Effect of chronic clonidine treatment on transmitter release from sympathetic varicosities of the guinea-pig vas deferens

1 Previous studies have demonstrated that chronic pre-synaptic inhibition of transmitter release by morphine evokes a counter-adaptive response in the sympathetic nerve terminals that manifests itself as an increase in transmitter release during acute withdrawal. In the present study we examined the possibility that other pre-synaptically acting drugs such as clonidine also evoke a counter-adaptive response in the sympathetic nerve terminals. 2 In chronically saline treated (CST) preparations, clonidine (0.5 muM) completely abolished evoked transmitter release from sympathetic varicosities bathed in an extracellular calcium concentration ([Ca2+](o)) of 2 mM. The inhibitory effect of clonidine was reduced by increasing [Ca2+](o) from 2 to 4 mM and the stimulation frequency from 0.1 to 1 Hz. 3 The nerve terminal impulse (NTI) was not affected by concentrations of clonidine that completely abolished evoked transmitter release. 4 Sympathetic varicosities developed a tolerance to clonidine (0.5 muM) following 7-9 days of chronic exposure to clonidine. 5 Acute withdrawal of preparations following chronic clonidine treatment (CCT) resulted in a significant (P

Effect of chronic morphine treatment on alpha(2)-adrenoceptor mediated autoinhibition of transmitter release from sympathetic varicosities of the mouse vas deferens

1 The effect of chronic morphine treatment (CMT) on sympathetic innervation of the mouse vas deferens and on alpha (2)-adrenoceptor mediated autoinhibition has been examined using intracellular recording of excitatory junction potentials (EJPs) and histochemistry. 2 In chronically saline treated (CST) preparations. morphine (1 muM) and the alpha (2)-adrenoceptor agonist (clonidine, 1 muM) decreased the mean amplitude of EJPs evoked with 0.03 Hz stimulation by 81+/-8% (n=16) and 92+/-6% (n=7) respectively. In CMT preparations, morphine (1 muM) and clonidine (1 muM) decreased mean EJP amplitude by 68+/-8% (n = 7) and 79+/-8% (n = 7) respectively. 3 When stimulating the sympathetic axons at 0.03 Hz. the mean EJP amplitude recorded from smooth muscles acutely withdrawn from CMT was four times greater than for CST smooth muscles (40.7+/-3.8 mV, n = 7 compared with 9.9+/-0.3 mV, n = 7). 4 Part of the increase in mean EJP amplitude following CMT was produced by a 31% increase in the density of sympathetic axons and varicosities innervating the smooth muscle. 5 Results from the present study indicate that the effectiveness of alpha (2)-adrenocrptor mediated autoinhibition is only slightly reduced in CMT preparations. Most of the cross tolerance which develops between morphine, clonidine and alpha (2)-adrenoceptor mediated autoinhibition occurs as a consequence of increased efficacy of neuromuscular transmission which is produced by an increase in the probability of transmitter release and an increase in the density of sympathetic innervation.

Spatial distribution and developmental appearance of postjunctional P2X(1) receptors on smooth muscle cells of the mouse vas deferens

P2X(1)-type purinoceptors, have been shown to mediate fast transmission between sympathetic varicosities and smooth muscle cells in the mouse vas deferens but the spatial organization of these receptors on the smooth muscle cells remains inconclusive. Voltage clamp techniques were used to estimate the amplitudes of spontaneous excitatory junction currents (SEJCs) in cells of the vas deferens longitudinal smooth muscle layer. These currents involved the activation of about 6% of the P2X-type channels present on the cell, as compared to whole cell currents produced when isolated smooth muscle cells were exposed to maximal concentrations of either ATP or alpha,beta -MeATP. Immunofluorescence staining of the vas deferens with antibodies against P2X(1) receptor showed a diffuse, grainy distribution over the entire membrane of each smooth muscle cell. Anti-P2X(1) staining was not markedly clustered beneath anti-SV2-stained sympathetic varicosities. Similar results were obtained for cells in the urinary bladder. During development, P2X(1) mRNA was detected as early as embryonic day 15 (E15). Increasing intensities of diffuse immunostaining for P2X(1) were observed in the walls of the bladder, tail artery, and aorta from E15 until 6 weeks postnatal. The vas deferens showed increasing intensities of diffuse staining of its smooth muscle layers between 2 and 6 weeks postnatal, consistent with the time-course of development of fast purinergic transmission described previously. Together, the results suggest that the response of smooth muscle of the vas deferens to ATP released from sympathetic varicosities relies on rapidly desensitizing P2X(1) receptors, distributed diffusely across the smooth muscle cell surface. Synapse 42:1-11, 2001. (C) 2001 Wiley-Liss, Inc.

Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens

Background: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. Methods: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. Results: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. Conclusions: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD. The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

Melatonin modulation of presynaptic nicotinic acetylcholine receptors located on short noradrenergic neurons of the rat vas deferens: a pharmacological characterization

Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on nerve terminals of the rat vas deferens. Recently we showed the presence of high affinity nicotine-binding sites during the light phase, and low and high affinity binding sites during the dark phase. The appearance of the low affinity binding sites was due to the nocturnal melatonin surge and could be mimicked by exposure to melatonin in vitro. The aim of the present research was to identify the receptor subtypes responsible for the functional response during the light and the dark phase. The rank order of potency of agonists was dimethylphenylpiperazinium (DMPP) = cytisine > nicotine > carbachol and DMPP = nicotine = cytisine > carbachol, during the light and dark phase, respectively, due to an increase in apparent affinity for nicotine. Mecamylamine similarly blocked the DMPP response during the light and the dark phase, while the response to nicotine was more efficiently blocked during the light phase. In contrast, methyllycaconitine inhibited the nicotine-induced response only at 21:00 h. Since a7 nicotinic acetylcholine receptors (nAChRs) have low affinity for nicotine in binding assays, we suggest that a mixed population composed of a3ß4 - plus a7-bearing nAChR subtypes is present at night. This plasticity in receptor subtypes is probably driven by melatonin since nicotine-induced contraction in organs from animals sacrificed at 15:00 h and incubated with melatonin (100 pg/ml, 4 h) is not totally blocked by mecamylamine. Thus melatonin, by acting directly on the short adrenergic neurons that innervate the rat vas deferens, induces the appearance of the low affinity binding site, probably an a7 nAChR subtype.

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 ~9.5), phentolamine (pA2 ~8.3) and yohimbine (pA2 ~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 (~9.5), benoxathian (~9.7), 5-methylurapidil (~8.5), indoramin (~8.7) and BMY 7378 (~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.

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

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

Can vitamins C and E restore the androgen level and hypersensitivity of the vas deferens in hyperglycemic rats?

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Immunolocalization of aquaporins 1, 2 and 7 in rete testis, efferent ducts, epididymis and vas deferens of adult dog

The transepithelial movement of water into the male reproductive tract is an essential process for normal male fertility. Protein water channels, referred to as aquaporins (AQPs), are involved in increasing the osmotic permeability of membranes. This study has examined the expression of AQP1, AQP2, and AQP7 in epithelial cells in adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis, initial segment, caput, corpus and cauda epididymidis, and vas deferens were investigated by immunohistochemistry and Western blotting procedures to show the localization and distribution of the AQPs. AQP1 was noted in rete testis, in efferent ducts, and in vessels in the intertubular space, suggesting that AQP1 participated in the absorption of the large amount of testicular fluid occurring characteristically in the efferent ducts. AQP2 expression was found in the rete testis, efferent ducts and epididymis, whereas AQP7 was expressed in the epithelium of the proximal regions of the epididymis and in the vas deferens. This is the first time that AQP2 and AQP7 have been observed in these regions of mammalian excurrent ducts, but their functional role in the dog male reproductive tract remains unknown. Investigations of AQP biology could be relevant for clinical studies of the male reproductive tract and to technologies for assisted procreation.

Vas Deferens Surface Epithelium of Agouti paca: Fine Structural Features

The surface epithelium of the vas deferens of Agouti paca, a wild and large South American rodent, was basically formed by principal and basal cells being only the principal cells related to endocytosis processes and also secretion taking base on their cytoplasmic ultrastructural features. Principal cell of vas deferens epithelium were characterized mainly by presence of vesicles with several shapes, sizes and internalized content at their apical cytoplasm occurring smaller pits and pale small vesicles seen next to the apical brush border of microvillus. Moreover, coated vesicles, smooth surface vesicles and great vesicles; multivesicular bodies, endosomes and lysosomes were seen. Presence of an apocrine secretory apparatus was also viewed, showing apical cytoplasmic expansions protruding into the vas deferens luminal compartment. The basal flattened cells, without luminal surface contact, occurred next to the basement membrane of the ductus, and did no exhibit special ultrastructural features.

Functional effects of castration on alpha(1)-adrenoceptors in rat vas deferens

The effects of castration on alpha(1)-adrenoceptors in rat vas deferens were investigated by determining the actions of selective antagonists against the contractions induced by noradrenaline. The results obtained in vas deferens from control rats suggest participation of alpha(1A)-adrenoceptors as judged by the pA(2) values for prazosin (9.6), benoxathian (9.5), 2(2,6-dimethoxyphenoxyethyl) amino-methyl-1,4-benzodioxone hydrochloride) (WB 4101) (9.6), phentolamine (8.4), 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dionedihydrochloride (BMY 7378) (6.7) and by the insensitivity to chloroethylclonidine (100 mu M, 45 min). In vas deferens from castrated rats, WE 4101 and spiperone showed slopes lower than 1.0 in the Schild plots, suggesting participation of multiple receptors. In these organs, noradrenaline contractions were partially inhibited by chloroethylclonidine (100 mu M, 45 min), indicating participation of alpha(1B)-adrenoceptors. After chloroethylclonidine treatment, WE 4101 showed a slope not different from 1.0 in the Schild plot, resulting in a pA(2) of 9.4, which indicates an interaction with alpha(1A)-adrenoceptors. It is suggested that castration modifies the functional alpha(1)-adrenoceptors subtypes in rat vas deferens. (C) 1998 Elsevier B.V. B.V. All rights reserved.

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

The contractions of the rat vas deferens in response to noradrenaline are mediated through alpha(1A)-adrenoceptors. We observed participation of alpha(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 pA(2) values (congruent to 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 ( approximate to 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 alpha(1B)-adrenoceptors are detectable in vas deferens from at least the 7th through the 30th day after castration and that testosterone prevents this plasticity. (C) 2003 Elsevier B.V. B.V. All rights reserved.

Functional characterisation of alpha(1)-adrenoceptors in denervated rat vas deferens

We investigated whether or not surgical denervation of the rat vas deferens changes the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline. Denervated vas deferens was approximate to22 times more sensitive to noradrenaline (pD(2)=7.35 +/- 0.04) than control vas (pD(2)= 6.01 +/- 0.03). This difference in noradrenaline potency was eliminated when cocaine (6 muM) was added to control vas (pD(2)=7.22 +/- 0.04). The noradrenaline-induced contractions of control and denervated vas deferens were insensitive to the alpha(1B)/alpha(1D)-adrenoceptor alkylating agent chloroethylclonidine (100 muM, 45 min). The concentration-response curves to noradrenaline in control and denervated vas deferens were competitively antagonised by prazosin (pA(2)approximate to9.6), WB-4101 (pA(2)approximate to9.5), 5-methyl urapidil (pA(2)approximate to8.4), phentolamine (pA(2)approximate to8.7), yohimbine (pA(2)approximate to6.9), BMY 7378 (pA(2)approximate to6.9) and indoramin (pA(2)approximate to8.7). After the treatment of control and denervated vas deferens with phenoxybenzamine, the partial agonist oxymetazoline antagonised competitively the concentration-response curves to noradrenaline showing pA(2) values approximate to7.4 in both groups. We conclude that noradrenaline-induced contractions in control and denervated rat vas deferens are mediated by alpha(1A)-adrenoceptors and that surgical denervation of the rat vas deferens is not able to change the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline.

Adrenergic response patterns in vas deferens isolated from rats under diurnal rhythms - Influence of stress

The aim of this study was to verify, by means of functional methods, whether the circadian rhythm changes adrenergic response patterns in the epididymal half of the vas deferens isolated from control rats as well as from rats submitted to acute stress. The experiments were performed at 9:00 a.m., 3:00 p.m., 9:00 p.m., and 3:00 a.m. The results showed a light-dark dependent variation of the adrenergic response pattern on organs isolated from control as well as from stressed rats. In the control group, only the phenylephrine sensitivity was changed throughout the circadian rhythm. Under the stress condition, both norepinephrine and phenylephrine response patterns were changed, mainly during darkness. The maximal contractile response to both alpha- and beta-agonist and alpha(1)-agonist was increased in the dark phase, corresponding to high plasmatic concentrations of endogenous melatonin. The vas deferens isolated from stressed rats during the light phase simultaneously incubated with exogenous melatonin showed the same pattern of response obtained in the dark phase, thus indicating a peripheric action of melatonin on this organ. Therefore, the circadian rhythms are important to the adrenergic response pattern in rat vas deferens from both control and stressed rats. In conclusion, we suggest a melatonin modulation on alpha(1)-postsynaptic adrenergic response in the rat vas deferens. (c) 2005 Elsevier B.V. All rights reserved.