Rat Vas Deferens Research Articles

Extracellular cAMP elicits contraction of rat vas deferens: Involvement of ecto-5′-nucleotidase and adenosine A1 receptors

AimsIt is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called “extracellular cAMP-adenosine pathway”. Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. Main methodsThe effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. Key findingsWhile 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5′-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SignificanceOur data show that activation of β-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.

Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility.

6-Cyanodopamine is a novel catecholamine released from rabbit isolated heart. However, it is not known whether this catecholamine presents any biological activity. Here, it was evaluated whether 6-cyanodopamine (6-CYD) is released from rat vas deferens and its effect on this tissue contractility. Basal release of 6-CYD, 6-nitrodopamine (6-ND), 6-bromodopamine, 6-nitrodopa, and 6-nitroadrenaline from vas deferens were quantified by LC-MS/MS. Electric-field stimulation (EFS) and concentration-response curves to noradrenaline, adrenaline, and dopamine of the rat isolated epididymal vas deferens (RIEVD) were performed in the absence and presence of 6-CYD and /or 6-ND. Expression of tyrosine hydroxylase was assessed by immunohistochemistry. The rat isolated vas deferens released significant amounts of both 6-CYD and 6-ND. The voltage-gated sodium channel blocker tetrodotoxin had no effect on the release of 6-CYD, but it virtually abolished 6-ND release. 6-CYD alone exhibited a negligible RIEVD contractile activity; however, at 10 nM, 6-CYD significantly potentiated the noradrenaline- and EFS-induced RIEVD contractions, whereas at 10 and 100 nM, it also significantly potentiated the adrenaline- and dopamine-induced contractions. The potentiation of noradrenaline- and adrenaline-induced contractions by 6-CYD was unaffected by tetrodotoxin. Co-incubation of 6-CYD (100 pM) with 6-ND (10 pM) caused a significant leftward shift and increased the maximal contractile responses to noradrenaline, even in the presence of tetrodotoxin. Immunohistochemistry revealed the presence of tyrosine hydroxylase in both epithelial cell cytoplasm of the mucosae and nerve fibers of RIEVD. The identification of epithelium-derived 6-CYD and its remarkable synergism with catecholamines indicate that epithelial cells may regulate vas deferens smooth muscle contractility.

Mechanism of action of <i>Pterorhachis</i> <i>zenkeri</i> (Meliaceae), a sexual enhancer: effect on vas deferens contractility

Background: Pterorhachis. zenkeri commonly known as “Ayilalou” or “Démareur” is an aphrodisiac plant used by Cameroonian traditional healers to enhance libido and sperm production. This study was undertaken to investigate the aphrodisiac mechanism of P. zenkeri on the contractile activity of vas deferens with regard to its function on ejaculation. Materials and Methods: The proximal parts of rat vas deferens were mounted in a 20 mL organ bath containing Krebs solution at 37°C. Cumulative dose-response curves were recorded with KCl (35-280 mM), adrenaline (7-70 µM) and aqueous extract of P. zenkeri (3,55-21,3 mg/mL). Prazosin (type 1 alpha-adrenergic receptor antagonist, 10 µM), Nifedipine (a specific L-type calcium channel antagonist, 5 µM), 2-AminoethoxydiPhenyl Borate (2-APB, IP3 receptor antagonist, 200 μM) and calcium-free medium were further used to study the mechanism of action. Results: KCl, adrenaline and aqueous extract of P. zenkeri showed a concentration-dependent effect on vas deferens contractibility. Nifedipine and Pazosin partially inhibited the effects of P. zenkeri. In calcium-free medium containing Ethylene Glycol-bis (2-aminoethylether)-N,N,N’N’-tetraacetic acid (EGTA, 1µM), P. zenkeri triggered vas deferens contraction which was completely abolished in the presence of 2-APB. These results suggest that the aqueous extract of P. zenkeri possesses bioactive compounds capable of inducing vas deferens contractions through type 1 alpha adrenergic receptors and mobilization of extra and intracellular calcium. Conclusion: These results confirm the aphrodisiac properties of P. zenkeri through the activation of the emissive phase of ejaculation.

Interaction of endocannabinoid system and cyclooxygenase metabolites with fatty acid amide hydrolase and cyclooxygenase enzyme activities on contractile responses in rat vas deferens tissue.

The endocannabinoid system and prostaglandins are important modulators in the genitourinary system. This study aimed to investigate the possible interactions between the endocannabinoid system and the cyclooxygenase (COX) pathway on rat vas deferens. For this purpose, the concentration responses of the endocannabinoid anandamide, prostaglandin F2α analog latanoprost, and prostaglandin E1 analog misoprostol on the electrical field stimulation (EFS)-induced contractile responses were obtained. The concentration responses to anandamide were obtained again in the presence of nonselective COX inhibitor flurbiprofen and prostaglandin analogs, while the concentration responses of latanoprost and misoprostol were obtained in the presence of cannabinoid receptor antagonists and fatty acid amide hydrolase (FAAH) enzyme inhibitor URB597. FAAH, COX-1, and COX-2 enzyme levels in vas deferens tissue samples were also determined. The cumulative addition of anandamide was not different from the vehicle; however, the EFS-induced contractile responses were significantly increased with the incubation of latanoprost or flurbiprofen in the prostatic portion. Flurbiprofen and misoprostol decreased FAAH enzyme levels in both portions of the vas deferens, while latanoprost induced the inhibition in the prostatic portion. The cumulative administration of latanoprost and misoprostol significantly enhanced the contractile responses in the prostatic portion. This effect of latanoprost was significantly antagonized by URB597 and AM251. The enhancing effect of misoprostol was antagonized by anandamide, URB597, AM251, and AM630. Anandamide, AM251, AM630, and URB597 decreased enzyme levels of COX-1 and COX-2 in both portions of the vas deferens. These results demonstrate an intricate crosstalk between endocannabinoids and prostaglandins in modulation of the vas deferens contractility.

Effects of RS17053 on α1 -adrenoceptors in rat vas deferens and aorta.

RS17053 is classed as an α1A -adrenoceptor selective antagonist. We have examined its profile of action at all subtypes of α1 -adrenoceptor. Noradrenaline (NA) evoked contractions of rat vas deferens involve α1D -adrenoceptors in phasic contractions and α1A -adrenoceptors in tonic contractions. Contractions of rat aorta to NA involve α1D - and α1B -adrenoceptors. RS17053 (10-5 M) shifted NA potency and virtually abolished tonic contractions to NA, with little or limited effect on phasic contractions. The α1D -adrenoceptor antagonist BMY7378 (3 × 10-7 M) significantly inhibited the remaining phasic component of the contractions, and the α1A -adrenoceptor antagonist RS100329 (10-7 M) inhibited further the residual tonic contraction. Hence, RS17053 shows high selectivity for α1A -adrenoceptors over α1D -adrenoceptors in rat vas deferens. However, RS17053 (10-5 M) produced a large shift in the potency of NA in rat aorta, with a pKB of 6.82. Large shifts of NA potency in rat aorta involve α1B -adrenoceptor blockade. Results in rat vas deferens demonstrate low potency of RS17053 at α1D -adrenoceptors, but results from rat aorta can only be explained as demonstrating α1B -adrenoceptor antagonism by RS17053. RS17053 may be a useful pharmacological tool when reclassified as a mainly α1A - and to a lesser extent α1B -adrenoceptor antagonist with little effect at α1D -adrenoceptors.

Effect of Ferula elaeochytris root extract on smooth muscle contraction of vas deferens gland in rat

Purpose: To evaluate the effect of Ferula elaeochytris (FE) at the prostatic and epididymal ends of rat vas deferens.
 Methods: The effects of cumulative concentrations of FE (10 μL; 31.25 mg/μL and 20 μL; 62.5 mg/μL) were investigated on prostatic and epididymal ends of rat vas deferens in the presence of prazosin (0.3 μM), suramin (100 μM), atropine (10 nM) and nitric oxide synthase inhibitor (L-NOARG; 100 μM). The muscle contractions were induced by electrical field stimulation (EFS; 4 Hz, 50 V, 0.15 ms). Calcium (3 and 6 mM) was added into the bath medium while electrical field stimulation (EFS) was in progress.
 Results: Ferula elaeochytris significantly inhibited the muscle contractions induced by electrical field stimulation (EFS) in a concentration-dependent manner. In the presence of prazosin or suramin, the contractile responses to EFS were significantly inhibited by FE at the prostatic and epididymal ends of vas deferens (p < 0.05). However, this inhibition was not affected by atropine and L- NOARG, suggesting that there is no direct interaction of FE with cholinergic and nitrergic responses. However, in the presence of prazosin or suramin, Ca2+ addition to the organ bath significantly reversed the inhibitory effect of FE at the prostatic and epididymal ends of vas deferens (p < 0.05).
 Conclusion: These results show an inhibitory effect for the extract of FE on neurogenic contractile activity of prostatic and epididymal ends of vas deferens. This effect of FE may be associated with Ca2+ channels.
 Keywords: Contractile activity; Electrical Field Stimulation (EFS); Ferula elaeochytris; Rat; Vas deferens

6-NitroDopamine is an endogenous modulator of rat heart chronotropism

6-Nitrodopamine (6-ND) is released by rat vas deferens and exerts a potent contractile response that is antagonized by tricyclic antidepressants and α1-, β1- and β1/β2-adrenoceptor antagonists. The release of 6-ND, noradrenaline, adrenaline and dopamine from rat isolated right atria was assessed by tandem mass spectrometry. The effects of the catecholamines were evaluated in both rat isolated right atria and in anaesthetized rats. 6-ND was the major catecholamine released from the isolated atria and the release was significantly reduced in nitric oxide synthase inhibitor L-NAME pre-treated atria or in atria obtained from L-NAME chronically treated animals, but unaffected by tetrodotoxin. 6-ND (1 pM) significantly increased the atrial frequency, being 100 times more potent than noradrenaline and adrenaline. Selective β1-blockers reduced the atrial frequency only at concentrations that prevented the increases in atrial frequency induced by 6-ND 1pM. Conversely, β1-blockade did not affect dopamine (10 nM), noradrenaline (100 pM) or adrenaline (100 pM) effect. The reductions in atrial frequency induced by the β1-adrenoceptor antagonists were absent in L-NAME pre-treated atria and in atria obtained from chronic L-NAME-treated animals. Tetrodotoxin did not prevent the reduction in atrial frequency induced by L-NAME or by β1-blockers treated preparations. In anaesthetized rats, at 1 pmol/kg, only 6-ND caused a significant increase in heart rate. Inhibition of 6-ND synthesis by chronic L-NAME treatment reduced both atrial frequency and heart rate. The results indicate that 6-ND is a major modulator of rat heart chronotropism and the reduction in heart rate caused by β1-blockers are due to selective blockade of 6-ND receptor.

Tasipimidine—the pharmacological profile of a novel orally active selective α2A-adrenoceptor agonist

The pharmacological profile of tasipimidine, a novel orally active α2-adrenoceptor agonist developed for situational anxiety and fear in dogs, was studied in various in vitro and in vivo models. In the cell assays, tasipimidine demonstrated binding affinity and full agonism on the human α2A-adrenoceptors with a pEC50 of 7.57, while agonism on the α2B-and α2C-adrenoceptors and the rodent α2D-adrenoceptor was weaker, resulting in pEC50 values of 6.00, 6.29 and 6.56, respectively. Tasipimidine had a low binding affinity on the human α1-adrenoceptors. It had no functional effects in the LNCaP cells expressing endogenously the human α1A-adrenoceptors but was a weak agonist in the Chem-1 cells coexpressing Gα15 protein and α1A-adrenoceptors. In the recombinant CHO cells, although tasipimidine was a weak partial agonist in the inositol monophosphate accumulation assay, it was a full agonist in the intracellular [Ca2+] assay. No functional effects were observed on the human α1B-adrenoceptor, whereas in the rat α1A and α1B-adrenoceptors, tasipimidine was a weak partial agonist. In the rat vas deferens preparations, tasipimidine was a full agonist on the α2D-adrenoceptor but weak partial agonist on the α1-adrenoceptor. The receptor profile of tasipimidine indicated few secondary targets, and no functional effects were observed. Sedative effects of tasipimidine were demonstrated in vivo by the reduced acoustic startle reflex in rats with subcutaneous doses and decreased spontaneous locomotor activity in mice with subcutaneous and higher oral doses. It may be concluded that tasipimidine is an orally active and selective α2A-adrenoceptor agonist.

Pharmacological characterisation of the CB1 receptor antagonist activity of cannabidiol in the rat vas deferens bioassay

Cannabidiol is increasingly considered for treatment of a wide range of medical conditions. Binding studies suggest that cannabidiol binds to CB1 receptors. In the rat isolated vas deferens bioassay, a single electrical pulse causes a biphasic contraction from nerve-released ATP and noradrenaline. WIN 55,212-2 acts on prejunctional CB1 receptors to inhibit release of these transmitters. In this bioassay, we tested whether cannabidiol and SR141716 were acting as competitive antagonists of this receptor. Monophasic contractions mediated by ATP or noradrenaline in the presence of prazosin or NF449 (P2X1 inhibitor), respectively, were measured to a single electrical pulse delivered every 30 min. Following treatment with cannabidiol (10–100 μM) or SR141716 (0.003–10 μM), cumulative concentrations of WIN 55,212–2 (0.001–30 μM) were applied followed by a single electrical pulse. The WIN 55,212–2 concentration-contraction curve EC50 values were applied to global regression analysis to determine the pKB. The antagonist potency of cannabidiol at the CB1 receptor in the rat vas deferens bioassay matched the reported receptor binding affinity. Cannabidiol was a competitive antagonist of WIN 55,212–2 with pKB values of 5.90 when ATP was the effector transmitter and 5.29 when it was noradrenaline. Similarly, SR141716 was a competitive antagonist with pKB values of 8.39 for ATP and 7.67 for noradrenaline as the active transmitter. Cannabidiol's low micromolar CB1 antagonist pKB values suggest that at clinical blood levels (1–3 μM) it may act as a CB1 antagonist at prejunctional neuronal sites with more potency when ATP is the effector than for noradrenaline.

Effect of vas ligation on testicular sperm extraction results in nonobstructive azoospermic rats: Shaeer's lock-in technique.

This study evaluates the efficacy of vas ligation in enhancing sperm retrieval in nonobstructive azoospermia cases, by accumulating intratesticular spermatozoa. Fifty-six mature male rats with equally sized testes were included in this study. Forty-six were in the study group, and 10 were in the control group. Bilateral testicular fine needle aspiration was performed for all, to confirm presence of spermatozoa in all testes. Nonobstructive azoospermia was induced in all 56 rats, using Dienogest (40mg/kg) + Testosterone Undecanoate (25mg/kg) every month for three months. Monthly aspirations confirmed nonobstructive azoospermia from all rats, within the three months treatment. This was followed by unilateral vas ligation and was performed for 46 rats of the study group, with no ligation performed in the control group. After a further period of 90days (2 spermatogenic cycles) with the same medical treatment maintained, bilateral testicular sperm extraction was performed. Sperm retrieval was evaluated, comparing the outcome of vas-ligated testicles to the nonligated. Upon evaluation, spermatozoa were found in 14/46 of the vas-ligated testes (30.4%), compared to none of the nonligated (0/66), p=.0005. Ligation of the vas deferens in rats with nonobstructive azoospermia may enhance the results of sperm retrieval via sperm accumulation.

Contribution of amniotic membrane to the healing of iatrogenic vas deferens injury

Background/aim Iatrogenic vas deferens injury is one of the most serious complications of operations in the inguinal region. Vasovasostomy is performed as treatment. However, stenosis is common after vasovasostomy. Oligospermia or azoospermia may develop and result in infertility. This study aimed to investigate the effect of amniotic membrane on healing in vas deferens injuries.Materials and methods Four groups consisting of 10 rats each were formed. No procedure was performed in Group-I. In Group-II, the left vas deferens was transected and left to spontaneous healing. In Group-III, the left vas deferens was transected, and end-to-end anastomosis was performed. In Group-IV, the left vas deferens was transected, end-to-end anastomosis was performed, and it was closed with a wrapping of amniotic membrane on the anastomosis line. Rats were sacrificed after 60 days, and each left vas deferens was evaluated. Lumen patency was checked by passing methylene blue through the vas deferens. Subsequently, the vas deferens was evaluated both macroscopically and histopathologically. Data were evaluated using SPSS version 21.0. p < 0.05 was considered statistically significant for all variables.Results The anastomosis lines in Group-IV healed better than those in Group-III, and less stenosis was observed. There were differences between the groups in terms of luminal patency (p = 0.009), adhesions to surrounding tissues (p = 0.02) and separation of the ends of the vas deferens (p = 0.03).ConclusionWe observed improvement on luminal patency and histology of rat vas deferens injury after surrounding human amniotic membrane on the transected and repaired surface. Further studies are needed to apply this promising result on human beings.

Neurotoxicity of Olindias sambaquiensis and Chiropsalmus quadrumanus extracts in sympathetic nervous system

Cnidarians are equipped with nematocysts, which are specialized organelles used to inoculate venom during prey capturing and defense. Their venoms are rich in toxins and a potential source of bioactive compounds, however, poorly explored so far. In this work, the activity of the methanolic extracts from the hydromedusa Olindias sambaquiensis and the cubozoan jellyfish Chiropsalmus quadrumanus were studied in sympathetic neurotransmission. For that, bisected rat vas deferens - a classic model of sympathetic neurotransmission - were incubated with the extracts for further myographic and histopathological analysis. The O. sambaquiensis extract, at 0.1 μg/mL, facilitated the neurogenic contractions of the noradrenergic-rich epididymal portion, while reducing the noradrenaline (NA) potency, which suggests an interaction with postsynaptic α1-adrenoceptors. On the other hand, a higher concentration (1 μg/mL) leads to time- and frequency-dependent blockade of nerve-evoked contractions without significantly changing the response to exogenous NA. In turn, the C. quadrumanus extract at 0.1 μg/mL induced blockade of nerve-evoked noradrenergic contractions while reducing the potency to exogenous NA. Both extracts did not affect the purinergic neurotransmission or induce muscle damages. Our results demonstrate that O. sambaquiensis and C. quadrumanus extracts significantly interfere with the noradrenergic neurotransmission without altering purinergic response or smooth muscle structure on rat vas deferens. Such results bring to light the pharmacological potential of O. sambaquiensis and C. quadrumanus molecules for therapeutics focusing on noradrenergic neurotransmission.

Immunolocalization of Adrenoceptors in the Reproductive Tract of Male Domestic Cats in Comparison to Rats.

Simple SummaryIn cats, semen is collected by pharmacological stimulation. The administration of a drug that stimulates α2-adrenoceptors causes the expulsion of spermatozoa into the urethra. However, as the results are not always satisfactory, this method needs to be improved. There are nine subtypes of adrenoceptors that are involved in the contraction of smooth muscle, including those in the reproductive tract, so adrenoceptors other than the α2-subtype are potential targets in any new, optimized protocol. The aim of this study was to analyze the immunolocalization of the adrenergic receptors in the reproductive tract of the male cat for the first time in this species. The expression of all adrenoceptor subtypes was noted in the peritubular smooth muscle in cats, indicating a potential clinical application for agonists of these receptors for the optimization of the pharmacological semen collection in felids. In a broader context, the development of a new procedure for semen collection in the male cat, using active substances from groups other than those currently used, will support the wider application of reproductive biotechnologies in felids.Adrenoceptors mediate the action of the sympathetic nervous system, including the contraction of the epididymis and vas deferens. The aim of this study was to immunolocalize the adrenergic receptors in the reproductive tract of the male cat, as this information is not yet available. The epididymis and vas deferens of domestic cats and rats (the biological controls) were analyzed by immunohistochemistry to determine the localization of the α1A-, α1B-, α1D-, α2A-, α2B-, α2C-, β1-, β2-, and β3-adrenoceptors. All the receptors were expressed in the peritubular smooth muscles of the cat, but the α1D-, α2C-, and β1-adrenoceptors were not detected in this tissue in the rat. For the α2A-adrenoceptor, the intensity of immunostaining differed significantly between the caput epididymis (weakest staining) and the vas deferens (strongest staining). The presence of all the types of the receptors was also detected in the cytoplasm of the epithelial cells in all the regions of the reproductive tract. The strong expression of the α2A-adrenoreceptor suggests it has a leading role in the contraction of the reproductive tract in the cat. The presence of other adrenergic receptors in the smooth muscle of the epididymis and vas deferens indicates a potential clinical application for α1-mimetics in the optimization of pharmacological semen collection in felids.

Neuropharmacological Exploration of Standardized Extract of Annona squamosa (L.) Fruit Pulp in Experimental Animals

Aims: The present study aimed to investigate the neuroprotective potential of standardized Annona squamosa Linn fruit pulp extract using various in-vitro and in-vivo models. Methodology: Neuroprotective potential of standardized extract was screened against dopamine-induced contraction of isolated rat vas deferens, serotonin-induced contractions of isolated rat fundus, acetylcholine-induced contractions of isolated goat tracheal chain. In-vivo models such as elevated plus maze, light and dark model, force swim test, tail suspension test, lithium-induced head twitches, haloperidol-induced catalepsy, PTZ induced seizure and foot shock-induced aggression were implemented to screen various doses intervals (50-200mg/kg) of extracts in experimental animals. Results: Standardization of extract showed content of polyphenols 65.37 mg/g of GAE, total flavonoid 5.33 mg/g of RE and HPLC fingerprinting of ASP-ME showed identical retention time as that of standard gallic acid, quercetine and rutin, viz 3.830, 5.765 and 3.830 respectively. Inhibition of DPPH radical reflected as 91.32±0.19 % while percent inhibition of RRI of DPPH was observed as 95.99±0.47 at 150 min. ASP-ME significantly inhibited dopamine and serotonin induced contraction on isolated rat vas deferens and rat fundus respectively at log dose (1.3, 2.5) for dopamine and log dose (2.2, 2.5) for serotonin. ASP-ME potentiated ach-induced contractions on goat tracheal chain preparation. Ach alone produces 106.90±4.6 % response, while ASP-ME in presence of Ach potentiates response and produces 141.80±10 % response. The extract demonstrated anxiolytic activity by increasing the time spent in open arms and light zone in elevated plus maze and light dark test respectively. The duration of immobility was significantly decreased in force swim &amp; tail suspension test respectively demonstrating antidepressant activity. Administration of ASP-ME shown antipsychotic effect in dose dependent manner by minimising aggression induced by foot shock (reduced number of flights), while potentiation of catalepsy induced by haloperidol. The extract also exhibited serotonergic system inhibitory effect by significantly reducing head twitches imparted by lithium. The ASP-ME significantly delayed the onset of first myoclonic and clonic spasms induced by PTZ indicating anticonvulsant effects. Extract also shown ability to decrease the behavior facilitated by the serotonergic and dopaminergic coordination, while potentiated the actions produced by GABA. Conclusion: Finding of the study suggests anxiolytic, antidepressant, antipsychotic effects of ASP-ME probably mediated through dopamine D2 and 5-HT receptors, with neuroprotective activity.

Synthesis of 2-phenyl-5,6,7,8-tetrahydroquinoxaline derivatives and screening for P2X1-purinoceptor antagonist activity in isolated preparations of rat vas deferens, for translation into a male contraceptive†.

Sympathetically mediated contractions of smooth muscle cells in the vasa deferentia are mediated by neuronally released adenosine 5′-triphosphate (ATP) and noradrenaline, which stimulate P2X1-purinoceptors and α1A-adrenoceptors, respectively. This process is crucial for sperm transport, as demonstrated in knockout mouse studies where simultaneous genetic deletion of P2X1-purinoceptors and α1A-adrenoceptors resulted in male infertility. We hypothesize that dual pharmacological antagonism of these two receptors could inhibit sperm transport sufficiently to provide a novel nonhormonal method of male contraception. To generate a suitable P2X1-purinoceptor antagonist, substituents were introduced on the phenyl moiety of 2-phenyl-5,6,7,8-tetrahydroquinoxaline to create a series of analogues that were tested for P2X1-purinoceptor antagonism in isolated preparations of rat vas deferens. Novel compounds were initially screened for their ability to attenuate contractile responses to electrical field stimulation (EFS: 60 V, 0.5 ms, 0.2 Hz). The addition of polar substituents to the meta, but not ortho, position markedly increased the inhibition of contractions, as did the addition of both polar and aliphatic substituents to the para position. Di-substituted compounds were also synthesized and tested, resulting in a compound 31 (2-hydroxy, 4-fluoro), which exhibited the greatest potency, with an IC50 of 14 μM (95% confidence limits: 12–16 μM). Additionally, compound 31 noncompetitively antagonized contractions mediated by exogenously administered αß-methylene ATP (10 nM–30 μM) but had no inhibitory effect on contractions mediated by exogenously administered noradrenaline (30 nM–100 μM) or acetylcholine (30 nM–100 μM). These results have contributed to a structure–activity relationship profile for the P2X1-purinoceptor that will inform future designs of more potent antagonists.

Cyclohexylamine, an active compound from Toddalia asiatica, contracts epididymal vas deferens via serotonergic receptors.

BackgroundToddalia asiatica of Rutaceae, a Taiwan folk medicine, is used as an analgesic and anti-inflammatory herb. Cyclohexylamine (CHA) is an active compound from T. asiatica. Previous reports indicate CHA contracts rat vas deferens. However, the contractile mechanism of CHA on rat vas deferens was not yet reported. The purpose of this study was to investigate the contractile mechanism of CHA on rat epididymal portion of vas deferens.MethodsMale S.D. rats weighting between 200 g to 250 g were used. The epididymal portion of vas deferens was isolated and was added with various concentrations of serotonin, serotonin antagonists and CHA.ResultsSerotonin elicited dose-dependent (1 × 10−7M~1 × 10−4M) contractions on rat epididymal vas deferens, which were inhibited by pretreatment with ketanserin (1 × 10−8 M ~ 1 × 10−6 M), methysergide (1 × 10−5 M) and propranolol (1 × 10−4 M), respectively. CHA elicited dose-dependent (1 × 10−8M~1 × 10−4M) contractions on rat epididymal vas deferens. The contractions of CHA (1 × 10−4M) on epididymal vas deferens were enhanced by serotonin in a dose-dependent manner. Methysergide (1 × 10−7 ~1 × 10−5 M) did not affect the contractions evoked by CHA. However, the CHA elicited contraction was almost completely inhibited by ketanserin (1 × 10−5 M) and was enhanced by propranolol. The effect of propranolol at the concentration of 1 × 10−4 M on CHA was likely as CHA at high concentration alone.ConclusionsFrom the above results, the contraction evoked by CHA on the isolated rat epididymal vas deferens might be mediated by serotonergic receptors through 5-HT2A subtype.

Resveratrol and quercetin attenuate depressive-like behavior and restore impaired contractility of vas deferens in chronic stress-exposed rats: involvement of oxidative stress and inflammation.

Chronic stress is associated with male sexual problems including ejaculatory dysfunctions. The aim of this study was to determine whether resveratrol (RS) or quercetin (QE) has protective effects on vas deferens (VD) contractility in the unpredictable chronic mild stress (UCMS) rat model of depression. Animals were separated into six groups: control, control + RS and control + QE, stress, stress + RS, and stress + QE. Stress groups were subjected to UCMS procedure for 5weeks. Animals in treatment groups were injected intraperitoneally with RS (20mg/kg) or QE (30mg/kg) for 5weeks during UCMS period. UCMS caused depressive-like behaviors and enhanced systemic levels of corticosterone. The nerve-evoked contractile responses of VD significantly impaired and, noradrenaline- and ATP-induced contractile responses of VD significantly increased in stressed rats. UCMS exposure also markedly enhanced oxidative stress and inflammation in VD tissues. Treatment with RS or QE significantly ameliorated all the aforementioned parameters. The current study demonstrated that RS or QE protected against chronic stress-induced VD dysfunction by their antioxidant and anti-inflammatory effects on VD, suggesting that oxidative stress and inflammation may be synergistic parts in the development of VD dysfunction associated with chronic stress-induced depression.

The effects of varying Mg2+ ion concentrations on contractions to the cotransmitters ATP and noradrenaline in the rat vas deferens.

The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ± 7% and raising Mg2+ to 3 mM decreased it to 39 ± 3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ± 3%) but was decreased to 63 ± 4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.

Castration Induces Down-Regulation of A-Type K+ Channel in Rat Vas Deferens Smooth Muscle

A-type K+ channels contribute to regulating the propagation and frequency of action potentials in smooth muscle cells (SMCs). The present study (i) identified the molecular components of A-type K+ channels in rat vas deferens SMs (VDSMs) and (ii) showed the long-term, genomic effects of testosterone on their expression in VDSMs. Transcripts of the A-type K+ channel α subunit, Kv4.3L and its regulatory β subunits, KChIP3, NCS1, and DPP6-S were predominantly expressed in rat VDSMs over the other related subtypes (Kv4.2, KChIP1, KChIP2, KChIP4, and DPP10). A-type K+ current (IA) density in VDSM cells (VDSMCs) was decreased by castration without changes in IA kinetics, and decreased IA density was compensated for by an oral treatment with 17α-methyltestosterone (MET). Correspondingly, in the VDSMs of castrated rats, Kv4.3L and KChIP3 were down-regulated at both the transcript and protein expression levels. Changes in Kv4.3L and KChIP3 expression levels were compensated for by the treatment with MET. These results suggest that testosterone level changes in testosterone disorders and growth processes control the functional expression of A-type K+ channels in VDSMCs.

Evaluation of Antidepressant Effects on Recovery of Electrical Field Stimulation-Induced Contractions that have been Suppressed by Clonidine in Isolated Rat Vas Deferens

Background: A report examining whether clinically available antidepressants increase urethral smooth muscle contraction via antagonistic effects on the α2-adrenoceptor (α2-AR) is lacking. Objectives: The present study was performed to evaluate the potential of clinically available antidepressants to reverse α2-AR-mediated contractile inhibition in rat vas deferens, in order to predict whether they can induce voiding impairment. Method: The effects of 18 antidepressants of different classes on electrical field stimulation (EFS)-induced contractions suppressed by 10–8 mol/L clonidine (a selective α2-AR agonist) in isolated rat vas deferens were investigated and related to their respective clinical blood concentrations. Results: The EFS-induced contractions suppressed by clonidine were recovered by amitriptyline (a tricyclic antidepressant), mirtazapine (a noradrenergic and specific serotonergic antidepressant), and trazodone (a serotonin 5-HT2A receptor antagonist) at concentrations close to the clinical blood levels. EFS-induced contractions were also recovered by trimipramine, clomipramine (tricyclic antidepressants), mianserin (a tetracyclic antidepressant), sertraline (a selective serotonin reuptake inhibitor [SSRI]), and sulpiride (a dopamine D2-receptor antagonist), albeit at concentrations that substantially exceeded their clinically-achievable blood levels. EFS-induced contractions were not significantly affected by imipramine, nortriptyline, amoxapine (tricyclic antidepressants), maprotiline (a tetracyclic antidepressant), fluvoxamine, paroxetine, escitalopram (SSRIs), milnacipran, duloxetine (serotonin and noradrenaline reuptake inhibitors), and aripiprazole (a dopamine partial agonist). Conclusions: These findings suggest that amitriptyline, mirtazapine, and trazodone induce voiding impairment caused by increased urethral resistance by enhancing sympathetic nerve activities attributed to α2-AR antagonism.