Prostatic Vas Deferens Research Articles

Novel naftopidil derivatives containing methyl phenylacetate and their blocking effects on α1D/1A-adrenoreceptor subtypes

α1-Adrenoceptor (α1-AR) antagonists are considered to be the most effective monotherapy agents for lower urinary tract symptoms associated with benign prostatic hyperplasia (LUTS/BPH). In this study, we synthesized compounds 2–17, which are novel piperazine derivatives that contain methyl phenylacetate. We then evaluated the vasodilatory activities of these compounds. Among them, we found that compounds 2, 7, 12, which contain 2-OCH3, 2-CH3 or 2, 5-CH3, respectively, exhibited potent α1-blocking activity similar to protype drug naftopidil (1). The antagonistic effects of 2, 7, and 12 on the (−)-noradrenaline-induced contractile response of isolated rat prostatic vas deferens (α1A), spleen (α1B) and thoracic aorta (α1D) were further characterized to assess the sub receptor selectivity. Compared with naftopidil (1) and terazosin, compound 12 showed the most desirable α1D/1A subtype selectivity, especially improved α1A subtype selectivity, and the ratios pA2 (α1D)/pA2 (α1B) and pA2 (α1A)/pA2 (α1B) were 17.0- and 19.5-fold, respectively, indicating less cardiovascular side effects when used to treat LUTS/BPH. Finally, we investigated the chiral pharmacology of 12. We found, however, that the activity of enantiomers (R)-12 and (S)-12 are not significantly different from that of rac-12.

The effect of cocaine on kinetics of α1-adrenergic contractile response in different portions of the rat vas deferens

The effect of cocaine (10 microM) on the kinetics of contractile response to noradrenaline (NA) was studied in the rat epididymal and prostatic vas deferens. Cocaine caused an acute increase in vas deferens adrenergic sensitivity primarily due to blockage of NA neuronal capture. The presynaptic action prevailed in the epididymal half: the EC50 value decreased 32-fold with a slight increase of the maximum adrenergic response more evident in the prostatic half. In the presence of cocaine, the prostatic contraction to NA was mediated not by a single pool of alpha1-adrenoceptors like in epididymal vas deferens but by two. Its high affinity pool had the same affinity as alpha1-adrenoceptors of the epididymal half, the affinity value of the low one was 36-fold less, and the total maximal response of both pools increased 4.5-fold. The differences in cocaine effect on the rat epididymal and prostatic vas deferens contractions to NA appear to be caused by the different sources of Ca2+ involved in these responses.

Identification of α 1-adrenoceptor subtypes involved in contraction of young CD rat epididymal vas deferens

Contraction of rat epididymal vas deferens is regulated via a release of neurotransmitters from autonomic nerves and is mediated by α 1-adrenoceptors. This study was directed to the characterization of α 1-adrenoceptors involved in the contraction of the epididymal portion of young CD rat vas deferens, that were selectively discriminated in two populations through the irreversible blockade of two β-chloroethyamines, 1 and 2. The antagonist activity of known subtype-selective α 1-adrenoceptor antagonists, WB4101, 5-MU, and RS17053 (α 1A), (+)-cyclazosin (α 1B), and BMY7378 (α 1D), was evaluated in the α 1-adrenoceptors of the studied tissue as such and after pre-treatment with a proper discriminating concentration of β-chloroetylamines 1 and 2, comparing the results with the affinities determined in classical Wistar rat models: prostatic vas deferens (α 1A), spleen (α 1B), and thoracic aorta (α 1D). The results suggested that two α 1A-adrenoceptors are involved in the contraction of the epididymal vas deferens of young CD rats. These may represent two α 1A-adrenoceptor isoforms that are selectively and irreversibly blocked by β-chloroetylamines 1 and 2, and reversibly antagonized by RS17053. The minor population, preferentially blocked by 1, seems correspond to a classical α 1A-adrenoceptor subtype, while the major population, preferentially blocked by 2 and antagonized by RS17053 with low affinity, seems to correspond to an α 1L-adrenoceptor.

"Ejaculatory disorders and α1-adrenoceptor antagonists therapy: clinical and experimental researches"

BackgroundIt is well known that the use of the α-adrenergic receptor antagonists in the BPH therapy may induce ejaculatory disorder. A review of clinical literature shows a greater incidence of ejaculatory disorder during the use of tamsulosin compared with alfuzosin. Anejaculation has been until now referred to retrograde ejaculation due to relaxation of prostatic and bladder neck smooth muscle tone. In a recent researches was evaluated the effect of tamsulosin and alfuzosin on rat vas deferent "in vitro", concluding that tamsulosin may "cause ejaculatory dysfunction by altering the progression and emission of sperm". An abnormal increase of contraction would be the cause of ejaculatory disorder. The aim of our paper is to compare human and rat vas deferens contractile activity and to evaluate with a clinical study if tamsulosin causes retrograde ejaculation disorder.MethodsWe have revaluated the human and rat vas deferens contractile activity in vitro according to our experience and literature. We have also performed a clinical study on 10 patients (48–72 y) affected by anejaculation. Post-coital urine was examined to search spermatozoa.ResultsHuman and rat vas deferens activity is not comparable. Contractile activity induced by norepinephrin after tamsulosin incubation in rat prostatic vas deferens strips is similar to the contractile activity evoked by norepinephrin in human strips. Spermatozoa were found in post coital urine of 6 patients.ConclusionIn our opinion the treatment with tamsulosin may induce retrograde ejaculation but not other ejaculatory disorder due to abnormal sperm progression.

Differential expression of caveolin-3 in mouse smooth muscle cells in vivo

Expression of caveolin-1 and -3 in mouse smooth muscle cells in vivo was examined by immunohistochemistry. Caveolin-1 was detected in almost all smooth muscles examined, except for the pupillary dilator muscle, whereas caveolin-3 was present only in smooth muscles of some specific tissues. In the eye, the pupillary sphincter muscle was intensely positive for caveolin-3, whereas the ciliary muscle and pupillary dilator muscle were negative. In the gastrointestinal tract, caveolin-3 was detected in the inner circular layer, but not in the outer longitudinal layer. Vascular smooth muscle cells of the resistance-sized artery in the uterus and corpus cavernosum were intensely positive for caveolin-3, whereas those of the aorta were only weakly positive and those of the vena cava were negative. Caveolin-3 was also detected in smooth muscle cells of the urinary bladder, ureter, prostatic vas deferens, and seminal vesicle. The different levels of caveolin-3 expression among various smooth muscle tissues were confirmed by Western blot analysis. Even within the same muscle, the relative expression levels of caveolin-1 and -3 were variable among neighboring cells, suggesting distinct fine regulation of expression of these two caveolins. Moreover, even in the same cell, caveolin-1 and -3 showed different distributions. These results indicate that the two caveolins form distinct caveolae in smooth muscles, and that caveolin-1 and -3 serve different functions. Their differential expression may therefore be related to the functional diversity of smooth muscles.

(+)-Cyclazosin, a selective α 1B-adrenoceptor antagonist: Functional evaluation in rat and rabbit tissues

To shed light on the discrepancy between reported binding and functional affinity and selectivity at α 1b/B-adrenoceptors, the antagonist (+)-cyclazosin was reinvestigated in rat and rabbit tissues. It displayed a competitive antagonism at α 1A and α 1D-adrenoceptors of rat prostatic vas deferens and aorta with p A 2 values 7.75 and 7.27, respectively. In rabbit thoracic aorta (+)-cyclazosin competitively antagonized noradrenaline-induced contractions at α 1B-adrenoceptors with a p A 2 value of 8.85, whereas its affinity at α 1L-adrenoceptors was markedly lower (p A 2 = 6.75 − 7.09). In conclusion, these data confirmed that (+)-cyclazosin is a selective α 1B-adrenoceptor antagonist also in functional assays, showing 13- and 38-fold selectivity for the α 1B-adrenoceptor over α 1A- and α 1D-subtypes, respectively. Furthermore, (+)-cyclazosin displayed a significant selectivity for α 1B-adrenoceptors relative to the α 1L-subtype.

Synthesis and structure-activity studies on N-[5-(1H-imidazol-4-yl)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide, an imidazole-containing alpha(1A)-adrenoceptor agonist.

Structure-activity studies were performed on the alpha(1A)-adrenoceptor (AR) selective agonist N-[5-(1H-imidazol-4-yl)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide (4). Compounds were evaluated for binding activity at the alpha(1A), alpha(1b), alpha(1d), alpha(2a), and alpha(2B) subtypes. Functional activity in tissues containing the alpha(1A) (rabbit urethra), alpha(1B) (rat spleen), alpha(1D) (rat aorta), and alpha(2A) (rat prostatic vas deferens) was also evaluated. A dog in vivo model simultaneously measuring intraurethral pressure (IUP) and mean arterial pressure (MAP) was used to assess the uroselectivity of the compounds. Many of the compounds that were highly selective in vitro for the alpha(1A)-AR subtype were also more uroselective in vivo for increasing IUP over MAP than the nonselective alpha(1)-agonists phenylpropanolamine (PPA) (1) and ST-1059 (2, the active metabolite of midodrine), supporting the hypothesis that greater alpha(1A) selectivity would reduce cardiovascular side effects. However, the data also support a prominent role of the alpha(1A)-AR subtype in the control of MAP.

Conformational restraints revealing bioactive beta-bend structures for halpha CGRP8-37 at the CGRP2 receptor of the rat prostatic vas deferens.

1. The main aim of this study was to identify putative beta-bends and the role of the N- and C-terminus in the CGRP receptor antagonist halpha CGRP8-37, which was measured against halpha CGRP inhibition of twitch responses in the rat prostatic vas deferens. 2. With a bend-biasing residue (proline) at position 16 in halpha CGRP8-37 (10(-5) M) an inactive compound was produced, while alanine at the same position retained antagonist activity (apparent pKB 5.6+/-0.1 at 10(-5) M). Proline at position 19 within halpha CGRP8-37 (10(-5) M) was an antagonist (apparent pKB 5.8+/-0.1). 3. Incorporation of a bend-forcing structure (beta-turn dipeptide or BTD) at either positions 19,20 or 33,34 in halpha CGRP8-37 (10(-5) M) antagonized halpha CGRP responses (apparent pKB 6.0+/-0.1 and 6.1+/-0.1, respectively). Replacement by BTD at both positions 19,20 and 33,34 within halpha CGRP8-37 competitively antagonized responses to halpha CGRP (pA2 6.2; Schild plot slope 1.0+/-0.1). 4. Halpha CGRP8-37 analogues (10(-5) M), substituted at the N-terminus by either glycine8, or des-NH2 valine8 or proline8 were all antagonists against halpha CGRP (apparent pKB 6.1+/-0.1, 6.5+/-0.1 and 6.1+/-0.1, respectively), while halpha CGRP8-37 (10(-5) M) substituted in three places by proline8 and glutamic acid10,14 was inactive. 5. Replacement of the C-terminus by alanine amide37 in halpha CGRP8-37 (10(-5) M) failed to antagonize halpha CGRP responses. 6. Peptidase inhibitors did not alter either the agonist potency of halpha CGRP or the antagonist affinities of halpha CGRP8-37 BTD19,20 and 33,34 and halpha CGRP8-37 Gly8 (against halpha CGRP responses). 7. In conclusion, two beta-bends at positions 18-21 and 32-35 are compatible with high affinity by BTD and is the first approach of modelling the bioactive structure of halpha CGRP8-37. Further, the N-terminus of halpha CGRP8-37 is not essential for antagonism, while the C-terminus interacts directly with CGRP receptor binding sites of the rat vas deferens.

Prejunctionally mediated inhibition of neurotransmission by isoprenaline in rat vas deferens

Effects of isoprenaline on monophasic contractions evoked by electric field stimulation were studied in rat isolated prostatic vas deferens. Isoprenaline reduced electrically evoked contractions (EC 50: 0.27 ± 0.05 μM) and propranolol concentration-dependently antagonized the effect of isoprenaline. In contrast, isoprenaline (0.3–3 μM) did not affect the contractile response induced by exogenous noradrenaline or ATP, while forskolin (100 nM) attenuated agonist-induced contraction. In some tissues, adrenergic and purinergic components of the electrically evoked contraction were isolated by exposure to α,β-methylene ATP (3 μM) and prazosin (3 μM), respectively. Isoprenaline induced a greater inhibition of purinergic than adrenergic component of the electrically evoked contraction. Iberiotoxin (50 nM), glibenclamide (3 μM), 4-aminopyridine (0.3 mM) and tetraethylammonium ions (1 mM) attenuated the effect of isoprenaline. These results indicate that isoprenaline-induced inhibition of the electrically evoked (both purinergic and adrenergic) contraction was mediated primarily through activation of prejunctional β-adrenoceptors, which probably inhibited release of contractile transmitters from sympathetic nerves supplying vas deferens. Lack of effect of isoprenaline on agonistinduced contraction does not favour a functional role of β-adrenoceptors in vas smooth muscle.

Search for α1-adrenoceptor subtypes selective antagonists: Design, synthesis and biological activity of cystazosin, an α antagonist

Two novel quinazolines (2 and 3) related to both prazosin and its open analogue 1 were synthesized, and their biological profile at alpha 1-adrenoceptor subtypes was assessed by functional assays in rat isolated tissues, namely prostatic vas deferens (alpha 1A), spleen (alpha 1B) and aorta (alpha 1D). Furthermore, the binding profile of 3 was assessed at native alpha 2 and D2 receptors, and cloned human 5-HT1A receptors, in comparison to prazosin, (+)-cyclazosin, 1 and BMY 7383. It turned out that the cystamine-bearing quinazoline 3 (cystazosin) has a reversed affinity profile relative to (+)-cyclazosin owing to a higher affinity for alpha 1D-adrenoceptors and a significantly lower affinity for the alpha 1A and alpha 1B subtypes. Furthermore, in comparison to BMY 7378, cystazosin (3) displays a much better specificity profile since it has lower affinity for D2 and 5-HT1A receptors.

Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens.

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP8-37. 2. In the pulmonary artery, human (h)alpha-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by NG-nitro-L-arginine (10(-5) M). The inhibitory effect of NG-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10(-4) M). Thus, CGRP acts via nitric oxide released from the endothelium. 3. In the endothelium-intact artery, halpha-CGRP, hbeta-CGRP and human adrenomedullin (10(-10) - 3 x 10(-7) M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with halpha-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM2,7)] halpha-CGRP (10(-7) - 10(-4) M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10(-4) M). 4 Human alpha-CGRP8-37 (3 x 10(-7) - 3 x 10(-6) M) antagonized halpha-CGRP (pA2 6.9, Schild plot slope 1.2+/-0.1) and hbeta-CGRP (apparent pKB of 7.1+/-0.1 for halpha-CGRP8-37 10(-6) M) in the pulmonary artery. Human beta-CGRP8-37 (10(-6) M) antagonized halpha-CGRP responses with a similar affinity (apparent pKB 7.1+/-0.1). Human adrenomedullin responses were not inhibited by halpha-CGRP8-37 (10(-6) M). 5. In the prostatic vas deferens, halpha-CGRP, hbeta-CGRP and rat beta-CGRP (10(-10) - 3 x 10(-7) M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10(-8) - 10(-5) M) was around 10 fold less potent and the linear analogue [Cys(ACM2,7)] halpha-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10(-4) M). 6 The antagonist effect of halpha-CGRP8-37 (10(-5) 3 x 10(-5)) in the vas deferens was independent of the agonist, with pA2 values against halpha-CGRP of 6.0 (slope 0.9+/-0.1), against hbeta-CGRP of 5.8 (slope 1.1+/-0.1), and an apparent pKB value of 5.8+/-0.1 against both rat beta-CGRP and rat amylin. Human beta-CGRP8-37 (3 x 10(-5) - 10(-4) M) competitively antagonized halpha-CGRP responses (pA2 5.6, slope 1.1+/-0.2). The inhibitory effect of halpha-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by halpha-CGRP8-37 (pA2 5.8 and 5.8, slope 1.0+/-0.2 and 1.0+/-0.3, respectively). 7 The effects of halpha-CGRP and halpha-CGRP8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10(-6) M). The weak agonist activity of [Cys(ACM2,7)] halpha-CGRP in the vas deferens was not increased by peptidase inhibitors. 8 These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP1 receptor subtype in the rat pulmonary artery (CGRP8-37 pA2 6.9), while the lower affinity for CGRP8-37 (pA2 6.0) in the vas deferens is consistent with a CGRP2 receptor.

Alpha1A-adrenoceptor mediated contraction of rat prostatic vas deferens and the involvement of ryanodine stores and Ca2+ influx stimulated by diacylglycerol and PKC.

1 The present study has investigated the alpha1-adrenoceptor subtype mediating contraction of the rat isolated prostatic vas deferens and the possible effector mechanisms involved in this response by use of functional experiments. 2 Contractions to noradrenaline in the rat isolated prostatic vas deferens were antagonized by prazosin (9.4, 1.04+/-0.19, pA2 and Schild plot slope), 5-methyl urapidil (8.9, 1.10+/-0.13), BMY 7378 (6.4, 1.53+/-0.07) and RS 17053 (8.3, 1.13+/-0.18). These affinities are consistent with the response being mediated by the alpha1A-adrenoceptor subtype. 3 The contraction to noradrenaline at 37 degrees C consisted of an initial phasic response, composed of many rhythmic contractile spikes and a more slowly developing tonic contraction. When the temperature was lowered to 25 degrees C the phasic contraction became a smooth single response which was increased in magnitude. 4 In Ca2+-free Krebs solution the tonic contraction to noradrenaline (10(-4) M) was abolished, suggesting that this response was dependent on influx of extracellular Ca2+. After 2 min in Ca2+-free Krebs solution at 37 degrees C and 25 degrees C the phasic response to noradrenaline (10(-4) M) was 38+/-2% and 91+/-4%, respectively, compared with the phasic contraction to noradrenaline (10(-4) M in normal Krebs solution) and after 30 min it was abolished at 37 degrees C and was 7+/-1% at 25 degrees C. Ryanodine abolished the noradrenaline response in Ca2+-free Krebs solution for 2 min at 25 degrees C, while cyclopiazonic acid reduced it to 36+/-2%. 5 In normal Krebs solution at 25 degrees C the protein kinase C inhibitor calphostin C reduced the tonic contraction to noradrenaline (10(-5) M) from 36+/-8% to 14+/-3% compared with the phasic contraction to noradrenaline (10(-4) M). The DAG kinase inhibitor R 59022 increased the contraction following the initial phasic response to a maximum of 107+/-17% after 35 s, before dropping down to a well maintained contraction which was still greater in magnitude compared with the control. Nifedipine (3x10(-7) M) reduced the tonic contraction from 49+/-6% to 7+/-1% but did not reduce the phasic response. Ryanodine (10(-4) M) reduced the phasic contraction from 50+/-2% to 7+/-1% and the tonic response from 47+/-5% to 27+/-5%. 6 The phorbol ester phorbol-12,13-dibutyrate at 25 degrees C produced a transient contraction of the rat prostatic vas deferens, maximum response (10(-5) M) 48+/-4%, compared with the maximum tonic response to noradrenaline. The contraction to PDBu (10(-5) M) was reduced to 23+/-2% by calphostin C (10(-6) M) and to 15+/-1% by nifedipine (3x10(-7) M) and was abolished after 2 min in Ca2+-free Krebs solution. 7 In conclusion, the alpha1A-adrenoceptor mediated contraction to noradrenaline of the rat prostatic vas deferens appears to consist of an initial phasic component due to the release of intracellular Ca2+ from ryanodine-sensitive stores. These stores are depleted in the absence of extracellular Ca2+ and this depletion is slower at 25 degrees C than at 37 degrees C. The phasic contraction is followed by a tonic contraction involving activation of protein kinase C by diacylglycerol and influx of Ca2+ through nifedipine-sensitive channels.

Expression and localization of endothelin converting enzyme in rat vas deferens.

Endothelins (ETs) are a family of vasoconstrictor and mitogenic peptides originally isolated from the endothelial cells. Three isoforms of ET, namely ET-1, ET-2 and ET-3, are generated from their respective intermediate precursors big ETs through specific endoproteolytic cleavage by endothelin converting enzyme (ECE). Using reverse-transcription polymerase chain reaction (RT-PCR), we have isolated a cDNA encoding for ECE from both the prostatic and epididymal halves of rat vas deferens. In situ hybridization using digoxigenin-labeled ECE cDNA probe demonstrated that ECE mRNA is preferentially localized in the inner longitudinal smooth muscle layer adjacent to submucosa region of rat vas deferens. Both ET-1 and big ET-1 at 30 nM potentiated electrically stimulated contractile response of prostatic vas deferens. Pre-incubation of tissue with a metalloprotease ECE inhibitor phosphoramidon (10 microM) strongly inhibited the response to big ET-1, but not to ET-1. On the other hand, big ET-1 failed to elicit contractile response of epididymal vas deferens. Phosphoramidon alone did not affect both the basal and electrically stimulated contractile responses in vas deferens. These data indicate that the circulating ET-1 and its immediate precursor big ET-1 could differentially regulate smooth muscle contractions in the prostatic and epididymal vas deferens of the rat.

Potentiation of purinergic transmission by angiotensin in prostatic rat vas deferens.

1. Angiotensin II (AII) elicited only a minute, if any, direct contractile response in smooth muscle cells of prostatic rat vas deferens, but it potentiated contractile responses to field stimulation. 2. Angiotensin-potentiated contractile response to field stimulation was concentration-dependent, and the order of potency was AII > AIII approximately AI. The EC50 of AII was 8.11 +/- 2.79 nM. 3. AII did not modify the contractile response of exogenous noradrenaline (NA) on non-stimulated prostatic vas deferens. Furthermore, the concentration-response curve for AII-potentiated contractile responses to field stimulation in reserpine-treated rats did not significantly differ from the control group. 4. Desensitization of purinoceptors with 30 microM alpha, beta-methylene-ATP almost completely abolished the potentiation of the contractile response to field stimulation by AII. 5. The response to AII in the prostatic rat vas deferens was blocked by the AT1 selective antagonist losartan, but not by the AT2 selective antagonist CGP 42112. Losartan acted as a competitive antagonist with a pA2 value of 8.75. 6. In conclusion, AII potentiated purinergic transmission in the prostatic rat vas deferens via the AT1 receptor.

Failure of the putative neuropeptide Y antagonists, benextramine and PYX-2, to inhibit Y2 receptors in rat isolated prostatic vas deferens.

1. The pharmacological activity of neuropeptide Y (NPY) and some analogues in inhibiting the twitch contractions induced by electrical stimulation (single pulses at 25 V, 0.15 Hz, 1 ms) in the prostatic portion of the rat isolated vas deferens was investigated. The rank order of agonist potency was: PYY > NPY2-36 > NPY >> NPY13-36 >> NPY18-36 >> [Leu31,Pro34]NPY = hPP, which is consistent with the activation of a Y2 receptor. 2. The putative Y1 and Y2 antagonist, benextramine (BXT), incubated at 100 microM for 10 or 60 min, was ineffective against PYY-induced inhibition of the twitch response, suggesting that the prejunctional Y2 receptor in this tissue is different from the postjunctional one reported in the literature to be sensitive to BXT blockade. 3. The putative NPY antagonist, PYX-2, incubated at 1 microM for 20 min, was completely ineffective in antagonizing PYY-induced inhibition of twitches. 4. The twitch response was totally inhibited by suramin (100 microM) but was little affected by prazosin (1 microM). Furthermore, NPY was without effect on the dose-response curve to ATP in resting conditions. Taken together, these results suggest that in our paradigm, NPY inhibits the release of a purinergic neurotransmitter which mediates contraction of the prostatic portion of the rat vas deferens.

Purinergic modulation of field stimulation responses of rat and human vas deferens smooth muscle

1. 1. Guanethidine at 5 × 10 −6 M strongly inhibited rat prostatic but not epididymal vas deferens, reflecting differences in innervation and the neurogenic field stimulation responses of these tissues. 2. 2. Adenosine and ATP inhibited the field stimulation responses of rat prostatic vas deferens by 56 and 50% respectively. A 10-min pretreatment with 10 −4 M caffeine partly reversed this inhibition, by 55% in the case of adenosine and 60% for ATP. 3. 3. Pretreatment for 10 min with 5 μM quinidine failed to significantly alter the extent of either adenosine or ATP inhibition of the field stimulation responses of rat prostatic vas deferens. 4. 4. 8-Phenyltheophylline, the selective blocker of the A 1 subtype of the P 1 receptor, partly reversed adenosine-induced inhibition of the vas deferens FS responses. NECA, the selective agonist of the A 2 subtype of the P 1 receptor, very strongly inhibited vas deferens FS responses. 5. 5. Field stimulation responses of human vas deferens were also inhibited by both adenosine and ATP but to a lesser extent and more variably than in rat tissue. 6. 6. Adenosine and ATP inhibition was reversed by caffeine pretreatment, but far more variably than in rat tissue, and quinidine was without significant effect on inhibition of the responses. 7. 7. It is concluded that in these tissues adenosine and ATP may operate via a P 1 type receptor of both A 1 and A 2 subtypes and that a P 2 type receptor may be lacking.

Prejunctional nicotinic receptors involved in facilitation of stimulation‐evoked noradrenaline release from the vas deferens of the guinea‐pig

In guinea-pig prostatic vas deferens loaded with [3H]-noradrenaline ([3H]-NA), nicotinic receptor agonists, nicotine and dimethylphenylpiperazinium (DMPP) enhanced the resting and facilitated the stimulation-evoked release of [3H]-NA in a concentration-dependent fashion. The effect of nicotine on both contraction of vas deferens and release of NA in response to field stimulation was stereospecific in favour of the naturally occurring (-)-enantiomer. Prolonged (15 min) exposure to (-)-nicotine resulted in a cessation of the facilitatory effect on NA release and on responses of the vas deferens to field stimulation. 2 The rank order of agonist potency in facilitating NA release was DMPP = (-)-nicotine greater than (+)-nicotine. Cytisine had no agonistic activity. The dissociation constants (KD) of antagonists were 9.3 +/- 0.6 and 31.4 +/- 2.4 microM for (+)-tubocurarine and hexamethonium, respectively, when (-)-nicotine was used as agonist. alpha-Bungarotoxin had no antagonistic activity. These findings suggest that nicotinic receptors located on noradrenergic axon terminals are different from those located postsynaptically in striated muscle or ganglia but seem similar to those present on cholinergic axon terminals at the neuromuscular junction. 3. Cotinine, the breakdown product of nicotine failed to have any agonistic activity indicating that nicotine itself is responsible for the effects observed on axon terminals. 4 Stimulation of presynaptic muscarinic receptors by oxotremorine prevented the nicotine-induced facilitation of [3H]-NA release, indicating the presence of both inhibitory muscarinic and facilitatory nicotinic receptors on noradrenergic axon terminals.

Voltage and time dependency of calcium mediated phasic and tonic responses in rat vas deferens smooth muscle—The effect of some calcium agonist and antagonist agents

1. Phasic and tonic components of the K+-induced contracture response were found to be expressed to different degrees in the prostatic and epididymal portions of the rat vas deferens. 2. With elevation of external potassium greater than 25 mM, the mechanisms underlying the phasic component operate only transiently before inactivation and replacement with tonic tension. 3. Both phasic and tonic components of the vas deferens response to potassium were markedly dependent upon external calcium ions. 4. Nifedipine and verapamil equally inhibited the phasic and tonic components of the K+ response in the prostatic vas deferens. However, inhibition by these agents was far more pronounced in the phasic components than in the tonic component of the epididymal vas deferens. 5. BAY K 8644 potentiated, in a dose-dependent manner, the phasic components of the K+ response, particularly in the prostatic vas deferens. 6. Abscisic acid also potentiated, in a dose-dependent manner the K+ response of rat vas deferens, but this action was far more pronounced in the tonic component of the epididymal portion. 7. Papaverine abolished the BAY K 8644 potentiated epididymal K+ response but did not affect the BAY K 8644 potentiated prostatic K+ response. 8. It is concluded that abscisic acid potentiated responses associated with the activation of voltage-dependent, non-inactivating slow calcium channels. 9. Papaverine, nifedipine and verapamil appear to be less selective than abscisic acid in that they equally inhibit phasic and tonic responses in prostatic vas deferens, but these may be interdependent, yet they more strongly inhibit phasic responses of epididymal vas deferens.

Inhibitory effect of adenosine on electrically evoked contractions in the rat vas deferens: Calcium dependence

The inhibitory effect of adenosine on the electrically evoked contractions in the rat prostatic vas deferens was markedly dependent on the calcium concentration in the medium, with adenosine being 20 times more potent at lower calcium levels (1.26 mM vs 3.6 mM). Similar calcium dependence of adenosine inhibition was observed in the presence of a potent nucleoside uptake inhibitor, nitrobenzylthioguanosine (NBTGR), suggesting that this calcium dependence of adenosine inhibition was not secondary to an alteration in adenosine uptake. Furthermore, Bay K8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate), a dihydropyridine ‘calcium entry activator’, reduced the potency of adenosine in inhibiting the electrically evoked contractions in a calcium-dependent manner. These results support the notion that adenosine might exert its inhibition on neurotransmission in the prostatic rat vas deferens by decreasing transmembrane calcium currents into the nerve terminals and/or the smooth muscles.