Julio Alejandro Rojas-Moscoso

Long-term oral treatment with BAY 41-2272 ameliorates impaired corpus cavernosum relaxations in a nitric oxide-deficient rat model.

Study Type – Therapy (case control) 
Level of Evidence 3b

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.

Characterization of the urinary bladder dysfunction in renovascular hypertensive rats

Association between arterial hypertension and urinary bladder dysfunction has been reported in humans and spontaneously hypertensive rats. However, no study exists evaluating the bladder dysfunction in conditions of renovascular hypertension. The purpose of this study was to characterize the bladder dysfunction in two kidney‐one clip (2K‐1C) hypertensive rats.

Vas deferens smooth muscle responses to the nitric oxide-independent soluble guanylate cyclase stimulator BAY 41‐2272

The nitric oxide-cGMP signaling pathway modulates the ejaculatory functions. The nitric oxide (NO)-independent soluble guanylate cyclase haem-dependent stimulator BAY 41-2272 potently relaxes different types of smooth muscles. However, no study investigated its effects in vas deferens smooth muscle. Therefore, we designed experiments to evaluate the in vitro relaxing responses of vas deferens to BAY 41-2272. The effects of prolonged oral intake with BAY 41-2272 in vas deferens contractions of rats treated chronically with the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) were also investigated. BAY 41-2272 (0.001-100 μM) produced concentration-dependent relaxations in the prostatic and epididymal portions of vas deferens, an effect markedly reduced by the soluble guanylate cyclase inhibitor ODQ (100 μM). BAY 41-2272 significantly increased cGMP levels that were fully prevented by ODQ. In separate protocols, rats received L-NAME (20mg/rat/day) concomitantly with BAY 41-2272 (10mg/kg/day, 4 weeks), after which vas deferens contractions to electrical-field stimulation and noradrenaline were achieved. Electrical-field stimulation (1-32 Hz) evoked frequency-dependent contractions that were significantly enhanced in L-NAME-treated rats. Co-treatment with BAY 41-2272 fully reversed the increased contractile responses in L-NAME group. Noradrenaline (0.01-100 μM)-induced contractions were also greater in L-NAME-treated rats, and that was normalized by BAY 41-2272. In conclusion, BAY 41-2272 potently relaxes in vitro rat vas deferens smooth muscle and elevates the cGMP levels in an ODQ-sensitive manner. Moreover, prolonged oral intake with BAY 41-2272 restores the enhanced contractile vas deferens activity in rats treated with L-NAME. NO-independent soluble guanylate cyclase stimulators may be an alternative treatment for premature ejaculation.

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.

Immunohistochemical and functional characterization of nitric oxide signaling pathway in isolated aorta from Crotalus durissus terrificus

We characterized the nitric oxide (NO)-cyclic GMP-phosphodiesterase-5 (PDE5) pathway in Crotalus durissus terrificus aorta. Concentration responses curves to acetylcholine (ACh), sodium nitroprusside (SNP), BAY41-2272 (soluble guanylyl cyclase [sGC] stimulator), BAY60-2770 (sGC activator) and tadalafil (PDE5 inhibitor) were constructed in phenylephrine (10 μM)-precontracted tissues with intact (E(+)) or denuded (E(-)) endothelium. ACh (0.0001-10 μM) and SNP (0.0001-10 μM) relaxed aorta, which were reduced by the NO synthase (L-NAME,100 μM) or the sGC inhibitors (ODQ, 10 μM). Tadalafil (0.0001-10 μM) relaxed E(+) rings with potency (pEC(50)) and maximal response (E(max)) values of 7.34±0.02 and 105±8%, respectively. E(-) or ODQ treatment significantly (P<0.05) reduced tadalafil relaxations (66±18% and 71±7%, respectively). BAY41-2272 (0.0001-300 nM) produced concentration-dependent relaxations in E(+) rings, which were reduced by addition of either ODQ or L-NAME (16.0- and 5.2-fold rightward shifts, respectively). The relaxation of BAY60-2770 was markedly potentiated by ODQ and L-NAME (41.0- and 9.7-fold leftward shifts, respectively), whereas in E(-) the pEC(50) values were shifted by 7-fold to the right. Immunohistochemistry, followed validation by transcriptomic analysis, revealed the presence of eNOS in endothelium, whereas nNOS was observed only in perivascular nerves. sGC and PDE5 were expressed in smooth muscle. Thus, NO-sGC-PDE5 pathway is evolutionarily present in Crotalus sp. vessels, and has a remarkable degree of functional similarity to mammalian vessels.

Micturition dysfunction in four-month old ovariectomized rats: Effects of testosterone replacement

Aims: Androgen deficiency has been implicated in urological complications of postmenopausal women. This study examined the effects of testosterone replacements on the lower urinary tract dysfunction in 4‐month old ovariectomized (OVX) rats. Main methods: Sprague‐Dawley female rats were OVX bilaterally. Three months later, rats received single intramuscular injections of testosterone undecanoate. Cystometric study, and bladder and urethra smooth muscle reactivities were evaluated. Key findings: Ovariectomy reduced by 65% (p < 0.05) the serum testosterone levels. Testosterone replacement at 5 mg/kg restored serum hormone levels to baseline, whereas 10 mg/kg produced 14‐fold higher testosterone levels. OVX rats exhibited significant increases of body weight, perigonadal fat and blood pressure, and reduced uterus weight, but none of these parameters were changed by testosterone replacements. OVX rats exhibited micturition dysfunction characterized by increases of basal pressure, threshold pressure, voiding frequency and post‐voiding pressure. In addition, the bladder contractions induced by electrical‐field stimulation (EFS) and carbachol were significantly reduced, whereas angiotensin II‐induced urethral contractions were significantly increased in OVX rats. Testosterone replacement at 10 mg/kg (but not at 5 mg/kg) dose fully normalized the in vivo micturition dysfunction, as well as the in vitro bladder and urethral alterations. Testosterone (10 mg/kg) also significantly potentiated the bladder relaxations induced by the &bgr;3‐adrenoceptor agonist mirabegron. The protective effects of testosterone were not modified by concomitant treatment with the aromatase inhibitor letrozole (2.5 mg/kg, 4 weeks). Significance: The improvement of micturition dysfunction by testosterone replacement suggests that androgen therapy might be of therapeutic benefit for urological complications associated with post‐menopause. Highlights:Four‐month old OVX rats proving a suitable experimental model for exploring the lower urinary function after menopauseTestosterone normalizes micturition dysfunction in 4‐month old OVX ratsWould be clinically relevant to examine testosterone administration in postmenopausal women with urological disorders; or testosterone administration should be examined in postmenopausal women with urological disorders.

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 ± 86.1 ng/mL ∗ h and 805.7 ± 252.4 ng/mL ∗ h, respectively. The calculated systemic bioavailability was 12.73% ± 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.

Pharmacokinetics of Riluzole in Beagle Dogs

BACKGROUND Riluzole is a benzothiazole anticonvulsant used in the treatment of patients with amyotrophic lateral sclerosis and it is being investigated for clinical use in patients with spinal cord injury. The present study evaluated the pharmacokinetics of riluzole in beagle dogs after oral dose administration. METHODS The oral doses (1.5, 5, 15 and 50 mg/kg) of riluzole were administered to beagle dogs and blood samples were collected from 0 h to 24 h post drug administration. Riluzole was quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RESULTS The method was sensitive, precise, accurate and selective to riluzole quantification in plasma of beagle dogs. The pharmacokinetics following oral administration was linear from 1.5 to 15 mg/kg and the t1/2 was 2.16, 1.5, 1.8 and 3.0 h after oral administration of 1.5, 5.0, 15 and 50 mg/kg riluzole. CONCLUSION The riluzole pharmacokinetics was linear up to 15 mg/kg and had a significantlyshorter t1/2 in beagle dogs than in humans.

L-Carnitine supplementation impairs endothelium-dependent relaxation in mesenteric arteries from rats

Abstract L-Carnitine (L-Car) is taken as fat burner. The risks of L-Car supplementation for the cardiovascular system are unclear. We evaluated the relaxing responses of the mesenteric and aorta rings from rats after four weeks of L-Car supplementation and/or physical training. Concentration response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as cyclic GMP levels, superoxide dismutase (SOD) activity and malondialdehyde (MDA) were evaluated. Physical training decreased body weight gain that was potentiated by L-Car. In mesenteric rings, L-Car impaired endothelium-dependent relaxation whereas endothelium independent relaxation was increased. In aorta, exercise improved endothelium-dependent relaxation; however, it was partially inhibited by L-Car. SNP-induced relaxation was similar in aorta of all groups. Basal cGMP were increased in aorta of exercised rats. SOD activity and MDA levels were unaltered. In conclusion, L-Car and physical exercise promotes body weight loss; however, it impairs endothelium-dependent vaso-relaxation possibly involving alterations in muscarinic receptors/eNOS/NO signalling pathway in mesenteric artery.