Mirabegron elicits rat corpus cavernosum relaxation and increases in vivo erectile response.

Mirabegron is the first ß3-adrenoceptor agonist approved on the market and may offer beneficial pharmacological action in patients with overactive bladder and erectile dysfunction. Here, we further investigate the mechanisms by which mirabegron induces rat corpus cavernosum (CC) relaxation. Adult male Wistar rats were used. The CC were isolated for in vitro functional assays and ß-adrenoceptors subtypes mRNA expression evaluation. Animals were treated orally with mirabegron (30 mg/kg, 3 h), tadalafil (10 mg/kg, 3 h) or both for intracavernous pressure (ICP). Intracellular levels of cAMP and cGMP were also determined. The ß1-, ß2- and ß3-adrenoceptors subtypes were expressed in rat CC. Mirabegron produced concentration-dependent CC relaxations that were unaffected by the ß1-, ß2- or ß3-adrenoceptor antagonists atenolol (1 µM), ICI-118,551 (1 µM) and L748,337 (10 µM), respectively. Mirabegron-induced relaxations were not affected by the phosphodiesterase type 4 inhibitor, rolipram, or the adenylyl cyclase selective inhibitor, SQ 22,536. Potassium channel- or calcium influx-blockade are not involved in mirabegron-induced relaxations. In contrast, mirabegron produced rightward shifts in the contractile response induced by the α1-adrenoceptor agonist, phenylephrine. Finally, cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly increased in rats treated with mirabegron in a similar degree of tadalafil-treated rat, without promoting a significant cAMP or cGMP accumulation. Together, our results demonstrate that mirabegron induced CC relaxation through α1-adrenoceptor blockade. Care should be taken to translate the effect of mirabegron into the clinic, especially when using rat as an animal model of erectile dysfunction.

Pharmacological and transcriptomic characterization of the nitric oxide pathway in aortic rings isolated from the tortoise Chelonoidis carbonaria.

In this study the nitric oxide (NO)-soluble guanylate cyclase (sGC) and phosphodiesterase-5 (PDE-5) pathway was characterized in tortoise Chelonoidis carbonaria aorta. Concentration response curves (CCR) to ATP, ADP, AMP, adenosine and histamine were performed in the presence and absence of L-NAME in aorta pre-contracted with ACh (3 µM). CCR to SNP, BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator) and tadalafil (PDE-5 inhibitor) were constructed in the presence and absence of ODQ (10 µM). ATP (pEC50 6.1 ±â€¯0.1), ADP (pEC50 6.0 ±â€¯0.2), AMP (pEC50 6.8 ±â€¯0.1) and histamine (pEC50 6.8 ±â€¯0.12) relaxed Chelonoidis aorta and the addition of L-NAME reduced their efficacy (p < .05). Adenosine effects (pEC50 6.6 ±â€¯0.1) were not changed in the presence of L-NAME. SNP (pEC50 7.5 ±â€¯0.7; Emax 102.2 ±â€¯2.5%), BAY 41-2272 (pEC50 7.3 ±â€¯0.2; Emax 130.3 ±â€¯10.2%), BAY 60-2770 (pEC50 11.4 ±â€¯0.1; Emax 130.3 ±â€¯6.5%) and tadalafil (pEC50 6.7 ±â€¯0.3; Emax 121.3 ±â€¯15.3%) relaxed Chelonoidis aorta. The addition of ODQ reduced the SNP and tadalafil maximum response (p < .05) and promoted 63 fold right shift on BAY 41-2272 curve. In contrast, no alteration was observed on BAY 60-2770 response. Transcriptomic analysis for eNOS and sGC were found in aorta and brain libraries with high homology when compared with human transcripts. The NO-sGC-PDE-5 is functionally present in Chelonoidis aorta with a functional and genomic similarity to mammalian vessels. Unlike most of mammalian vessels, ACh did not cause endothelium-dependent relaxation in Chelonoidis carbonaria aortic rings.

The rabbit vagina as an in vivo model for vaginal fenticonazole permeability and toxicity.

INTRODUCTION: Vaginal route is often used in topical antifungal formulations. Vaginal permeability assays are generally performed as in vitro tests. METHOD: An in vivo vaginal permeability assay was developed using female rabbits. Fenticonazole permeability was evaluated by assessing fenticonazole bioavailability in plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS). Toxicity was monitored histopathologically after 8 consecutive days of antifungal treatment (20 mg/animal). RESULTS: The method of quantification was linear with a lower limit of quantification (LLOQ) of (0.1 ng/mL). The area-under-the-curves (AUC) of fenticonazole on day 1 and 8 of treatment were 280.3 ±â€¯86.1 ng/mL ∗ h and 805.7 ±â€¯252.4 ng/mL ∗ h, respectively. The calculated systemic bioavailability was 12.73% ±â€¯0.14%. No signs of toxicity were observed both macroscopically and histologically after 8 days fenticonazole treatment. DISCUSSION: The plasma levels of fenticonazole observed in rabbits are similar to that observed in human. Rabbit vagina may be a suitable model to evaluate vaginal antifungal formulations.

Tetrodotoxin-insensitive electrical field stimulation-induced contractions on Crotalus durissus terrificus corpus cavernosum.

Reptiles are the first amniotes to develop an intromitent penis, however until now the mechanisms involved in the electrical field stimulation-induced contraction on corpora cavernosa isolated from Crotalus durissus terrificus were not investigated. Crotalus and rabbit corpora cavernosa were mounted in 10 mL organ baths for isometric tension recording. Electrical field stimulation (EFS)-induced contractions were performed in presence/absence of phentolamine (10 µM), guanethidine (30 µM), tetrodotoxin (1 µM and 1mM), A-803467 (10 µM), 3-iodo-L-Tyrosine (1 mM), salsolinol (3 µM) and a modified Krebs solution (equimolar substitution of NaCl by N-methyl-D-glucamine). Immuno-histochemistry for tyrosine hydroxylase was also performed. Electrical field stimulation (EFS; 8 Hz and 16 Hz) caused contractions in both Crotalus and rabbit corpora cavernosa. The contractions were abolished by previous incubation with either phentolamine or guanethidine. Tetrodotoxin (1 µM) also abolished the EFS-induced contractions of rabbit CC, but did not affect EFS-induced contractions of Crotalus CC. Addition of A-803467 (10 µM) did not change the EFS-induced contractions of Crotalus CC but abolished rabbit CC contractions. 3-iodo-L-Tyrosine and salsolinol had no effect on EFS-induced contractions of Crotalus CC and Rabbit CC. Replacement of NaCl by N- Methyl-D-glucamine (NMDG) abolished EFS-induced contractions of rabbit CC, but did not affect Crotalus CC. The presence of tyrosine hydroxylase was identified in endothelial cells only of Crotalus CC. Since the EFS-induced contractions of Crotalus CC is dependent on catecholamine release, insensitive to TTX, insensitive to A803467 and to NaCl replacement, it indicates that the source of cathecolamine is unlikely to be from adrenergic terminals. The finding that tyrosine hydroxylase is present in endothelial cells suggests that these cells can modulate Crotalus CC tone.

Effects of nitric oxide inhibitors in mice with bladder outlet obstruction

ABSTRACT Purpose To investigate the lower urinary tract changes in mice treated with L-NAME, a non-selective competitive inhibitor of nitric oxide synthase (NOS), or aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), after 5 weeks of partial bladder outlet obstruction (BOO), in order to evaluate the role of constitutive and non-constitutive NOS in the pathogenesis of this experimental condition. Materials and Methods C57BL6 male mice were partially obstructed and randomly allocated into 6 groups: Sham, Sham + L-NAME, Sham + aminoguanidine, BOO, BOO + L-NAME and BOO + aminoguanidine. After 5 weeks, bladder weight was obtained and cystometry and tissue bath contractile studies were performed. Results BOO animals showed increase of non-voiding contractions (NVC) and bladder capacity, and also less contractile response to Carbachol and Electric Field Stimulation. Inhibition of NOS isoforms improved bladder capacity and compliance in BOO animals. L-NAME caused more NVC, prevented bladder weight gain and leaded to augmented contractile responses at muscarinic and electric stimulation. Aminoguanidine diminished NVC, but did not avoid bladder weight gain in BOO animals and did not improve contractile responses. Conclusion It can be hypothesized that chronic inhibition of three NOS isoforms in BOO animals leaded to worsening of bladder function, while selective inhibition of iNOS did not improve responses, what suggests that, in BOO animals, alterations are related to constitutive NOS.

Effects of nitric oxide inhibitors in mice with bladder outlet obstruction.

PURPOSE: To investigate the lower urinary tract changes in mice treated with L-NAME, a non-selective competitive inhibitor of nitric oxide synthase (NOS), or aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), after 5 weeks of partial bladder outlet obstruction (BOO), in order to evaluate the role of constitutive and non-constitutive NOS in the pathogenesis of this experimental condition. MATERIALS AND METHODS: C57BL6 male mice were partially obstructed and randomly allocated into 6 groups: Sham, Sham + L-NAME, Sham + aminoguanidine, BOO, BOO + L-NAME and BOO + aminoguanidine. After 5 weeks, bladder weight was obtained and cystometry and tissue bath contractile studies were performed. RESULTS: BOO animals showed increase of non-voiding contractions (NVC) and bladder capacity, and also less contractile response to Carbachol and Electric Field Stimulation. Inhibition of NOS isoforms improved bladder capacity and compliance in BOO animals. L-NAME caused more NVC, prevented bladder weight gain and leaded to augmented contractile responses at muscarinic and electric stimulation. Aminoguanidine diminished NVC, but did not avoid bladder weight gain in BOO animals and did not improve contractile responses. CONCLUSION: It can be hypothesized that chronic inhibition of three NOS isoforms in BOO animals leaded to worsening of bladder function, while selective inhibition of iNOS did not improve responses, what suggests that, in BOO animals, alterations are related to constitutive NOS.

The soluble guanylyl cyclase activator BAY 60-2770 potently relaxes the pulmonary artery on congenital diaphragmatic hernia rabbit model.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage, and a significant cause of morbidity and mortality. In this study, we have used a rabbit model of CDH to evaluate the effects of BAY 60-2770 on the in vitro reactivity of left pulmonary artery. METHODS: CDH was performed in New Zealand rabbit fetuses (n = 10 per group) and compared to controls. Measurements of body, total and left lung weights (BW, TLW, LLW) were done. Pulmonary artery rings were pre-contracted with phenylephrine (10 µM), after which cumulative concentration-response curves to glyceryl trinitrate (GTN; NO donor), tadalafil (PDE5 inhibitor) and BAY 60-2770 (sGC activator) were obtained as well as the levels of NO (NO3/NO2). RESULTS: LLW, TLW and LBR were decreased in CDH (p < 0.05). In left pulmonary artery, the potency (pEC50) for GTN was markedly lower in CDH (8.25 ± 0.02 versus 9.27 ± 0.03; p < 0.01). In contrast, the potency for BAY 60-2770 was markedly greater in CDH (11.7 ± 0.03 versus 10.5 ± 0.06; p < 0.01). The NO2/NO3 levels were 62 % higher in CDH (p < 0.05). CONCLUSION: BAY 60-2770 exhibits a greater potency to relax the pulmonary artery in CDH, indicating a potential use for pulmonary hypertension in this disease.

The Evolutionary Implications of Hemipenial Morphology of Rattlesnake Crotalus durissus terrificus (Laurent, 1768) (Serpentes: Viperidae: Crotalinae).

Most amniotes vertebrates have an intromittent organ to deliver semen. The reptile Sphenodon and most birds lost the ancestral penis and developed a cloaca-cloaca mating. Known as hemipenises, the copulatory organ of Squamata shows unique features between the amniotes intromittent organ. They are the only paired intromittent organs across amniotes and are fully inverted and encapsulated in the tail when not in use. The histology and ultrastructure of the hemipenes of Crotalus durissus rattlesnake is described as the evolutionary implications of the main features discussed. The organization of hemipenis of Crotalus durissus terrificus in two concentric corpora cavernosa is similar to other Squamata but differ markedly from the organization of the penis found in crocodilians, testudinata, birds and mammals. Based on the available data, the penis of the ancestral amniotes was made of connective tissue and the incorporation of smooth muscle in the framework of the sinusoids occurred independently in mammals and Crotalus durissus. The propulsor action of the muscle retractor penis basalis was confirmed and therefore the named should be changed to musculus hemipenis propulsor.The retractor penis magnus found in Squamata has no homology to the retractor penis of mammals, although both are responsible for the retraction of the copulatory organ.

Increased contractility and impaired relaxation of the left pulmonary artery in a rabbit model of congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage and a significant cause of morbidity and mortality. Our aim was to study the pulmonary artery reactivity in an animal model of CDH. METHODS: To investigate the reactivity of the aorta and left pulmonary artery in a rabbit model of CDH, we studied the in vitro responses to the α1-adrenoceptor agonist phenylephrine (PE) and to both the muscarinic receptor agonist (ACh) and the nitric oxide (NO) donor sodium nitroprusside (SNP). Rabbits underwent surgery at 25 days of gestation. CDH was created in one fetus per horn (n = 8). Remaining fetuses were considered controls (n = 18). At term (30 days), the lung, left pulmonary artery, and aorta were dissected. In a separate group, endothelium was mechanically removed. RESULTS: There were no differences in the contractile and relaxing responses of aorta in all groups. In left pulmonary artery, PE-induced contractions were significantly greater (p < 0.05) in CDH when compared with control group. The increased responsiveness to PE in CDH group was similar to that found in pulmonary artery without endothelium. The ACh-induced pulmonary artery relaxation was markedly reduced in CDH when compared with control group (p < 0.05), whereas no differences were found for SNP. CONCLUSION: Our results show increased contractility and impairment in endothelium-dependent relaxation of pulmonary artery in CDH, mimicking an endothelial dysfunction, with preserved response to endothelium-independent mechanism.

The cholinergic response is increased in isolated ileum from gastroschisis rat model.

INTRODUCTION: Babies with gastroschisis (G) have high morbidity rate and long hospital stay due to bowel hypomotility caused by chronic exposure of the bowel to the amniotic fluid. Our aim was to evaluate the reactivity of isolated ileum in fetal rats selected for experimental gastroschisis. METHOD: G was surgically created at 18.5 days of gestation (term = 22 days). Concentration-dependent curve to the muscarinic agonist methacholine (1-30 µM) and contractions induced by electrical field stimulation (EFS, 1-16 Hz, 50 V, 1 ms) were carried out in isolated ileum of groups control (C), sham (S) and gastroschisis (G) (n = 30). Protein expression for M(3) was assessed by western blot analysis. RESULTS: The frequency and amplitude of spontaneous contractions were decreased in G (p < 0.001). Methacholine produced concentration-dependent contractions being the maximal response values higher in G (p < 0.01). EFS-induced frequency-dependent contractions showed 1.8 times higher in G as well as an increase of M(3) expression. CONCLUSION: The frequency and the amplitude of rhythmic contractions were reduced along with an increase in the contraction induced by mucarinic agonist and by EFS in G. These results suggest the occurrence of an adaptative supersensitivity to cholinergic response via increases in the protein expression for M(3) receptor.