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.

BACKGROUND: RS17053 is classed as an α1A -adrenoceptor selective antagonist. OBJECTIVES: We have examined its profile of action at all subtypes of α1 -adrenoceptor. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

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.

Stereoselectivity of butylidenephthalide on non-adrenergic prejunctional voltage-dependent Ca(2+) channels in prostatic portion of rat vas deferens.

The naturally occurring and synthetic butylinenephthalide (Bdph) has two geometric isomers. Z- and E-Bdph were reported to have geometric stereoselectivity for voltage-dependent calcium channels (VDCCs) in guinea-pig ileum. The aim of this study was to investigate whether the binding of Z- and E-Bdph on prejunctional VDCCs of rat vas deferens (RVD) is stereoselective. The twitch responses to electrical field stimulation (EFS, supramaximal voltage, 1 ms, 0.2Hz) were recorded on a polygraph. Z- and E-Bdph concentration-dependently inhibited the twitch responses to EFS in full tissue, prostatic portion and epididymal portion of RVD. The pIC50 value of Z-Bdph was greater than that of E-Bdph in the electrically stimulated prostatic portion of RVD, suggesting that the binding of Bdph on the non-adrenergic prejunctional VDCCs of cell membrane is stereoselective. In the prostatic portion, exogenous Ca(2+) only partially reversed the twitch inhibition by Z-Bdph, but effectively reversed those by Ca(2+) channel blockers, such as verapamil, diltiazem and aspaminol, suggesting that the action mechanisms may be different from those of Ca(2+) channel blockers. K(+) channel blockers, such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP), may prolong duration of action potential to allow greater Ca(2+) entry and induced more release of transmitters. Therefore both blockers via their prejunctional actions reversed the twitch inhibition induced by Z-Bdph in all preparations of RVD by a non-specific antagonism.

Effects of desipramine on prazosin potency at α1A- and α1D-adrenoceptors in rat vas deferens: implications for the α1L-adrenoceptor subclassification.

This study investigates the interaction between cocaine, desipramine and prazosin at α1-adrenoceptor subtypes mediating contractions to noradrenaline in epididymal portions of rat vas deferens. Noradrenaline potency was not significantly affected by desipramine (0.1-1.0 µM) and reduced by desipramine (10 µM), but was increased by the presence of cocaine (3.0-30 µM), particularly in terms of phasic contractions to low concentrations of noradrenaline. In vehicle experiments, prazosin exhibited relatively low potency as an antagonist against the predominantly α1A-adrenoceptor mediated response (pKB 8.50). In the presence of cocaine, prazosin exhibited higher potency against the revealed α1D-adrenoceptor mediated component (e.g. pKB 9.12). In the presence of desipramine, the potency of prazosin was either unchanged or indeed decreased. Cocaine (0.3-30 µM) significantly increased the single pulse nerve-stimulation-evoked contraction, with a maximum increase to 156±12% of control (n=9). In contrast, desipramine in low concentrations (0.1-0.3 µM) produced a small but significant increase to 126.6±5.5% (n=11), but higher concentrations failed to increase the response. In conclusion, desipramine fails to produce sufficient noradrenaline transporter block in low concentrations (0.1 µM) and produces α1-adrenoceptor antagonism in slightly higher concentrations (0.3-1 µM), and so is unsuitable for use in α1-adrenoceptor subclassification studies. Contractions of rat vas deferens are mediated by α1A- and α1D-adrenoceptors, and prazosin has selectivity for α1D- over α1A-adrenoceptors. The α1L-adrenoceptor previously identified in rat vas deferens is the native α1A-adrenoceptor. The range of prazosin potencies and receptor subtypes previously reported in rat vas deferens may be explained by the choice of cocaine or desipramine as noradrenaline transporter blocker.

Huwentoxin-XVI, an analgesic, highly reversible mammalian N-type calcium channel antagonist from Chinese tarantula Ornithoctonus huwena.

N-type calcium channels play important roles in the control of neurotransmission release and transmission of pain signals to the central nervous system. Their selective inhibitors are believed to be potential drugs for treating chronic pain. In this study, a novel neurotoxin named Huwentoxin-XVI (HWTX-XVI) specific for N-type calcium channels was purified and characterized from the venom of Chinese tarantula Ornithoctonus huwena. HWTX-XVI is composed of 39 amino acid residues including six cysteines that constitute three disulfide bridges. HWTX-XVI could almost completely block the twitch response of rat vas deferens to low-frequency electrical stimulation. Electrophysiological assay indicated that HWTX-XVI specifically inhibited N-type calcium channels in rat dorsal root ganglion cells (IC50 ∼60 nM). The inhibitory effect of HWTX-XVI on N-type calcium channel currents was dose-dependent and similar to that of CTx-GVIA and CTx-MVIIA. However, the three peptides exhibited markedly different degrees of reversibility after block. The toxin had no effect on voltage-gated T-type calcium channels, potassium channels or sodium channels. Intraperitoneal injection of the toxin HWTX-XVI to rats elicited significant analgesic responses to formalin-induced inflammation pain. Toxin treatment also changed withdrawal latency in hot plate tests. Intriguingly, we found that intramuscular injection of the toxin reduced mechanical allodynia induced by incisional injury in Von Frey test. Thus, our findings suggest that the analgesic potency of HWTX-XVI and its greater reversibility could contribute to the design of a novel potential analgesic agent with high potency and low side effects.

Prazosin has low potency at α1A-adrenoceptors and high potency at α1D -adrenoceptors in rat vas deferens.

(1) We have investigated α1 -adrenoceptor subtypes mediating contractions to noradrenaline in epididymal portions of rat vas deferens. (2) Contractions to noradrenaline were investigated in the absence or presence of the noradrenaline transporter blocker cocaine. (3) In the absence of cocaine, contractions to noradrenaline were potently antagonized by RS100329, but not by BMY7378, and so are mediated mainly by α1A -adrenoceptors. (4) In the presence of cocaine, noradrenaline potency was increased, particularly in terms of low concentrations and phasic contractions. Contractions to low concentrations of noradrenaline in the presence of cocaine were resistant to RS100329 but potently antagonized by BMY7378, demonstrating that α1D-adrenoceptors are additionally involved in contractions amplified by cocaine. (5) In the absence of cocaine, prazosin exhibited relatively low potency as an antagonist against the α1A-adrenoceptor-mediated component to the response. In the presence of cocaine, prazosin exhibited higher potency against the α1D-adrenoceptor-mediated component. (6) In conclusion, prazosin has previously unreported selectivity for α1D-over α1A -adrenoceptors in functional studies of rat vas deferens. Contractions of rat vas deferens are mediated by α1A-and α1D -adrenoceptors. The range of prazosin potencies and of receptor subtypes previously reported in rat vas deferens may be explained by the presence of these two subtypes.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

Relaxant effect of the aqueous extract of Erythrina vellutina leaves on rat vas deferens

The effect of the Aqueous Extract from the leaves of Erythrina vellutina (AE) on rat vas deferens preparation was evaluated in this work. The AE inhibited the muscle contractions induced by electrical field stimulation (EFS) in a concentration-dependent manner. This inhibition was not affected by atropine (10-5M), propanolol (10-5M), prazosin (10-5M) or yohimbine (10-5M), suggesting that there is no direct interaction of the AE with cholinergic nor adrenergic receptors. Incubation of vas deferens with the K+ channel antagonists, tetraethylamonium (10-6M) or 4-aminopyridine (10-6M) had also no effect on the AE-induced inhibition. On the other hand, glibenclamide (10-6) significantly attenuated the effect of the AE, suggesting a possible involvement of ATP-dependent K+ channels. The AE (0.15 mg/mL) did not alter the contractions induced by noradrenaline (10-5M), ATP (10-4M) nor KCl (80 mM), against an interaction of the extract with post-synaptic sites. The data presented suggests that the inhibition of the electrically driven muscle twitches by the AE could be due to a pre-synaptic interaction of the extract with ATP-dependent K+ channels from vas deferens sympathetic neurons.

O objetivo deste trabalho foi avaliar o efeito do extrato aquoso das folhas de Erythrina vellutina (AE) sobre ducto deferente de rato. Nesta preparação, o AE inibiu as contrações induzidas por estímulo elétrico de campo de maneira dependente da concentração. Esta inibição não foi afetada após atropina (10-5M), propanolol (10-5M), prazosin (10-5M) ou yohimbina (10-5M), sugerindo uma ação indireta do AE sobre receptores colinérgicos ou adrenérgicos. A incubação da preparação com os antagonistas de canais de K+, tetraetilâmonio (10-6M) ou 4-aminopiridina (10-6M) não alterou o efeito inibitório induzido pelo AE. Entretanto, a glibenclamida (10-6M) atenuou significantemente este efeito, sugerindo um possível envolvimento de canais de K+ dependentes de ATP. Além disso, o AE (0.15 mg/mL) não alterou as contrações induzidas por noradrenalina (10-5M), ATP (10-4M) ou KCl (80 mM), descartando uma interação do AE com um sítio pós-sináptico. Em conclusão, estes resultados demonstram que o efeito inibitório do AE pode ser devido a uma interação pré-sináptica com canais de K+ dependentes de ATP em neurônios simpáticos de ducto deferente de rato.

The effects of vasectomy on the autonomic innervation of the rat vas deferens / 대한비뇨기과학회지

Immunohistochemical techniques have been employed to observe the arrangement and distribution or the autonomic nervous systems in the normal rat vas deferens and to observe periodic alterations or the innervation patterns following surgical division of the vas. 18 male Sprague-Dawley rat were divided into 6 groups: normal control group, 1 week after vasectomy group, 3 weeks after vasectomy group. 5 weeks after vasectomy group. 8 weeks after vasectomy group and 16 weeks after vasectomy group. Samples from sites on the testicular and urethral sides of the original vasectomy have been compared with the other group specimens as to the arrangement and distribution of autonomic nerves containing vasoactive intestinal polypeptide (VIP) and catecholamine. The VIPergic and adrenergic innervation patterns of the normal control group is almost identical with that of a normal human vas. In contrast with tissues from the testicular portions and from the controls. the testicular specimens were devoid of autonomic nerve fibers, which were neither re-innervated nor re distributed in every vasectomy group. In the urethral portion of the vas, it was observed that the adrenergic nerve fibers had few changes after vasectomy and the VIPergic nerve fibers became thicker from 3 weeks after vasectomy. showing axonal growth into the smooth muscle coat from 6 weeks after it. and showing axonal growth into the deep muscle coat from 16 weeks after it. These findings indicate firstly that the autonomic nerve fibers run from the urethral portion towards the testis ; secondly that VIPergic nerve fibers have high a possibility to be re-innervated by axonal growth from the urethral portion to the testicular portion after vasovasostomy; thirdly that injuring autonomic nerves to the vas deferens during vasectomy could result in poor function of the vas after vasovasostomy in relation to the contractile and secretory activities of the vas that may be of importance to the maturation and fertility of spermatozoa.