both precontracted and resting segments. In precontracted arterial rings treated with the H, receptor antagonist mepyramine, histamine evoked a vasodilatation, followed by contraction at higher concentrations, The vasoconstrictor effect of histamine and the H 1 receptor agonist, 2-pyridylethylamine (PEA) on preparations under conditions of basal tone, was competitively antagonized by mepyramine (10 -9 -10 -8 M), The relaxant effect of histamine, unmasked by mepyramine, was abolished by cimetidine. Dimaprit, the H 2 receptor agonist, provoked a relaxation of precontracted segments that was also competitively inhibited by cimetidine (10 -6 -10 -5 M). Selective H 3 receptor activation with the agonist (R)α-methylhistamine (10 -10 -10 -4 M) produced no effect in penile dorsal artery. The biphasic response to histamine was unaffected by endothelium removal or the nitric oxide inhibitor N G -nitro-L-arginine methyl ester (L-NAME) (3 x 10 -4 M) and its precursor, L-arginine (3 x 10 -4 M). Similarly, the cyclooxygenase inhibitor, indomethacin (3 x 10 -6 M) and a combination of Ca 2+ -activated K + channel blockers apamin (5 x 10 -7 M) and charybdotoxin (10 -7 M) showed no effect on the histamine-induced relaxation or contraction. In conclusion, contraction, the predominant effect of histamine, is mediated by the activation of H, receptors that mask the relaxant effect brought about by H 2 receptors. Both these effects appear to be mediated by direct action on the smooth muscle, with no participation of nitric oxide or cyclooxygenase products or Ca 2+ -activated K + channels.