Isolated Vas Deferens Research Articles

Effect of Atomoxetine on Mouse Isolated Vas Deferens Contractility

Objective: Atomoxetine (ATX), a selective noradrenaline re-uptake inhibitor, is a preferred drug with sufficient efficacy and favorable safety profile for the treatment of attention-deficit hyperactivity disorder. Ejaculatory dysfunctions have been reported in the patients receiving ATX as sexual side effect, of which underlying mechanisms are largely unknown. The present study aimed to investigate the effect of ATX on mouse isolated vas deferens (VD) contractility as a potential mechanism of ATX-induced ejaculatory dysfunction. Material and Methods: Isolated organ bath studies were performed on prostatic parts of VD obtained from adult male Balb/c mice. The effect of ATX (10-6, 10-5, 3x10-5 and 10-4 M) on KCl (80 mM), phenylephrine (PhE, 3x10-4 M), adenosine 5’-triphosphate (ATP, 10-2 M) and electrical field stimulation (EFS; 100 V, 64 Hz)-induced contractions of VD strips were evaluated in concentration dependent manner. Results: ATX at 10-6 and 10-5 did not alter the contractile responses (p>0.05), however, higher concentrations of ATX (3x10-5 or 10-4 M) significantly inhibited the KCl, PhE, ATP and EFS-induced contractions of VD strips (p<0.05). Conclusion: The present study demonstrated for the first time that ATX decreased the contractile responses of mouse isolated VD concentration-dependently. Our results suggest that ejaculatory dysfunction might be related to the inhibitory effect on ATX on VD.

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.

Neuronal nitric oxide synthase is the main isoform responsible for 6-nitrodopamine release from mouse isolated vas deferens

Rat and human vas deferens (VD) present basal release 6-nitrodopamine (6-ND), which is significantly reduced when the VD is pre-incubated with the nitric oxide (NO) synthase inhibitor L-NAME, or in the case of the rat, when the VD was obtained from rats chronically treated with L-NAME. 6-Nitrodopamine is considered a major mediator of both rat and human VD contractility. Since the origin of 6-ND is not clear in the VD, here it was investigated the basal release of 6-ND from control, eNOS−/−, nNOS−/− and iNOS−/− mice VD to ascertain which NOS isoform(s) play(s) an essential role in 6-ND biosynthesis in the mouse VD. Euthanasia was performed by isoflurane overdose and both VD were removed and immediately placed in Krebs-Henseleit solution (KHS). The release of 6-ND was measured by LC-MS/MS. To evaluate contractility, the mice VD strips were submitted to EFS (60 V for 20 sec, at 2-16 Hz in square-wave pulses, 0.3 ms pulse width, and 0.1 ms delay), using a Grass S88 stimulator (Astro-Medical, Industrial Park, RI, USA). The tissues were allowed to equilibrate under a resting tension of 10 mN, and the isometric tension was registered using a PowerLab system. Isolated VD from control, nNOS−/−), eNOS−/−, and iNOS−/− mice present basal release of both 6-ND and dopamine, as detected by LC-MS/MS. The basal release of 6-ND from isolated VD of nNOS−/− mice was significantly reduced when compared to that obtained from control mice VD, whereas that of dopamine was significantly increased. The basal release of both 6-ND and dopamine from eNOS−/− mice VD was not different compared to that obtained from control mice VD. The basal release of 6-ND from iNOS−/− mice was significantly increased compared to that observed in control mice VD. Electric-field stimulation (EFS) caused frequency-dependent (2Hz - 32Hz) contractions of the mouse isolated VD. The contractions induced by EFS were significantly decreased in nNOS−/− mouse isolated VD, but unaltered in isolated VD obtained from either eNOS−/− or iNOS−/− mice. The results clearly demonstrate that neuronal nitric oxide synthase is the main isoform responsible for 6-ND synthesis/release from mouse isolated VD. The results obtained with the contractions induced by EFS in the mouse VD further support the concept that 6-ND is a major modulator of VD contractility, since the contractions of the VD was only reduced in tissue obtained from nNOS−/− mice. The reduction in the EFS-induced contractions observed in the vas deferens obtained from nNOS−/− mice could be attributed to the reduction observed in the basal release of 6-ND. This research is supported by the São Paulo Research Foundation (FAPESP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of nerve-evoked contractions of the murine vas deferens

Stimulation of sympathetic nerves in the vas deferens yields biphasic contractions consisting of a rapid transient component resulting from activation of P2X1 receptors by ATP and a secondary sustained component mediated by activation of α1-adrenoceptors by noradrenaline. Noradrenaline can also potentiate the ATP-dependent contractions of the vas deferens, but the mechanisms underlying this effect are unclear. The purpose of the present study was to investigate the mechanisms underlying potentiation of transient contractions of the vas deferens induced by activation of α1-adrenoceptors. Contractions of the mouse vas deferens were induced by electric field stimulation (EFS). Delivery of brief (1s duration) pulses (4 Hz) yielded transient contractions that were inhibited tetrodotoxin (100 nM) and guanethidine (10 µM). α,β-meATP (10 µM), a P2X1R desensitising agent, reduced the amplitude of these responses by 65% and prazosin (100 nM), an α1-adrenoceptor antagonist, decreased mean contraction amplitude by 69%. Stimulation of α1-adrenoceptors with phenylephrine (3 µM) enhanced EFS and ATP-induced contractions and these effects were mimicked by the phorbol ester PDBu (1 µM), which activates PKC. The PKC inhibitor GF109203X (1 µM) prevented the stimulatory effects of PDBu on ATP-induced contractions of the vas deferens but only reduced the stimulatory effects of phenylephrine by 40%. PDBu increased the amplitude of ATP-induced currents recorded from freshly isolated vas deferens myocytes and HEK-293 cells expressing human P2X1Rs by 93%. This study indicates that: (1) potentiation of ATP-evoked contractions of the mouse vas deferens by α1-adrenoceptor activation were not fully blocked by the PKC inhibitor GF109203X and (2) that the stimulatory effect of PKC on ATP-induced contractions of the vas deferens is associated with enhanced P2X1R currents in vas deferens myocytes.

The pivotal role of neuronal nitric oxide synthase in the release of 6-nitrodopamine from mouse isolated vas deferens

6-Nitrodopamine (6-ND) is released from rat and human vas deferens and is considered a major mediator of both tissues contractility. The contractions induced by 6-ND are selectively blocked by both tricyclic antidepressants and α1-adrenoceptor antagonists. Endothelial nitric oxide synthase (eNOS) is the major isoform responsible for 6-ND release in mouse isolated heart, however the origin of 6-ND in the vas deferens is unknown. Here it was investigated by LC-MS/MS the basal release of 6-ND from isolated vas deferens obtained from control, eNOS−/−, nNOS−/−, and iNOS−/− mice. In addition, it was evaluated in vitro vas deferens contractility following electric field stimulation (EFS).Basal release of 6-ND was significantly reduced in nNOS−/− mice compared to control mice, but not decreased when the vas deferens were obtained from either eNOS−/− or iNOS−/− mice. Pre-incubation of the vas deferens with tetrodotoxin (1 μM) significantly reduced the basal release of 6-ND from control, eNOS−/−, and iNOS−/− mice but had no effect on the basal release of 6-ND from nNOS−/− mice. EFS-induced frequency-dependent contractions of the vas deferens, which were significantly reduced when the tissues obtained from control, eNOS−/− and iNOS−/− mice, were pre-incubated with l-NAME, but unaltered when the vas deferens was obtained from nNOS−/− mice. In addition, the EFS-induced contractions were significantly smaller when the vas deferens were obtained from nNOS−/− mice.The results clearly demonstrate that nNOS is the main NO isoform responsible for 6-ND release in mouse vas deferens and reinforces the concept of 6-ND as a major modulator of vas deferens contractility.

6-Nitrodopamine potentiates contractions of rat isolated vas deferens induced by noradrenaline, adrenaline, dopamine and electric field stimulation.

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that is released from the rat isolated vas deferens, and has been characterized as a major modulator of the contractility of rat isolated epididymal vas deferens (RIEVD). Drugs such as tricyclic antidepressants, α1 and β1β2 adrenoceptor blockers, act as selective antagonists of the 6-ND receptor in the RIEVD. In the rat isolated atria, 6-ND has a potent positive chronotropic action and causes remarkable potentiation of the positive chronotropic effects induced by dopamine, noradrenaline, and adrenaline. Here, whether 6-ND interacts with the classical catecholamines in the rat isolated vas deferens was investigated. Incubation with 6-ND (0.1 and 1 nM; 30min) caused no contractions in the RIEVD but provoked significant leftward shifts in the concentration-response curves to noradrenaline, adrenaline, and dopamine. Pre-incubation of the RIEVD with 6-ND (1 nM), potentiated the contractions induced by electric-field stimulation (EFS), whereas pre-incubation with 1 nM of dopamine, noradrenaline or adrenaline, did not affect EFS-induced contractions. In tetrodotoxin (1 μM) pre-treated (30 min) RIEVD, pre-incubation with 6-ND (0.1 nM) did not cause leftward shifts in the concentration-dependent contractions induced by noradrenaline, adrenaline, or dopamine. Pre-incubation of the RIEVD with the α2A-adrenoceptor antagonist idazoxan (30 min, 10 nM) did not affect dopamine, noradrenaline, adrenaline, and EFS-induced contractions. However, when idazoxan (10 nM) and 6-ND (0.1 nM) were simultaneously pre-incubated (30 min), a significant potentiation of the EFS-induced contractions of the RIEVD was observed. 6-nitrodopamine causes remarkable potentiation of dopamine, noradrenaline, and adrenaline contractions on the RIEVD, due to activation of adrenergic terminals, possibly via pre-synaptic adrenoceptors.

6-nitrodopamine is a major endogenous modulator of human vas deferens contractility.

Rat isolated vas deferens releases 6-nitrodopamine (6-ND), and the spasmogenic activity of this novel catecholamine is significantly reduced by tricyclic compounds such as amitriptyline, desipramine, and carbamazepine and by antagonists of the α1 -adrenergic receptors such as doxazosin, tamsulosin, and prazosin. To investigate the liberation of 6-ND by human epididymal vas deferens (HEVDs) and its pharmacological actions. The in vitro liberation of 6-ND, dopamine, noradrenaline, and adrenaline from human vas deferens was evaluated by LC-MS/MS. The contractile effect of the catecholamines in HEVDs was investigated in vitro. The action of tricyclic antidepressants was evaluated on the spasmogenic activity ellicited by the catecholamines and by the electric-field stimulation (EFS). The tissue was also incubated with the inhibitor of nitric oxide (NO) synthase L-NAME and the release of catecholamines and the contractile response to EFS were assessed. 6-ND is the major catecholamine released from human vas deferens and its synthesis/release is inhibited by NO inhibition. The spasmogenic activity elicited by EFS in the human vas deferens was blocked by tricyclic antidepressants only at concentrations that selectively antagonize 6-ND induced contractions of the human vas deferens, without affecting the spasmogenic activity induced by dopamine, noradrenaline, and adrenaline in this tissue. Incubation of the vas deferens with L-NAME reduced both the 6-ND release and the contractions induced by EFS. 6-ND should be considered a major endogenous modulator of human vas deferens contractility and possibly plays a pivotal role in the emission process of ejaculation. It offers a novel and shared mechanism of action for tricyclic antidepressants and α1 -adrenergic receptor antagonists.

Chronic Effects of Atypic Antipsychotics on Mice Isolated Vas Deferens Contractility: An In vitro Study

Aims: Antipsychotic drugs are known to be commonly associated with sexual dysfunction. The aim of this study to investigate the effects of sertindole, asenapine and ziprasidone on serotonin (5-HT), noradrenaline (NA), adenosine triphosphate (ATP) and potassium chloride (KCl)-induced contractions of the isolated vas deferens in mice.
 Study design: All the drugs were administered intraperitoneally (i.p.) in a volume of 0.1 ml per 10 g body weight of mice.
 Place and Duration of Study: Department of Pharmacology, Kocaeli University, Animal Research Center, between May 2018 and August 2019.
 Methodology: The mice were randomly divided into groups (n=7) as follows: saline; sertindole 1.3 mg/kg; sertindole 2.5 mg/kg; asenapine 0.05 mg/kg; asenapine 0.075 mg/kg; ziprasidone 1 mg/kg; ziprasidone 2 mg/kg once daily for 21 days. Mice receiving only the vehicle (0.9% saline, i.p.) served as control group. After 21 days of treatment, the effects of drugs were investigated on 5-HT, NA, ATP and KCl-induced contractions of isolated vas deferens.
 Results: The results showed that serotonin-induced contractions of vas deferens were affected by the chronic treatments of sertindole, asenapine and ziprasidone, however these drugs had no significant effect on NA, ATP, and KCl-induced contractions of mice vas deferens.
 Conclusion: Serotonergic receptors may contribute to changes in vas deferens contractions in mice with chronic treatment of sertindole, asenapine and ziprasidone. Thus, our results may explain one of the causes of sexual dysfunction of sertindole, asenapine and ziprasidone.

Chronic Effects of Loxapine, Iloperidone, Paliperidone on Mice Isolated Vas Deferens Contractility

Objective: Erectile dysfunction is a usual side effect of antipsychotic medications; this causes patients to avoid using drugs. The aim of this study to investigate the effects of iloperidone, paliperidone and loxapine on serotonin (5-HT), noradrenaline (NA), adenosine triphosphate (ATP) and potassium chloride (KCl)-induced contractions of the vas deferens in mice.Materials and Methods: The mice were randomly divided into experimental groups and treated by ip injection of drugs for 21 days. After the treatment, the effects of drugs were investigated on 5-HT, ATP, NA and KCl-induced contractile responses in the epididymal and prostatic portions of mice vas deferens strips. Results: 5-HT-induced contractile responses were significantly increased while ATP-induced contractile responses were significantly decreased in the both portions of the vas deferens obtained from the iloperidone, paliperidone and loxapine-treated groups. However, these drugs had no significant effect on NA- and KCl-induced contractions of mice vas deferens. Conclusion: These results showed that serotonin and ATP-induced contractions of vas deferens were affected by the chronic treatments of iloperidone, paliperidone, and loxapine. In mice chronically treated with these drugs, serotonergic and purinergic receptors may contribute to changes in vas deferens contractions that cause erectile dysfunction.

Alpha1-adrenergic antagonists block 6-nitrodopamine contractions on the rat isolated epididymal vas deferens

6-nitrodopamine (6-ND) is released from rat isolated vas deferens and modulates electrical-field stimulation (EFS) contractions of the rat isolated epididymal vas deferens (RIEVD) via a specific receptor which is blocked by tricyclic antidepressants. Here, the effects of selective α1-adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline and EFS were investigated. Doxazosin and tamsulosin (3–10 nM) caused significant rightward shifts of the concentration-response curve to 6-ND, but had no effect on dopamine-, noradrenaline- and adrenaline-induced contractions. Alfuzosin (10 nM) produced rightward shifts on concentration-response curves to all catecholamines. Silodosin (10 nM) and terazosin (100 nM) displaced to the right the noradrenaline, dopamine and adrenaline curves, but higher concentrations of both antagonists (100 and 300 nM, respectively) were required to displace the 6-ND curves. The EFS-induced contractions were significantly inhibited only at the concentrations that the α1-adrenergic receptor antagonists caused rightward shifts on the 6-ND concentration-response curves. The inhibition of EFS-induced contractions by doxazosin (10 nM), tamsulosin (10 nM), alfuzosin (10 nM), silodosin (100 nM) and terazosin (300 nM), were not observed in RIEVD obtained from animals chronically treated with L-NAME. This work demonstrates that α1-adrenoceptor antagonists act as 6-ND receptor antagonists in RIEVD, opening the possibility that many actions previously attributed to noradrenaline could be due to 6-ND antagonism. In addition, blockade of the 6-ND receptors by both tricyclic antidepressants and α1-adrenergic receptor antagonists may represent the common mechanism of action responsible for their therapeutic use in the treatment of premature ejaculation.

6-Nitrodopamine is an endogenous mediator of rat isolated epididymal vas deferens contractions induced by electric-field stimulation

6-nitrodopamine (6-ND) is released from human umbilical cord vessels and modulates vascular reactivity by acting as a dopamine antagonist. Here we investigate whether 6-ND is released by the rat isolated vas deferens and its effect on this tissue. Dopamine, noradrenaline, adrenaline and 6-ND levels were quantified in rat isolated vas deferens by LC-MS-MS. Electric-field stimulation (EFS) and concentration-response curves to 6-ND, noradrenaline, dopamine and adrenaline were performed in the absence and in the presence (30 min) of L-NAME, SCH-23390, haloperidol, PG-01037, sonepiprazole, desipramine, clomipramine, amitriptyline, cyclobenzaprine, carbamazepine, maprotiline, paroxetine, oxcarbazepine and ketanserin in the rat isolated epididymal vas deferens (RIEVD).Basal releases of 6-ND and noradrenaline were detected from the rat isolated vas deferens. 6-ND release was reduced by tissue incubation with L-NAME and from the vas deferens obtained from L-NAME-treated rats.SCH-23390 caused leftward shifts on concentration-response curves to 6-ND without affecting dopamine- or EFS-induced RIEVD contractions. Haloperidol, PG-01037 and sonepiprazole caused significant rightward shifts on concentration-response curves to dopamine but had no effect on either the 6-ND or EFS-induced RIEVD contractions.The tricyclic compounds desipramine, clomipramine, amitriptyline, cyclobenzaprine and carbamazepine induced rightward shifts on 6-ND concentration-response curve but did not reduce the noradrenaline, dopamine and adrenaline contractile responses. They also reduced the EFS-induced RIEVD contractions in control but not in tissues obtained from L-NAME-treated animals. Maprotiline, oxcarbazepine, paroxetine and ketanserin had no effect in either 6-ND or EFS-induced RIEVD contractions.Thus, 6-ND modulates RIEVD contractility, and desipramine, clomipramine, amitriptyline, cyclobenzaprine and carbamazepine act as selective 6-ND receptor antagonists.

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.

Effects of long-term treatment with haloperidol, clozapine and aripiprazole on mice isolated vas deferens.

Sexual dysfunction is a common condition in patients taking antipsychotics and is the most bothersome symptom and adverse drug effect, resulting in a negative effect on treatment compliance. Pharmacology research into human ejeculatory disorders is limited to clinical studies with registered drugs affecting the ejaculation process; therefore, animal research has become the need. We aimed to investigate the effects of haloperidol, clozapine and aripiprazole on serotonin, noradrenaline, adenosine triphosphate (ATP) and potassium chloride (KCl)-induced contractions of the vas deferens in order to evaluate the effect of haloperidol, clozapine and aripiprazole on the contraction of the vas deferens. Male inbred BALB/c ByJ mice aged 7weeks upon arrival to the laboratory were used in this study. Haloperidol, clozapine, aripiprazole, serotonin, noradrenaline, ATP and KCl were dissolved in 0.9% physiological saline. The mice were randomly divided into experimental groups as follows: saline; haloperidol 0.125mg/kg; haloperidol 0.25mg/kg; clozapine 1.25mg/kg; clozapine 2.5mg/kg; aripiprazole 3mg/kg; aripiprazole 6mg/kg. Mice were treated by ip injection of drugs during 21days. Mice receiving only the vehicle ip (0.9% saline) during 21days served as control group (n=7). Each experimental group consisted of 7 mice. After 21days of treatment, epididymal and prostatic portions of vas deferens were surgically dissected free and immersed in 20-mL organ baths containing Krebs' solution. The effects of chronic treatment with haloperidol (0.125 and 0.25mg/kg), clozapine(1.25 and 2.5mg/kg) and aripiprazole (3 and 6mg/kg) were investigated on serotonin [10 (-8) to 10 (-4) M], noradrenaline [10 (-8) to 10 (-4) M], ATP [10 (-8) to 10 (-4) M] and 80mM KCl-induced contractile responses in the epididymal and prostatic portions of mice isolated vas deferens strips. Statistical comparison between the groups was performed using ANOVA supported by Dunnett's post hoc test. Serotonin-induced contractile responses were significantly increased in the epididymal and prostatic portions of the vas deferens obtained from the haloperidol-treated group and clozapine-treated group. However, aripiprazole treatment had no effect on serotonin responses in both epididymal and prostatic portions of mice vas deferens. On the other hand, haloperidol and clozapine treatments significantly inhibited both noradrenaline and ATP-induced contractions of the prostatic and epididymal portions of the mice vas deferens, but had no effect on KCl-induced contractions of the vas deferens in both portions. There were no significant differences in KCl-induced contractile responses among the groups. These results revealed that induced contractions of vas deferens were affected after chronic treatment with haloperidol and clozapine but not aripiprazole. Serotonergic, noradrenergic and purinergic receptors may, at least in part, contribute to changes in vas deferens contractions in mice with chronic treatment of haloperidol and clozapine but not aripiprazole.

Enantiospecific Allosteric Modulation of Cannabinoid 1 Receptor.

The cannabinoid 1 receptor (CB1R) is one of the most widely expressed metabotropic G protein-coupled receptors in brain, and its participation in various (patho)physiological processes has made CB1R activation a viable therapeutic modality. Adverse psychotropic effects limit the clinical utility of CB1R orthosteric agonists and have promoted the search for CB1R positive allosteric modulators (PAMs) with the promise of improved drug-like pharmacology and enhanced safety over typical CB1R agonists. In this study, we describe the synthesis and in vitro and ex vivo pharmacology of the novel allosteric CB1R modulator GAT211 (racemic) and its resolved enantiomers, GAT228 (R) and GAT229 (S). GAT211 engages CB1R allosteric site(s), enhances the binding of the orthosteric full agonist [3H]CP55,490, and reduces the binding of the orthosteric antagonist/inverse agonist [3H]SR141716A. GAT211 displayed both PAM and agonist activity in HEK293A and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in mouse-brain membranes rich in native CB1R. GAT211 also exhibited a strong PAM effect in isolated vas deferens endogenously expressing CB1R. Each resolved and crystallized GAT211 enantiomer showed a markedly distinctive pharmacology as a CB1R allosteric modulator. In all biological systems examined, GAT211's allosteric agonist activity resided with the R-(+)-enantiomer (GAT228), whereas its PAM activity resided with the S-(-)-enantiomer (GAT229), which lacked intrinsic activity. These results constitute the first demonstration of enantiomer-selective CB1R positive allosteric modulation and set a precedent whereby enantiomeric resolution can decisively define the molecular pharmacology of a CB1R allosteric ligand.

Antipsychotic-like activity of scopoletin and rutin against the positive symptoms of schizophrenia in mouse models.

In an earlier report, we demonstrated an antipsychotic-like activity of a methanolic extract of Morinda citrifolia Linn fruit in mouse models and postulated the contribution of its bioactive principles, scopoletin and rutin. Moreover, the antidopaminergic activities of scopoletin and rutin were reported in isolated vas deferens preparations. In the present study, scopoletin and rutin were assessed for antipsychotic-like activity using apomorphine-induced climbing behavior and methamphetamine-induced stereotypy in mice. The results of this study revealed that scopoletin and rutin (0.05, 0.1, 0.5, and 1 mg/kg, p.o.) had a “U-shaped” dose-dependent effect on climbing and stereotyped behaviors induced by apomorphine and methamphetamine, respectively, in mice. A significant reduction in climbing and stereotyped behaviors caused by scopoletin and rutin was observed only at a dose 0.1 mg/kg. This study suggests that scopoletin and rutin can alleviate positive symptoms of schizophrenia only at a specific dose. Further studies evaluating the effects of scopoletin and rutin on animal models for negative symptoms of schizophrenia are required for a novel drug discovery in the treatment of neuropsychiatric diseases.

Regulation of nerve-evoked contractions of rabbit vas deferens by acetylcholine.

Stimulation of intramural nerves in the vas deferens of many species yields a classical biphasic contraction comprised of an initial fast component, mediated by P2X receptors and a second slower component, mediated by α1-adrenoceptors. It is also recognized that sympathetic nerve-mediated contractions of the vas deferens can be modulated by acetylcholine (Ach), however there is considerable disagreement in the literature regarding the precise contribution of cholinergic nerves to contraction of the vas deferens. In this study we examined the effect of cholinergic modulators on electric field stimulation (EFS)-evoked contractions of rabbit vas deferens and on cytosolic Ca2+ levels in isolated vas deferens smooth muscle cells (VDSMC). The sustained component of EFS-evoked contractions was inhibited by atropine and by the selective M3R antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP). EFS-evoked contractions were potentiated by Ach, carbachol (Cch), and neostigmine. The sustained phase of the EFS-evoked contraction was inhibited by prazosin, an α1-adrenoceptor antagonist and guanethidine, an inhibitor of noradrenaline release, even in the continued presence of Ach, Cch or neostigmine. The soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one enhanced the amplitude of EFS-evoked contractions and reduced the inhibitory effects of 4-DAMP. Isolated VDSMC displayed spontaneous Ca2+ oscillations, but did not respond to Cch. However, the α1-adrenoceptor agonist, phenylephrine, evoked a Ca2+ transient and contracted the cells. These data suggest that EFS-evoked contractions of the rabbit vas deferens are potentiated by activation of M3 receptors and reduced by activation of a sGC-dependent inhibitory pathway.

Interpositional substitution of free vas deferens segment autografts in rat: feasibility and potential implications

BackgroundInsufficient vas length for performing a tension-free vasovasostomy is a problem occasionally encountered by microsurgeons. Herein we evaluated utilization of a non-vascularized vas deferens autograft in a rat model.MethodsSegments of isolated vas deferens, 2.5 cm in length, were used as bilateral autografts in 15 rats. Each autograft was implanted between the two transected ends of vas deferens using end-to-end anastomosis. Fertility, sperm motility, and graft survival was evaluated and compared with the control group.ResultsAt the end of the 3 months, 9/15 (60%) rats were able to breed successfully and 24 (80%) vas grafts were patent and viable. Large granulomata developed at the proximal anastomosis sites in 6 (20%) autografts that failed. Unilateral minimal fluid leakage was observed in 6 (20%) of the proximal (testicular end) anastomosis sites in those rats that were able to breed. Histological evaluations demonstrated that graft survival was associated with mild to severe changes in the structure of the vas autograft. On semen analysis 76% of the sperms in the experimental group had forward motility compared to 78% in the control group (p > 0.05).ConclusionsVas autograft can successfully be performed in a rat model with ultimate breeding capability.

Effect of Boerhaavia diffusa in experimental prostatic hyperplasia in rats

Objective:Present investigation was undertaken to study the effectiveness of hydroalcoholic extract of roots of Boerhaavia diffusa in experimental benign prostatic hyperplasia (BPH) in rats using various animal models.Materials and Methods:BPH in rats was induced by subcutaneous injection of testosterone (5 mg/kg) daily for 28 days. Rats were divided in to five groups (six rats each). A negative control group received arachis oil (1 ml/kg s.c.) and four groups were injected testosterone. These four groups were further divided into reference group (finasteride 1 mg/kg), model group (testosterone), study group A (B. diffusa 100 mg/kg), and study group B (B. diffusa 250 mg/kg). On the 29th day, rats were sacrificed and body weight, prostate weight, bladder weight, and serum testosterone level were measured and histological studies were carried out. Further in vitro analysis of B. diffusa extract on contractility of isolated rat vas deferens and prostate gland, produced by exogenously administered agonists were carried out. All results were expressed as mean ± SEM. 0 Data were analyzed by one-way analysis of variance followed by Tukey's test.Results:B. diffusa (100 mg/kg) treatment for 28 days resulted in significant inhibition of prostate growth (P < 0.05). Drug extract did not have significant change on serum testosterone level. Histopathological analysis of prostate gland supported above results. Results of in vitro experiment suggest that extracts had attenuated the contractile responses of isolated vas deferens and prostate gland to exogenously applied agonists.Conclusion:The results suggested that treatment with B. diffusa may improve symptoms of disease and inhibit the increased prostate size. In vitro study implies that herbal extracts has the machinery to produce beneficial effect on prostatic smooth muscle, which would relieve the urinary symptoms of disease. B. diffusa could be a potential source of new treatment of prostatic hyperplasia.

Effects of Angiogenesis Inhibition by Spironolactone on Isolated Vas Deferens Contractility in an Experimental Varicocele Model in Rats

To investigate the effect of spironolactone, as an angiogenesis inhibitor, on the isometric contractile responses in isolated vas deferens strips from left varicocele-induced rats. Twenty-four adult (12-14 months) male Wistar albino rats were randomly assigned to 4 groups (n = 6 in each): (1) Control group, (2) sham-operated group, (3) experimental left varicocele group, and (4) Spironolactone (20 mg/kg/d)-treated experimental left varicocele group. Histopathologic and immunohistochemical (CD31 staining) findings in the rat testis and functional findings in the rat isolated vas deferens were investigated. Angiogenesis increased in the varicocele group and the spironolactone inhibited angiogenesis in the spironolactone-treated group. Spironolactone seemed to change phenylephrine and serotonin responses in the left vas deferens. It is possible that by inhibiting angiogenesis, spironolactone treatment negatively impairs testicular morphology and functional (vas deferens) pathways. Varicocele formation seems to elicit an increase to 5-HT sensitivity in rat vas deferens, and this process is prevented by spironolactone pretreatment.

Muscarinic properties of compounds related to arecaidine propargyl ester.

A series of new analogues of the arecaidine propargyl ester (CAS 35516-99-5), APE, 1a) with alcohols consisting of 4 or 5 carbon atoms were investigated at muscarinic receptor subtypes. The muscarinic activity of the quaternary and tertiary salts of the APE-related compounds were assayed on the isolated guinea-pig ileum (M3 receptor subtype) and guinea-pig left atria (M2 receptor subtype) as well as on rabbit isolated vas deferens (M1 receptor subtype). The structural variations made in the APE molecule, replacing the triple bond in the ester side chain with structures such as double bond, an allene moiety, a single bond, a cyclopropyl group or two triple bonds should alter the selectivity and potency in favour of the M2 subtype. Enhanced, though modest, selectivity for M2 receptors was achieved with the 2-butynyl ester 2a. The other structural variations resulted in a loss of potency, but not necessarily of efficacy.