Human Vas Deferens Research Articles

Effects of nociceptin and endomorphin 1 on the electrically stimulated human vas deferens.

To examine the effects of nociceptin (NC) and endomorphin 1 (EM1) on electrical field stimulation (EFS)-induced contractions of the human vas deferens (hVD). Concentration-response curves to NC and EM1 were constructed in the absence and in presence of peptidase inhibitors (PI). In some experiments a NC receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 [F/G], 10 microM) or naloxone (1 microM) were included. All data are mean(95%CI). In the presence of PI, NC inhibited twitches (Emax = 67(44,90)%; pEC50 = 7.28(6.95,7.61)). NC inhibition was sensitive to [F/G]. EM1 also inhibited twitches both in the absence (Emax = 82(73,91)% pEC50 = 7.07(6.92,7.22)) and presence (Emax = 83(76,90)%; pEC50 = 7.00(6.91, 7.09)) of PI. EM1 inhibition was sensitive to naloxone. These data suggest that hVD express NC and opioid receptors that inhibit neurogenic contractions.

Inhibition of neuroeffector transmission in human vas deferens by sildenafil.

Sildenafil (0.1 - 30 microM), a cyclic GMP phosphodiesterase 5 (PDE 5) inhibitor, induced inhibition of electrically evoked contractions of ring segments of human vas deferens from 34 vasectomies. Zaprinast (0.1 - 100 microM), another PDE 5 inhibitor, and the nitric oxide (NO) donor sodium nitroprusside (SNP) (0.1 - 100 microM) had no effect on neurogenic contractions. The inhibition induced by sildenafil was not modified by the inhibitor of guanylate cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) (1 - 30 microM) but it was abolished by the K(+) channel blockers tetraethylammonium (TEA, 1 mM), iberiotoxin (0.1 microM) and charybdotoxin (0.1 microM). Sildenafil, zaprinast and SNP did not affect the contractions induced by noradrenaline. SNP (10 microM) caused elevation of cyclic GMP levels that was potentiated by sildenafil (10 microM) and zaprinast (100 microM). ODQ (10 microM) inhibited the increase in cyclic GMP. Sildenafil inhibits adrenergic neurotransmission in human vas deferens. The inhibition is not related to accumulation of cyclic GMP but is probably due to activation of prejunctional large-conductance Ca(2+)-activated K(+) channels.

Effects of antidepressants in adrenergic neurotransmission of human vas deferens

Objectives. To evaluate the effects of sertraline, fluoxetine, and amitriptyline on the contractile responses of the human vas deferens muscle elicited by norepinephrine, electrical field stimulation, and KCl, because the therapeutic action of antidepressants may be accompanied by sexual dysfunction related to the contractility of the vas deferens smooth muscle. Methods. Ring segments of the epididymal part of the vas deferens were taken from 32 elective vasectomies and mounted in organ baths for isometric recording of tension. We then studied the effects of sertraline, fluoxetine, and amitriptyline on the neurogenic and agonist-induced contractile responses. Results. Amitriptyline caused concentration-dependent inhibition of neurogenic and norepinephrine-induced contractions. In contrast, only the highest concentration (10 −5 M) of sertraline and fluoxetine reduced the adrenergic contractions. The dihydropyridine calcium antagonist nifedipine (10 −6 M) completely prevented the inhibitory effect of sertraline and fluoxetine on neurogenic and norepinephrine-induced contractions but did not change the inhibition caused by amitriptyline. Sertraline, fluoxetine, and amitriptyline (all at 10 −5 M) attenuated contractions elicited by KCl and reduced contractions induced by CaCl 2 in KCl-depolarized preparations. Conclusions. The results indicate that sertraline and fluoxetine inhibit vas deferens motility through inhibition of Ca 2+ entry, with no effect on the adrenergic receptors, and amitriptyline acts as an adrenoceptor antagonist and inhibitor of the entry of calcium.

Rank-order inhibition by ω-conotoxins in human and animal autonomic nerve preparations

The inhibitory effects of the ω-conotoxins GVIA, MVIIA and MVIIC on electrically-evoked, tetrodotoxin (10 −7 M)-sensitive, autonomic nerve activity were studied using human, rat or guinea-pig vas deferens and intestinal tissues. In each preparation from each species, nM concentrations of ω-conotoxins GVIA and MVIIA prevented the neuronally-mediated contractions, whereas ω-conotoxin MVIIC was either markedly less potent (IC 50's 1.4 or 2.9 log units more than for ω-conotoxin GVIA in guinea-pig ileum and rat vas deferens, respectively) or was without significant activity (human vas deferens, human Taenia coli) when tested at similar concentrations. In contrast the differences in potency between ω-conotoxins GVIA and MVIIC were considerably less when assayed directly on Ca 2+ channel currents evoked from rat superior cervical ganglion neurons in culture (approximately 0.1 log unit difference) and from a stable cell line expressing rat α 1B, α 2δ, β 1b Ca 2+ channel subunits (approximately 0.9 log unit). These different rank-orders of inhibitory activity of the conotoxins support the suggestion that there are pharmacologically distinct N-type Ca 2+ channels in the peripheral nervous system, and that this tissue-dependent difference is seen in man.

The pharmacological perspective: role for the sympathetic nervous system in micturition and sexual function.

The predominant effects of the sympathetic innervation of the lower urinary tract in man are mediation of contraction of the bladder base and the urethra and inhibition of the parasympathetic pathways at spinal and ganglion levels. Sympathetic tone also mediates the contractility of smooth muscle fibres in the prostatic stroma, and it has an important role in maintaining the penis in a flaccid state and in ejaculation. In the detrusor muscle of most species, beta-adrenoceptors (beta-ARs) mediating relaxation normally dominate over alpha-ARs mediating contraction, and the effect of noradrenaline (NA) is relaxation. The human detrusor is able to express beta(1)-, beta(2)- and beta(3)-ARs and stimulation of beta(3)-ARs causes relaxation. Low, but reproducible expression of alpha(1)-ARs (alpha(1a) and alpha(1d)) has been found in human detrusor muscle, but the alpha(1)-AR subtypes mediating contraction have not been established. The sympathetic pathways to the penis may mediate anti-erectile as well as erectile effects. Although alpha(1) and alpha(2)-ARs have been demonstrated in human corpus cavernosum tissue, available information supports the view of a functional predominance of alpha(1)-ARs. The human vas deferens also has an important adrenergic innervation and alpha(1A)-ARs seem to mediate contraction.

Colocalisation of neuropeptides, nitric oxide synthase and immunomarkers for catecholamines in nerve fibres of the adult human vas deferens.

Single and double-label immunofluorescence methods were used to determine the distribution and patterns of colocalisation of various neuropeptides and nitric oxide synthase (NOS) with the catecholamine synthesising enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH) in nerve fibres within specimens of adult human vas deferens obtained at vasectomy (age range 28 to 83 y). Cholinergic nerve fibres were immunolabelled with an antiserum to vesicular acetylcholine transporter (VAChT). Using the general nerve marker protein gene product 9.5 (PGP) the density of intramural nerve fibres was found to be similar irrespective of age. Many of these axons, especially in the outer 2 muscle layers were TH and DbetaH-immunoreactive (IR) and were thus confirmed as noradrenergic. Fewer such axons were seen in the inner longitudinal muscle layer. All the noradrenergic nerve fibres also displayed NPY-immunoreactivity with minor populations containing galanin (GAL) or somatostatin (SOM). Nerve fibres lacking TH and DbetaH-IR were immunoreactive for VAChT and were sparsely distributed throughout the 2 outer muscle layers but more numerous in the inner muscle layer. Nerves lacking TH and DbetaH were immunoreactive for NPY and some also contained NOS, VIP or CGRP. These results have been compared with those obtained previously from specimens of human neonatal and infant vas deferens where, in contrast to the present results, NOS and VIP were shown to be colocalised with TH in many of the intramuscular nerve fibres. It thus appears that NOS and VIP cease their coexistence with TH in intramuscular nerve fibres of the human vas deferens between the pre- and postpubertal states. In addition to the intramuscular nerve fibres a VAChT-IR subepithelial nerve plexus occurs in the vas deferens and may control the secretory activity of the lining epithelium. Most of these subepithelial nerve fibres were immunoreactive for NPY and many also contained VIP while minor populations were immunoreactive for NOS, GAL, SOM or SP although fibres containing CGRP were not observed. The neuropeptide content of the subepithelial nerve plexus was similar to that observed in the infant, except for an increased density of VIP-IR nerves, which may reflect greater activity of the lining epithelial cells in the adult vas deferens.

Functional characterization of α 1-adrenoceptor subtypes in longitudinal and circular muscle of human vas deferens

The α 1-adrenoceptor subtype(s) mediating contraction to noradrenaline in longitudinal and circular muscle of human epididymal vas deferens was studied using competitive antagonists. The effects of the alkylating agents, phenoxybenzamine and chloroethylclonidine were also investigated. Noradrenaline evoked concentration-dependent contractions of longitudinal and circular muscle with comparable potencies (pD 2; 5.6 and 5.5 respectively). The contractions in longitudinal and circular muscle respectively were inhibited by prazosin (pA 2, 8.6 and pK B, 9.2), 5-methylurapidil (pK B, 8.7 and 9.1) and less potently by spiperone (pA 2, 7.1) or BMY 7378 (pK B, 6.3 and 6.6). Contractions of the circular but not longitudinal muscle was comparatively insensitive to pretreatment with phenoxybenzamine. In contrast pretreatment with chloroethylclonidine reduced the contractions in both muscle types and also enhanced phenoxybenzamine-sensitivity in longitudinal but not circular muscle. The results suggest that contractions evoked by noradrenaline in both muscle types of human vas deferens is mediated via activation of α 1-adrenoceptors with pharmacological profile of the α 1A-subtype. However the involvement of α 1A-adrenoceptor variants, such as the hypothesised α 1L-subtype may underlie the differential effects of phenoxybenzamine in longitudinal and circular muscle. Factors contributing to chloroethylclonidine-sensitivity are discussed.

Structure and autonomic innervation of the human vas deferens: a review.

The motor innervation of the smooth muscle coat of the human vas deferens is predominantly noradrenergic in type while a less dense and differently distributed presumptive cholinergic innervation is also in evidence, although the precise role of the latter is undetermined. Immunohistochemical studies have confirmed the presence of catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) in the majority of fine, varicose intramuscular nerves, about two-thirds of which also contain neuropeptide Y (NPY). Minor populations of noradrenergic nerves contain enkephalin (ENK), galanin (GAL), somatostatin (SOM), or nitric oxide synthase (NOS). The presumptive cholinergic intramuscular nerves contain vasoactive intestinal polypeptide (VIP) and NPY. The subepithelial nerves of the vas deferens are assumed to have a secretomotor function and are rich in acetylcholinesterase and NPY, many also containing either VIP or NOS. The muscle coat of the human vas deferens is poorly differentiated until after birth, the intramuscular nerves in the fetus being relatively thick and non-varicose. Development of a subepithelial nerve plexus lags behind that in the muscle coat but its density in the neonatal vas deferens resembles that seen in the adult. Observations on specimens of human vas deferens obtained at vasovasostomy carried out 1 to 15 years after vasectomy have shown a marked reduction in the density of noradrenergic nerves in the muscle coat of the testicular portion while that in the urethral portion remains unaltered. Furthermore, the subepithelial secretomotor nerves degenerate in the testicular portion. These long-term changes in the pattern of innervation of the vas deferens consequent upon vasectomy may have profound effects upon the outcome of vasovasostomy with respect to subsequent sperm maturation, transport, and viability.

Caffeine- and Noradrenaline-Induced Contractions of Human Vas Deferens: Contrasting Effects of Procaine, Ryanodine and W-7

1. The effects of ryanodine, procaine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on noradrenaline (NA)- and caffeine-induced contractions of human vas deferens were investigated. 2. In the presence of nifedipine (1 μM), NA (100 μM) evoked biphasic contractions. Caffeine (20 mM) evoked repeatable tonic contractions. 3. Ryanodine (30 μM) inhibited the initial but not the secondary component of NA contractions. Procaine (1 and 10 mM) inhibited both components. Contractions induced by caffeine were unaffected by ryanodine or procaine. 4. The calmodulin antagonist W-7 (100 μM) reduced, in a reversible manner, both components of NA-induced response. Caffeine-induced contractions were also reduced in most preparations (8 of 11). In all preparations, contractions induced by caffeine were markedly inhibited after the washout of W-7. Higher doses of W-7 (300 μM) induced an increase in basal tension. 5. These results indicate that NA contracts the longitudinal muscle of human vas deferens by a ryanodine-sensitive calcium-induced calcium release (CICR) mechanism and, in addition, a ryanodine-insensitive pathway: both are sensitive to procaine. In contrast, contraction induced by caffeine is mediated by a pathway that is atypically insensitive to either ryanodine or procaine. The sensitivity of NA- and caffeine-induced contraction to W-7 suggests a role for calcium and its interaction with calmodulin in the response to both agents. The paradoxical action of W-7 is discussed.

Vasopressin receptors involved in adrenergic neurotransmission in the circular muscle of the human vas deferens

We studied the effects of vasopressin on the adrenergic responses of in vitro preparations of circular muscle from the vas deferens obtained from 28 men undergoing elective vasectomy. Vasopressin (3×10 −9–3×10 −8 M) enhanced the phasic contractions elicited by electrical field stimulation and noradrenaline. This potentiation was blocked by the vasopressin V 1 receptor antagonist d(CH 2) 5Tyr(Me)vasopressin (10 −6 M) but not by the vasopressin V 2 receptor antagonist [d(CH 2) 5, d-Ile 2,Ile 4,Arg 8]vasopressin (10 −6 M). The Ca 2+ antagonist nifedipine (10 −6 M) did not affect the potentiation of electrical field stimulation induced by vasopressin and noradrenaline but reduced KCl-induced contractions and abolished the induction of phasic activity by vasopressin in the presence of KCl. The results demonstrate that vasopressin, in addition to its direct contractile effects, strongly potentiates contractions of human vas deferens elicited by adrenergic stimulation. Both the direct and indirect effects of vasopressin appear to be mediated by vasopressin V 1 receptor stimulation and are independent of Ca 2+ entry through dihydropyridine Ca 2+ channels.

Ryanodine- and cyclopiazonic acid-sensitive components in human vas deferens contractions to noradrenaline.

1. The role of calcium stores in noradrenaline- (NA) and caffeine-induced contractions of human vas deferens were investigated using ryanodine and cyclopiazonic acid (CPA) in the presence of the calcium antagonist, nifedipine (1 microM) or in calcium-free/EGTA (1 mM) medium. 2. In either media, NA (100 microM) evoked biphasic contractions of longitudinal muscle and tonic circular muscle contractions. Caffeine (20 mM) evoked longitudinal but not circular muscle contractions. 3. Ryanodine (1-30 microM) or CPA (1-30 microM) inhibited contractions of circular muscle, and the initial but not secondary component of longitudinal muscle contraction to NA. 4. In the presence of nifedipine, pre-exposure to caffeine caused a potentiation of circular muscle, and the initial but not secondary longitudinal muscle contractions to NA. The presence of ryanodine or CPA during the caffeine pre-exposures effectively blocked the potentiation of the initial component and reduced the secondary component of the subsequent responses to NA in longitudinal muscle. 5. In calcium-free media, caffeine pre-exposures had little effect on subsequent NA-induced contractions in circular muscle, but reduced both components in longitudinal muscle. The presence of ryanodine or CPA during caffeine pre-exposures produced no further effects on either component of the subsequent NA-induced contraction in longitudinal muscle. 6 In the presence of nifedipine or in calcium-free media, repeated applications of caffeine evoked contractions in longitudinal muscle which were not blocked by either ryanodine or CPA. 7. These results suggest that circular muscle contraction by NA and the initial component of longitudinal muscle to NA both utilize an intracellular pool of calcium that is triggered via a ryanodine-sensitive mechanism and replenished via a CPA-sensitive Ca2+-ATPase. 8. In longitudinal muscle, both the secondary component of its response to NA and contraction to caffeine appear to involve an unusual but pharmacologically distinct (ryanodine- and CPA-insensitive) pathway. 9. The quiescence to caffeine of circular muscle may be caused by a relative absence of the ryanodine- and CPA-insensitive pathway.

Lectin histochemistry study in the human vas deferens.

The oligosaccharide sequences of glycoconjugates in the normal human vas deferens and the nature of the saccharide linkage were studied by lectin histochemistry. The cytoplasm of all epithelial cell types (principal cells, basal cells, and mitochondria-rich cells) and luminal contents reacted positively with WGA, MAA, PNA, DSA, LTA, UEA-I, AAA, and ConA. The reaction was more intense in the stereocilia of principal cells. Cytoplasmic staining was diffuse except for PNA and DSA labeling which was limited to the apical cytoplasm and stereocilia of columnar cells. The cytoplasm of all cell types also reacted diffusely with HPA, although staining was weak and was not observed in the stereocilia. Positive reaction with SBA only was encountered in the stereocilia of principal cells. SNA, LTA, and DBA were unreactive. GNA-labeling showed a granular distribution in the supranuclear cytoplasm of columnar epithelial cells. Reactions with MAA, PNA, DSA, AAA, HPA and SBA disappeared after the beta-elimination reaction. Reactions with WGA and UEA-I decreased after beta-elimination or Endo-F digestion. Reactions with ConA and GNA were suppressed by Endo-F digestion. Reactions with PNA, HPA, and SBA increased after desialylation. Of all the lectins that label the luminal contents of the vas deferens, only UEA-I was not found in the luminal contents of seminiferous tubules and epididymis and, thus, this lectin would probably bind to glycoproteins secreted by the vas deferens. The chemical treatments used suggest that this secretion contains fucose residues located in both N- and O-linked oligosaccharides. The other lectins may label secreted proteins, but also structural proteins or proteins reabsorbed from the luminal fluid. The lectin- binding pattern of mitochondria-rich cells in the vas deferens differed from that found in the epididymis.

A Novel Type of Internal Barium Block of a Maxi-K + Channel from Human Vas Deferens Epithelial Cells

We have recently shown that a maxi-K + channel from vas deferens epithelial cells contains two Ba 2+-binding sites accessible from the external side: a “flickering” site located deep in the channel pore and a “slow” site located close to the extracellular mouth of the channel. Using the patch-clamp technique, we have now studied the effect of internal Ba 2+ on this channel. Cytoplasmic Ba 2+ produced a voltage- and concentration-dependent “slow” type of block with a dissociation constant of ∼100 μM. However, based on its voltage dependence and sensitivity to K + concentration, this block was clearly different from the external “slow” Ba 2+ block previously described. Kinetic analysis also revealed a novel “fast flickering” block restricted to channel bursts, with an unblocking rate of ∼310 s −1, some 10-fold faster than the external “flickering” block. Taken together, these results show that this channel contains multiple Ba 2+-binding sites within the conduction pore. We have incorporated this information into a new model of Ba 2+ block, a novel feature of which is that internal “slow” block results from the binding of at least two Ba 2+ ions. Our results suggest that current models for Ba 2+ block of maxi-K + channels need to be revised.

Morphometric and immunocytochemical study of the fetal, infant, and adult human vas deferens.

The human vas deferens (VD) is often considered simply as a conduit to transfer mature sperm from the epididymis to the ejaculatory duct. The cells that make up the epithelium of the VD, however, exhibit many characteristics of cells found in more complex epithelia, which are involved in absorption and/or secretion. In the present investigation, morphometry was utilized to characterize in detail the changes incurred by the human VD during its development, growth, and aging and to determine if these changes correlate with testicular maturation. In addition, the specific types of keratins present in the epithelial cells were defined, as well as desmin distribution in the muscular layers, during the various phases of the development, growth, and involution of the human VD. Results of the morphometric study are consistent with the interpretation that the development, growth, and aging of the VD are delayed, but parallel to, the identical phases exhibited by the human testis. Further, a differential expression of distinct keratin types was observed in the VD during the various phases examined in this study. Taken together, these two correlations may suggest that the VD is unlikely to function solely as a conduit for sperm. The rationale for this interpretation is as follows: 1) the complex developmental and maturational changes measured in the present investigation in the human VD are common to other absorptive and/or secretory epithelia; and 2) these changes parallel developmental changes observed in other androgen-dependent epithelia of the male reproductive tract, which also function to contribute components to seminal fluid as well as to provide a conduit for sperm.

Co-localization of nitric oxide synthase, neuropeptides and tyrosine hydroxylase in nerves supplying the human post-natal vas deferens and seminal vesicle.

To determine the distribution and patterns of co-localization of nitric oxide synthase (NOS), neuropeptides and tyrosine hydroxylase (TH) in intrinsic nerves of the human post-natal vas deferens and seminal vesicle. Double and triple immunolabelling methods were used in tissue from 10 male infants and children (age range 2 months to 3 years) obtained at post-mortem examinations carried out within 12 h of death. Most nerves supplying the muscle coat of either organ were TH-immunoreactive (-IR), most of which also contained neuropeptide Y (NPY) while a smaller proportion contained both NPY and NOS. Minor populations of the TH/NPY-IR intramuscular nerves contained calcitonin gene-related peptide (CGRP), galanin (GAL), met-enkephalin (m-ENK) or vasoactive intestinal polypeptide (VIP). Non-TH-IR intramuscular nerves were relatively infrequent and most contained NPY and either VIP or NOS. Presumptive secretomotor nerves formed subepithelial plexuses in both organs, most of which contained NPY co-localized with either VIP or NOS, with minor populations containing CGRP and/or GAL. TH- and substance P (SP) -IR nerves were not observed subepithelially. Perivascular nerve plexuses were mainly formed by TH-IR varicose nerves, most of which contained co-localized NPY and CGRP, with a smaller proportion containing NPY and NOS and minor populations containing VIP, m-ENK, SP or GAL. These results indicate that the autonomic control of the human vas deferens and seminal vesicle is provided by several immunohistochemically distinct nerve populations. Furthermore, NOS is present in a proportion of both the noradrenergic and non-noradrenergic nerves. Pharmacological studies are now required to elucidate the precise roles of nitric oxide and neuropeptides in the functional control of these organs.

The effects of tamsulosin, a high affinity antagonist at functional alpha 1A- and alpha 1D-adrenoceptor subtypes.

1. The actions of the alpha 1-adrenoceptor antagonist tamsulosin have been examined at functional alpha 1-adrenoceptor subtypes and compared with those at the human prostate receptor. 2. At the alpha 1D-adrenoceptors of the rat aorta, tamsulosin acted as a competitive antagonist with a high affinity (pKB = 10.1). 3. At the alpha 1B-adrenoceptor of the rat spleen and rabbit corpus cavernosum penis, tamsulosin again acted as a competitive antagonist but with a significantly lower affinity (pKB = 8.9-9.2). 4. Tamsulosin acted as an unsurmountable antagonist of the alpha 1A-adrenoceptor-mediated responses of the rat and human vas deferens, reducing maximal responses to phenylephrine by 20% and 50%, respectively, at an antagonist concentration of 1 nM. Responses of depolarized (100 mM KCl) rat vas deferens preparations were unaffected by 10 nM tamsulosin but this concentration reduced maximal responses to 5-hydroxytryptamine (5-HT) in this tissue. 5. When longer antagonist incubation periods (> or = 60 min) were used, tamsulosin behaved as a competitive antagonist on the human prostate with a significantly higher affinity (pKB = 10.0) than obtained at the alpha 1B-adrenoceptor. 6. The data demonstrate that tamsulosin is a high affinity antagonist at functional alpha 1-adrenoceptors with a selectivity alpha 1D > or = alpha 1A > alpha 1B. In some tissues the compound exhibits an additional unsurmountable antagonist action, the clinical significance of which is unknown.

Volume-sensitive chloride currents in primary cultures of human fetal vas deferens epithelial cells.

Using the patch-clamp technique, we have identified a large, outwardly rectifying, Cl--selective whole-cell current in primary cultures of human vas deferens epithelial cells. Whole-cell currents were time- and voltage-dependent and displayed inactivation following depolarising pulses >/= 60 mV. Currents were equally permeable to bromide (PBr/PCl = 1.05 +/- 0.04), iodide (PI/PCl = 1. 06 +/- 0.07) and Cl-, but significantly less permeable to gluconate (PGluc /PCl = 0.23 +/- 0.03). Currents spontaneously increased with time after establishing a whole-cell recording, but could be inhibited by exposure to a hypertonic bath solution which reduced inward currents by 68 +/- 4%. Subsequent exposure of the cells to a hypotonic bath solution led to a 418 +/- 110% increase in inward current, indicating that these currents are regulated by osmolarity. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid (100 microM) produced a rapid and reversible voltage-dependent block (60 +/- 5% and 10 +/- 7% inhibition of current, measured at +/- 60 mV, respectively). Dideoxyforskolin (50 microM) also reduced the volume-sensitive Cl- current, but with a much slower time course, by 41 +/- 13% and 32 +/- 16% (measured at +/- 60 mV, respectively). Tamoxifen (10 microM) had no effect on the whole-cell Cl- current. These results suggest that vas deferens epithelial cells possess a volume-sensitive Cl- conductance which has biophysical and pharmacological properties broadly similar to volume-sensitive Cl- currents previously described in a variety of cell types.

Two barium binding sites on a maxi K+ channel from human vas deferens epithelial cells

Using the patch clamp technique, we have investigated the blockade of maxi-K+ channels present on vas deferens epithelial cells by extracellular Ba2+. With symmetrical 140 mM K+ solutions, Ba2+ produced discrete blocking events consisting of both long closings of seconds duration (slow block) and fast closings of milliseconds duration (flickering block). Kinetic analysis showed that flickering block occurred according to an "open channel blocking" scheme and was eliminated by reducing external K+ to 4.5 mM. Slow block showed a complex voltage-dependence. At potentials between -20 mV and 20 mV, blockade was voltage-dependent; at potentials greater than 20 mV, blockade was voltage-independent, but markedly sensitive to the extracellular K+ concentration. These data reveal that the vas deferens maxi-K+ channel has two Ba2+ binding sites accessible from the extracellular side. Site one is located at the cytoplasmic side of the gating region and binding to this site causes flickering block. Site two is located close to the extracellular mouth of the channel and binding to this site causes slow block.

α1A‐Adrenoceptor‐mediated contractile responses of the human vas deferens

1. The predominant alpha 1-adrenoceptor mediating contractions of the human vas deferens has been characterised in vitro by use of subtype selective antagonists. 2. Responses of human epididymal vas deferens were obtained to phenylephrine in the presence of amine uptake inhibitors and propranolol. The effects of the alpha 1-adrenoceptor antagonists, 5-methylurapidil, oxymetazoline, WB4101, prazosin and chloroethylclonidine were examined and also the L-type calcium channel blocker, nifedipine. 3. 5-Methylurapidil, WB4101, oxymetazoline and prazosin acted as competitive antagonists of the responses to phenylephrine, yielding pA2 values of 8.8, 9.2, 7.7 and 8.8 respectively. All four antagonists produced Schild plots with slopes similar to unity and maximum responses to phenylephrine were not altered in the presence of any of the antagonists. 4. Tamsulosin (1 nM) caused rightward shifts of phenylephrine concentration-response curves yielding an apparent pKB value of 10.0. However, maximum responses were also reduced by 51% with this concentration of antagonist. 5. Incubation of tissues with chloroethylclonidine (100 microM for 40 min) failed to alter responses significantly but the presence of nifedipine (1 microM) reduced maximum responses to phenylephrine by 32%. 6. The high affinity of 5-methylurapidil, oxymetazoline and WB4101, together with the failure of chloroethylclonidine to antagonize responses, indicate that the predominant alpha 1-adrenoceptor mediating contraction of the human vas deferens has the characteristics previously described for the pharmacologically-defined alpha 1A-adrenoceptor. The data are also consistent with those described for the cloned alpha 1c-adrenoceptor subtype thereby supporting the hypothesis that the two receptors are identical. The human vas deferens therefore represents a readily accessible preparation for functional studies of the human alpha 1A-adrenoceptor.

Differential inhibition in the human vas deferens by phenoxybenzamine: a possible mechanism for its contraceptive action

The effect of phenoxybenzamine on in vitro contractile responses of human vasectomy tissues was investigated. Phenoxybenzamine (0.01-10 mumol l-1) produced a differential inhibition of contractions induced by noradrenaline (1-300 mumol l-1) in the human vas deferens in that it blocked the longitudinal but not the circular muscle. Contractions of both muscle types induced by noradrenaline (100 mumol l-1) were inhibited by either prazosin (0.01-10 mumol l-1), a competitive alpha 1-selective adrenoceptor antagonist or benextramine (0.01-10 mumol l-1), an irreversible alpha 1-adrenoceptor blocker, but were unaffected by chloroethylclonidine (0.1-100 mumol l-1), an irreversible alpha 1B-adrenoceptor blocker. In calcium-free/EGTA (1 mmol l-1) medium, contractions of the longitudinal but not circular muscle induced by noradrenaline (100 mumol l-1) were inhibited by phenoxybenzamine (0.01-1 mumol l-1). Chloroethylclonidine (10-100 mumol l-1) was ineffective on both muscle types in calcium-free medium, but, on readmission of calcium, in markedly inhibited the recovery of longitudinal but not the circular muscle. These results suggest a mechanism for the male contraceptive action of phenoxybenzamine by a selective blockade of longitudinal but not circular muscle contractions. The results are discussed in relation to (i) the possible involvement of receptor reserves for noradrenaline and of alpha 1-adrenoceptor subtypes coupled in the longitudinal and circular muscle to different mechanisms for increasing cytosolic calcium, (ii) a role in the longitudinal muscle for chloroethylclonidine-sensitive alpha 1-adrenoceptors in replenishment of a functional pool of intracellular calcium and (iii) the inhibition of sperm emission by phenoxybenzamine.