Contractions Of Vas Deferens Research Articles

Inhibitory effect of mitragynine, an analgesic alkaloid from Thai herbal medicine, on neurogenic contraction of the vas deferens

The effect of an indole-alkaloid mitragynine isolated from the Thai medicinal herb kratom ( Mitragyna speciosa) on neurogenic contraction of smooth muscle was studied in guinea-pig vas deferens. Mitragynine inhibited the contraction of the vas deferens produced by electrical transmural stimulation. On the other hand, mitragynine failed to affect the responses to norepinephrine and ATP. Mitragynine did not reduce KCl-induced contraction in the presence of tetrodotoxin, prazosin and α,β-methylene ATP. Mitragynine inhibited nicotine- or tyramine-induced contraction. By using the patch-clamp technique, mitragynine was found to block T- and L-type Ca 2+ channel currents in N1E-115 neuroblastoma cells. In the Ca 2+ measurement by a fluorescent dye method, mitragynine reduced KCl-induced Ca 2+ influx in neuroblastoma cells. The present results suggest that mitragynine inhibits the vas deferens contraction elicited by nerve stimulation, probably through its blockade of neuronal Ca 2+ channels.

Spasmolytic Effect of Vitis Vinifera Leaf Hydroalcoholic Extract on Rat's Isolated Vas Deferens

Introduction: Many reports have shown that the grape (Vitis vinifera) seed extract induces antioxidant, hypotensive hypolipidemic, and vasodilatory effects. We have recently shown the relaxatory effect of grape leaf extract on rat's aorta, ileum, uterus and frog's heart rate and contractility. The objective of this study was to investigate the spasmolytic effect of Vitis Vinifera Leaf Hydroalcoholic extract (VLHE) on rat's isolated vas deferens. Materials & Methods: Vas deferens of male adult rat (Sprague Dawley) was removed, placed in an organ bath containing Tyrode’s solution (30oC, pH 7.4) and the bath bubbled with air. Then its contractions under 1 g initial tension, were recorded isometrically. The vas deferens was contracted either by KCl (80 mM) or adrenaline (2 μg/ml). Results: The results demonstrated that the VLHE at 1, 2, 4 and 8 mg/ml reduced the vas deferens contractions evoked by KCl (80 mM) significantly (P<0.0001) and dose-dependently. VLHE (1, 2 and 4 mg/ml) also reduced the adrenaline-induced vas deferens contractions (P<0.0001). In calcium free Tyrod’s solution, KCl-induced contractions were occurred only after adding normal calcium (1.77 mM) to the organ bath solution. VLHE (3mg/ml) reduced the calcium-induced contraction by KCl too. The VLHE-induced vas deferens relaxation was unaffected by the β- adrenoceptor antagonist (propranolol, 3 μM for 5 min). Nitric oxide synthase inhibitor (L-NAME, 300 μM for 10 min) was unable to modify either KCl-induced contraction or the vas deferens relaxation induced by the extract. Conclusion: These results suggested that the spasmolytic effect of Vitis vinifera leaf hydroalcoholic extract on rat's vas deferens occurred through the blockage of voltage dependent calcium channels (VDCC). Also, neither NO nor β-adrenoceptors were involved in this inhibitory effect of the extract.

Effects of Heptanol on Neurogenic Contractions of Vas Deferens: A Comparative Study of Stimulation Frequency in Guinea-pig and Rat

This study examines the role of gap junctional communication in smooth muscle in relation to the frequency of stimulation and the innervation density of the tissue in the generation of neurogenic contractions. Toward this end the effects of heptanol, a gap junctional blocker, on the neurogenic contractions of guinea pig and rat vas deferens at different frequencies of stimulation (single pulse, 5, 10, 20, 40, 60, and 80 Hz) were studied. In both the prostatic and epididymal halves of these tissues, heptanol abolished the neurogenic contractions at the lower frequencies of stimulation. At higher frequencies, contractions were resistant to heptanol action. The effect of heptanol on the neurogenic contractions was found to decrease with increasing stimulation frequency. The neurogenic contractions of rat vas deferens were more resistant to heptanol than those of guinea pig vas deferens. Our data indicate that gap junctional communication is significant in the generation of neurogenic contractions in both guinea pig and rat vas deferens in a frequency-dependent manner, and we discuss the mechanisms underlying these findings.

Effect of D-004, a Lipid Extract from??the Cuban Royal Palm Fruit, on In??Vitro and In Vivo Effects Mediated by????-Adrenoceptors in Rats

Benign prostatic hyperplasia (BPH) is the non-malignant, uncontrolled growth of glandular and stromal elements of the prostate gland. Lipid extracts from Saw palmetto (Arecaceae) fruits are widely used to treat BPH. The Cuban royal palm (Roystonea regia) is a member of the same family. Previous studies have found that D-004, a lipid extract from the R. regia fruit, administered orally at 200-800 mg/day for 14 days, prevented testosterone- but not dihydrotestosterone-induced prostate hyperplasia in rats. To determine whether D-004 can inhibit noradrenaline (NA) [norepinephrine]- and acetylcholine (ACh)-induced smooth muscle contraction in rat vas deferens and to investigate the in vivo effects of D-004 on NA pressure-elevating effects in rats, an effect mediated by vascular alpha1-adrenoceptors. In vitro effects were investigated by adding D-004 (125-500 microg/mL) to preparations of rat vas deferens suspended in an organ bath containing Tyrode's solution, in which in vitro contractions were induced by NA or ACh. Negative and positive controls containing Tyrode's solution alone or with Saw palmetto extracts (125-500 microg/mL), respectively, were included. To assess the in vivo effects of D-004 on arterial blood pressure, rats were randomly distributed to one of five groups (ten rats/group): these consisted of a negative control group receiving the vehicle, two groups treated with D-004 (400 and 800 mg/kg) and two other groups treated with Saw palmetto (400 and 800 mg/kg). All treatments were orally administered. Rats were anaesthetised with sodium thiopental. Heart rate and blood pressure were registered in baseline conditions. Immediately afterwards, rats were injected intravenously over 5 seconds with successive doses of NA (1, 2 and 4 microg/kg) [0.1mL/100g], with 5 minutes' interval between doses. D-004 and Saw palmetto (125-500 microg/mL) significantly (p < 0.05) and dose dependently inhibited contractions induced by NA in rat vas deferens versus control. D-004 was more effective in inhibiting NA-induced contractions than Saw palmetto. The contractions induced by NA in preparations with D-004 (500 microg/mL) were weaker (p < 0.05) than in preparations containing Saw palmetto (500 microg/mL). At 125 microg/mL, D-004 inhibited the contractions induced by NA 1 and 32 x 10(-6) mol/L by 70.8% and 28.5%, respectively, and Saw palmetto by 56.2% and 10.7%, respectively. At 500 microg/mL, D-004 inhibited these contractions by 100.0% and 71.3%, and Saw palmetto by 80.0% and 42.7%, respectively. The inhibitory concentrations of 50% (IC50) for NA contractions were 148.34 (D-004) and 188.38 (Saw palmetto) microg/mL. D-004 and Saw palmetto significantly (p < 0.05) and to a similar extent inhibited ACh-induced contractions, but less effectively than contractions induced by NA, since at 125 microg/mL they were ineffective. At a dose of 800 mg/kg, but not at 400 mg/kg, D-004 and Saw palmetto inhibited the pressure-elevating effects induced with low (1 microg/kg) but not with high doses (2 and 4 microg/kg) of NA. D-004 and Saw palmetto extracts inhibited in vitro the contractile responses to NA and ACh in rat vas deferens, and were more effective in inhibiting NA than ACh contractions. The in vivo effects of D-004 and Saw palmetto on the hypertensive response induced by NA were significant but modest. These results are preliminary as the relevance of the effects of D-004 on alpha1-adrenoceptors deserves further investigation, including comparative studies versus specific defined alpha1-adrenoceptor antagonists.

Differential antagonism by conotoxin ρ-TIA of contractions mediated by distinct α 1-adrenoceptor subtypes in rat vas deferens, spleen and aorta

The ability of the conotoxin ρ-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of α 1A-adrenoceptors in rat vas deferens, α 1B-adrenoceptors in rat spleen and α 1D-adrenoceptors in rat aorta, and to inhibit the binding of [ 125I]HEAT (2-[[β-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat α 1-adrenoceptors was investigated. ρ-TIA (100 nM to 1 μM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (p A 2∼7.2, n=4). This suggests that ρ-TIA is a competitive antagonist of α 1A- and α 1D-adrenoceptors with no selectivity between these subtypes. Incubation of ρ-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that ρ-TIA is a non-competitive antagonist at α 1B-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of ρ-TIA in inhibiting contractions was examined with similar occupancies (∼25%) at each subtype. Its potency (pIC 50) was 12 times higher in spleen (8.3±0.1, n=4) than in vas deferens (7.2±0.1, n=4) or aorta (7.2±0.1, n=4). In radioligand binding assays, ρ-TIA decreased the number of binding sites ( B max) in membranes from HEK293 cells expressing the rat α 1B-adrenoceptors without affecting affinity ( K D). In contrast, in HEK293 cells expressing rat α 1A- or α 1D-adrenoceptors, ρ-TIA decreased the K D without affecting the B max. It is concluded that ρ-TIA will be useful for distinguishing the role of particular α 1-adrenoceptor subtypes in native tissues.

Facilitation of the Purinergic Contractile Response of the Guinea Pig Vas Deferens by Sodium Orthovanadate

Experiments were carried out to examine the effects of protein tyrosine kinase (PTK) and protein tyrosine phosphatase inhibitors on the purinergic contraction of the guinea pig vas deferens. Sodium orthovanadate (SOV) produced a robust increase of the amplitude of contractions evoked by both neurogenic electrical field stimulation and exogenous ATP. This effect of SOV was concentration- and time-dependent, as well as, reversible and reproducible. Genistein, a PTK inhibitor, but not its inactive structural analog daidzein, inhibited the SOV-induced facilitation of the purinergic contraction. Another PTK inhibitor, 2,5-dihydroxycinnamic acid methyl ester, which is structurally unrelated to genistein, also inhibited the facilitation effects of SOV. Although an application of as low as 3 microM of these inhibitors significantly decreased the effect of SOV, other PTK inhibitors, namely, butein, levandustin C, and thyrphostin 23, were less effective even at concentrations of 100 microM. Western blot experiments showed that the facilitation of the purinergic contraction by SOV is associated with a prominent increase in the level of tyrosine phosphorylation of proteins with estimated molecular sizes of 180 and 123 kDa, which was reversed in the presence of genistein. Evidence is also presented that argue against the possibility that inhibition of the Na(+)/K(+)-ATPase or ATPases, responsible for the clearance of ATP is involved in the SOV-induced facilitation of the purinergic contraction. Together, these results suggest that the responsiveness of the smooth muscle of the vas deferens to the actions of ATP is modulated via a previously unidentified mechanism, which may involve protein tyrosine phosphorylation.

Opioid profiles of Cys 2-containing enkephalin analogues

To elucidate the structural features determining δ-opioid receptor properties of enkephalin analogues containing Cys(O 2NH 2) in position 2, a series of Cys 2-containing derivatives were synthesized and tested for their effectiveness in depressing electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective δ-opioid receptors) and the guinea-pig ileum (μ- and κ-opioid receptors). The peptidase resistance of the compounds was also tested. The ratio IC 50 in the guinea-pig ileum/IC 50 in the mouse vas deferens, indicating selectivity for δ-opioid receptors, was high for Cys(O 2NH 2) 2-containing analogues and especially for [Cys(O 2NH 2) 2, Leu 5]enkephalin, which was about seven times more selective than δ-opioid receptor selective ligand cyclic [ d-Pen 2, d-Pen 5]enkephalin (DPDPE). The dissociation constant ( K A) and relative efficacy ( e rel) of the compounds in the mouse-isolated vas deferens were determined using explicit formulae derived by fitting of the data points with two-parametric hyperbolic function. The obtained values for K A and e rel suggest that: (i) incorporation of Cys(O 2NH 2) 2 in the molecule of [Leu 5]enkephalin highly increases the efficacy and does not change significantly the affinity of the respective analogues to δ-opioid receptors; [Cys(O 2NH 2) 2, Leu 5]enkephalin has higher affinity than DPDPE, but is less resistant to enzyme degradation; the effect of this modification on the efficacy is decreased when methionine is in position 5; (ii) d-configuration of Cys(O 2NH 2) 2-containing analogues increases their peptidase resistance, but reduces efficacy and affinity of the peptides towards δ-opioid receptors; (iii) the substitution of Cys(O 2NH 2) with Hcy(O 2NH 2) reduces the efficacy, affinity and potency of the respective analogues and maintains their sensitivity to endogenous peptidases; (iv) the substitution of the sulfonamide group with benzyl group in the molecule of Cys in position 2 decreases their efficacy and affinity toward δ-opioid receptors, but attaches resistance to enzyme degradation. The results obtained in this study allow: (i) to involve the receptor affinity and agonist efficacy as drug-design consideration for δ-opioid receptor properties of newly synthesized compounds and (ii) to characterize some of the structural features, which set the pattern for their opioid profiles.

SR147778 [5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide], a new potent and selective antagonist of the CB1 cannabinoid receptor: biochemical and pharmacological characterization.

Based on binding, functional, and pharmacological data, this study introduces SR147778 [5-(4-bromophenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide] as a highly potent, selective, and orally active antagonist for the CB1 receptor. This compound displays nanomolar affinity (Ki = 0.56 and 3.5 nM) for both the rat brain and human CB1 recombinant receptors, respectively. It has low affinity (Ki = 400 nM) for both the rat spleen and human CB2 receptors. Furthermore, it shows no affinity for any of the over 100 targets investigated (IC50 > 1 microM). In vitro, SR147778 antagonizes the inhibitory effects of CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] on both the mouse vas deferens contractions (pA2 value = 8.1) and on forskolin-stimulated adenylyl cyclase activity in the U373 MG cell lines (pA2 value = 8.2) but not in Chinese hamster ovary (CHO) cells permanently expressing the human peripheral cannabinoid receptor (hCB2). SR147778 is able to block the mitogen-activated protein kinase activity induced by CP 55,940 in the CHO cell line expressing human brain cannabinoid receptor (IC50 = 9.6 nM) but was inactive in cells expressing hCB2. After oral administration, SR147778 displaced the ex vivo [3H]-CP 55,940 binding to mouse brain membranes (ED50 = 3.8 mg/kg) with a long duration of action, whereas it did not interact with the CB2 receptor expressed in the mouse spleen. Using different routes of administration, SR147778 (0.3-3 mg/kg) is shown to antagonize pharmacological effects (hypothermia, analgesia, and gastrointestinal transit) induced by R-(+)-(2,3-dihydro-5-methyl-3-[[4-morpholinyl]methyl] pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone in mice. Finally, per se, SR147778 (0.3-10 mg/kg) is able to reduce ethanol or sucrose consumption in mice and rats and food intake in fasted and nondeprived rats.

Effects of Echis carinatus Venom on the Haemodynamy and Contractility of Vascular and Visceral Smooth Muscle of Rats

Echis carinatus is considered to be one of the most dangerous snakes in the world because of its venom toxicity and high population densities in rural agriculture areas. However, little information is available on the pharmacological effects of venom from this snake on haemodynamy and vasculature smooth muscle contractility. The haemodynamic alterations induced by the intravenous administration of Echis carinatus venom was investigated in anaesthetized rats. Injection of several doses of venom (1, 3, 5, and 7 μ g kg − 1) decreased the arterial blood pressure. For instance, injection of 3 μ g kg−1 of Echis carinatus venom reduced the mean arterial blood pressure from 107 ± 4 to 71 ± 4 mmHg (p < 0.05). Exposure of the rat isolated vas deferens preparations to Echis carinatus venom (1 μ g ml−1) significantly reduced the contractile responses to epinephrine (10 μ g ml− 1) without changing the response to KCl (50 mM). To investigate the effects of venom on peripheral vascular resistance, mesenteric bed was removed and perfused with Krebs solution. Addition of different concentrations of venom (1, 3, and 10 μ g ml−1) to the precontracted mesenteric bed (with phenylephrine, 5 × 10− 4 M) decreased the contraction in a concentration-dependent manner. However, preincubation of mesenteric bed with atropine (0.1 μ M), L-NAME (10 −5 M) or indomethacin (10−5 M) prevented this effect of the venom. These results may suggest that the inhibitory effect of the venom on nonvascular smooth muscle (such as vas deferens) contractions is a postsynaptic phenomenon. Furthermore, it could be suggested that the cardiovascular effects of Echis carinatus venom are mediated through multiple mechanisms such as activation of muscarinic receptors as well as production of nitric oxide and prostaglandins.

No evidence for activation of alpha(2)-adrenoceptors by methanolic extracts of bovine brain and lung containing clonidine-displacing substance.

Methanolic extracts of bovine brain and lung are capable of displacing [(3)H]-clonidine from alpha(2)-adrenoceptor binding sites, indicating the presence of a clonidine-displacing substance (CDS). We have examined alpha(2)-adrenergic responses and the extracts in three models: [(3)H]-cyclic AMP accumulation in miniprisms of guinea pig cerebral cortex, isometric tension measurements of isolated segments of the rat vas deferens, and porcine palmar lateral vein. The selective alpha(2)-adrenoceptor agonist, 5-bromo-6-2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304) inhibited forskolin-stimulated [(3)H]-cyclic AMP accumulation in the cerebral cortex and elicited contractions of the porcine isolated palmar lateral vein. Clonidine (0.1-30 nM) inhibited neurogenic contractions of the rat vas deferens. Responses to both agonists were inhibited by the alpha(2)-adrenoceptor antagonists, idazoxan or rauwolscine. Brain CDS (5 units/mL) reduced forskolin-stimulated [(3)H]-cyclic AMP accumulation in the guinea pig cerebral cortex, whereas lung CDS (1 unit/mL) increased the accumulation of the cyclic nucleotide. Neither response to the extracts was inhibited by 1 microM idazoxan. Low concentrations of both extracts (0.05 unit/mL) reduced electrically evoked contractions of the rat vas deferens by approximately 20%, but higher concentrations enhanced neurogenic contractions by approximately 50%. Again, the effect of the brain extract was not altered by 1 microM idazoxan. Lung CDS (0.02-1 unit/mL) induced contractions of the porcine palmar lateral vein that were also insensitive to rauwolscine. The results suggest that brain and lung CDS do not activate either central or peripheral alpha(2)-adrenoceptors.

Effect of cyproterone acetate on alpha1-adrenoceptor subtypes in rat vas deferens.

Gonadal hormones regulate the expression of alpha1-adrenoceptor subtypes in several tissues. The present study was carried out to determine whether or not cyproterone acetate, an anti-androgenic agent, regulates the alpha1-adrenoceptor subtypes that mediate contractions of the rat vas deferens in response to noradrenaline. The actions of subtype selective alpha1-antagonists were investigated in vas deferens from control and cyproterone acetate-treated rats (10 mg/day, sc, for 7 days). Prazosin (pA2 approximately 9.5), phentolamine (pA2 approximately 8.3) and yohimbine (pA2 approximately 6.7) presented competitive antagonism consistent with activation of alpha1-adrenoceptors in vas deferens from both control and treated rats. The pA2 values estimated for WB 4101 ( approximately 9.5), benoxathian ( approximately 9.7), 5-methylurapidil (approximately 8.5), indoramin ( approximately 8.7) and BMY 7378 ( approximately 6.8) indicate that alpha1A-adrenoceptors are involved in the contractions of the vas deferens from control and cyproterone acetate-treated rats. Treatment of the vas deferens from control rats with the alpha1B/alpha1D-adrenoceptor alkylating agent chloroethylclonidine had no effect on noradrenaline contractions, supporting the involvement of the alpha1A-subtype. However, this agent partially inhibited the contractions of vas deferens from cyproterone acetate-treated rats, suggesting involvement of multiple receptor subtypes. To further investigate this, the actions of WB 4101 and chloroethylclonidine were reevaluated in the vas deferens from rats treated with cyproterone acetate for 14 days. In these organs WB 4101 presented complex antagonism characterized by a Schild plot with a slope different from unity (0.65 0.05). After treatment with chloroethylclonidine, the complex antagonism presented by WB 4101 was converted into classical competitive antagonism, consistent with participation of alpha1A-adrenoceptors as well as alpha1B-adrenoceptors. These results suggest that cyproterone acetate induces plasticity in the alpha1-adrenoceptor subtypes involved in the contractions of the vas deferens.

Effects of castration and of testosterone replacement on α 1-adrenoceptor subtypes in the rat vas deferens

The contractions of the rat vas deferens in response to noradrenaline are mediated through α 1A-adrenoceptors. We observed participation of α 1B-adrenoceptors in these contractions after castration. We now investigated the time course of this plasticity and the effects of testosterone by determining the actions of competitive antagonists on noradrenaline-induced contractions after 7, 14, 21 and 30 days of castration. BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride) antagonised noradrenaline-induced contractions in control and castrated rats with low p A 2 values (≅6.8). In control vas deferens, WB 4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) had a slope in the Schild plot no different from 1.0, while slopes lower than 1.0 (≈0.6) were observed for vas deferens from castrated rats. Chloroethylclonidine was ineffective in the control vas while it inhibited noradrenaline-induced contractions in vasa from castrated rats and converted the complex antagonism by WB 4101 into simple competitive antagonism. Treatment of castrated rats with testosterone prevented the effects of castration. The results suggest that α 1B-adrenoceptors are detectable in vas deferens from at least the 7th through the 30th day after castration and that testosterone prevents this plasticity.

Members of the acid blue 129 family as potent and selective P2Y‐receptor antagonists

AbstractWe previously reported a structure–activity study which identified Acid blue 129 (1‐amino‐4‐(2,4,6‐trimethylphenylamino)‐9,10‐dioxo‐9,10‐dihydroanthracene‐2‐sulfonic acid) as a highly selective antagonist at the P2Y‐receptor of the guinea pig taenia coli. In contrast to the less selective P2Y‐receptor antagonist Acid blue 25 (1‐amino‐4‐phenylamino‐9,10‐dioxo‐9,10‐dihydroanthracene‐2‐sulfonic acid) with its unsubstituted phenyl ring, Acid blue 129 does not show any effect at the P2X‐receptor of the rat vas deferens. It was hypothesized that the nonplanar orientation of the substituted phenyl ring vs. the anthraquinone core, caused by the buttressing methyl groups in 2′,6′‐position, prevents binding of Acid blue 129 to the P2X‐receptor. To prove this assumption we synthesized new compounds structurally related to Acid blue 129. The effects of all compounds were studied on contractions of the rat vas deferens elicited by α,β‐methylene ATP (α,β‐MeATP; mediated by P2X‐receptors) and relaxations of the carbachol‐precontracted guinea pig taenia coli elicited by adenosine 5′‐O‐(2‐thiodiphosphate) (ADPβS; mediated by P2Y‐receptors). All compounds were more or less P2Y‐ vs. P2X‐selective. In contrast to our assumption, it is the 4′‐methyl group of Acid blue 129 and of its new analog MG 32 (1‐amino‐4‐(4‐methylphenylamino)‐9,10‐dioxo‐9,10‐dihydroanthracene‐2‐sulfonic acid) which completely prevents binding to the P2X‐receptor up to 100 μM antagonist concentration, whereas the P2X‐receptor is blocked by related compounds lacking the 4′‐methyl group. Acid blue 129 and MG 32 were the most interesting compounds. Both turned out to be potent P2Y‐receptor antagonists with IC50‐values of approximately 3 μM. They are highly selective vs. both P2X‐receptor and non‐P2‐receptor effects. Drug Dev. Res. 59:64–71, 2003. © 2003 Wiley‐Liss, Inc.

Constitutional isomers of Reactive Blue 2 – selective P2Y-receptor antagonists?

The anthraquinone derivative Reactive Blue 2 (RB 2) is one of the most widely used P2-receptor antagonists, still claimed to be P2Y-selective. RB 2 is defined as a mixture of two constitutional isomers and commercially available in different identity and purity. A sample of RB 2, offered for sale by RBI, purchased from Biotrend, Köln, Germany, was chromatographically purified and identified by 1H- and 13C-NMR studies as a 35:65 mixture of the terminal ring F meta and para constitutional isomers. The two constitutional isomers of RB 2 were synthesised and tested alongside with the ortho isomer Cibacron Blue 3GA (CB 3GA) on contractions of the rat vas deferens (RVD) elicited by α,β-methylene ATP (α,β-MeATP), mediated by P2X 1-receptors, and relaxations of the carbachol-precontracted guinea pig taenia coli elicited by adenosine 5′- O-(2-thiophosphate) (ADPβS), mediated by P2Y 1-like-receptors. All compounds inhibited the α,β-MeATP induced contraction of the RVD and the ADPβS induced relaxation of the carbachol precontracted guinea-pig taenia coli. The IC 50-values at P2X 1-R were 9.1 μM for CB 3GA, 28.4 μM for RB 2, 19.7 μM for RB 2 meta, and 35.5 μM for RB 2 para. The IC 50-values at P2Y 1-like-R were 17.4 μM for CB 3GA, 7.7 μM for RB 2, 12.0 μM for RB 2 meta, and 2.6 μM for RB 2 para. The results clearly show that neither RB 2 as a mixture nor the pure ortho and meta isomer are P2Y 1-like- versus P2X 1-selective antagonists. In contrast the pure para-isomer of RB 2 is a moderately P2Y 1-like- versus P2X 1-selective antagonist.

Investigation of postjunctional alpha1- and alpha2-adrenoceptor subtypes in vas deferens from wild-type and alpha(2A/D)-adrenoceptor knockout mice.

1. The subtypes of alpha(1)- and alpha(2)-adrenoceptor mediating contractions of vas deferens have been examined in wild-type and alpha(2A/D)-adrenoceptor knockout mice. 2. Maximum contractions to noradrenaline but not phenylephrine were significantly greater in vas from wild-type. The alpha(1A)-adrenoceptor antagonist RS100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione) (10 nM) significantly shifted the potency of noradrenaline. The alpha(2D)-adrenoceptor antagonist BRL 44408 (2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole) significantly reduced the maximum contraction to noradrenaline in wild-type but not in knockout. 3. Following prazosin (0.1 micro M), a component of the contraction to noradrenaline in wild-type but not in knockout was sensitive to the alpha(2)-adrenoceptor antagonist yohimbine. 4. Nifedipine (10 micro M) or suramin (100 micro M) reduced the contraction to 10 Hz stimulation for 4 s to an early peak and small maintained response. The peak was abolished by the alpha(1)-adrenoceptor antagonist prazosin. 5. RS100329 or prazosin inhibited 10 Hz stimulation evoked contractions with a U-shaped concentration-response curve: inhibiting responses up to 0.1 micro M, with a reversal of inhibition above this concentration. In the presence of suramin or nifedipine, the reversal of inhibition by high concentrations of prazosin was reduced. 6. The alpha(1D)-adrenoceptor selective antagonist BMY7378 (8-[2-(4-(2- methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione) produced inhibition of 10 Hz evoked contractions only in high concentrations. 7. In conclusion, contractions to nerve stimulation in mouse vas deferens involve largely alpha(1A)-adrenoceptors and purinoceptors. alpha(1)-Adrenoceptor antagonists in high concentrations increase the purinergic response presumably by blocking prejunctional alpha(2)-adrenoceptor-mediated inhibition. In the presence of nifedipine, responses are predominantly alpha(1)-adrenoceptor mediated. Contractions to exogenous noradrenaline involved both alpha(1A)- and alpha(2A/D)-adrenoceptors in wild-type mice.

The purinergic component of human vas deferens contraction

The purinergic component of human vas deferens contraction

Regional differences in the adenosine A 2 receptor-mediated modulation of contractions in rat vas deferens

Adenosine receptors involved in modulation of contractions were characterized in the bisected rat vas deferens by combining pharmacological and immunohistochemical approaches. In both portions, noradrenaline-elicited contractions were enhanced by the adenosine A 1 receptor agonist N 6-cyclopentyladenosine (CPA), and inhibited by the non-selective adenosine receptor agonist 5′- N-ethylcarboxamidoadenosine (NECA) in the presence of the adenosine A 1 receptor antagonist 1,3-dipropyl-8-cyclopentyl-l,3-dipropylxanthine (DPCPX). The adenosine A 2A receptor agonist 2- p-(2-carboxyethyl)phenethyl-amino-5′- N-ethylcarboxamidoadenosine (CGS 21680) also inhibited noradrenaline-elicited contractions but only in the prostatic portion. Contractions elicited by the stable ATP analogue α,β-methyleneATP (α,β-MeATP) were inhibited only by NECA in the presence of DPCPX and only in the prostatic portion. This study provides functional evidence for the presence, in both portions of the rat vas deferens, of an adenosine A 1 receptor-mediated enhancement and of an adenosine A 2 receptor-mediated inhibition of contractions. The latter effect is mediated by both A 2A and A 2B subtypes in the prostatic portion but only by the A 2B subtype in the epididymal portion. This regional variation is supported by the immunohistochemical results that revealed an adenosine A 2A receptor immunoreactivity not co-localized with nerve fibres more abundant in the prostatic than in the epididymal portion.

Effects of Thymoquinone, the Major Constituent of Nigella sativa Seeds, on the Contractile Responses of Rat vas Deferens

We have evaluated the effects of thymoquinone on smooth muscle contraction in the isolated rat epididymal vas deferens using tension recording technique. The contractile responses to norepinephrine (NE), KCl, and electrical field stimulation were recorded using an isometric transducer. Thymoquinone inhibited the contractile responses to exogenous NE (100 µM) and KCl (80 mM) in a concentration-dependent manner. Moreover, thymoquinone reduced the amplitude of electrically-evoked contraction of vas deferens in a concentration-dependent manner. Cumulative addition concentrations of CaCl2 (0.1–10 mM) to tissue bath failed to increase the amplitude of contractile responses to electrical field stimulation in the presence of thymoquinone (80 µM). These results indicate that thymoquinone induced non-selective and concentration-dependent inhibition of contractile responses to NE, KCl, and electrical field stimulation. This action may be due to the ability of this alkaloid to interfere with the mobilization of Ca2+ required for smooth muscle contraction.

Long-Acting Fentanyl Analogues: Synthesis and Pharmacology of N-(1-Phenylpyrazolyl)- N-(1-phenylalkyl-4-piperidyl)propanamides

The synthesis of new fentanyl analogues in which the benzene ring of the propioanilido group has been replaced by phenylpyrazole is described. Antinociceptive activity was evaluated using the writhing and hot plate tests in mice. Two compounds, 3b and 3d , showed interesting analgesic properties, being more potent than morphine and less than fentanyl but with longer duration of action. These compounds inhibited the electrically evoked muscle contraction of guinea pig ileum and mouse vas deferens but not that of rabbit vas deferens and the effects could be reversed by antagonists (naloxone and/or CTOP), thus indicating that the compounds acted as μ agonists. Finally, the binding data confirmed that the compounds had high affinity and selectivity for the μ receptor.

Structure-activity studies of the Phe(4) residue of nociceptin(1-13)-NH(2): identification of highly potent agonists of the nociceptin/orphanin FQ receptor.

A total of 32 compounds was prepared to investigate the functional role of Phe(4) in NC(1-13)-NH(2), the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe(4) was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [(3)H]-NC-NH(2) to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP(4) receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH(3))Phe(4)]NC(1-13)-NH(2), which was only 3-fold less potent than NC(1-13)-NH(2). Compounds of the third series showed higher, equal, or lower potencies than NC(1-13)-NH(2). In particular, [(pF)Phe(4)]NC(1-13)-NH(2) (pF) and [(pNO(2))Phe(4)]NC(1-13)-NH(2) (pNO(2)) were more active than NC(1-13)-NH(2) by a factor of 5. In the mVD, these compounds showed the following order of potency: (pF) = (pNO(2)) > or = (pCN) > (pCl) > (pBr) > (pI) = (pCF(3)) = (pOCH(3)) > (pCH(3)) > (pNH(2)) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r(2) > or = 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe(4) and inversely with their size.