Presynaptic Regulation by ACh of the NE Mediated Responses in the Rat Vas Deferens

Abstract

In the isolated electrically stimulated vas deferens preparation the effect of exogenous acetylcholine (ACh) was studied. It was possible to differentiate two separate sites for the action of ACh. A postsynaptic effect (M₁) which is revealed as a sudden decrease in the basal tension of the muscular twitch, antagonized competitively by atropine (pA₂ = 8.44 ± 0.79), potentiated at a ratio of 10.24 by neostigmine, and not altered by hexamethonium, yohimbine, clonidine, theophylline or prazosin. Treatment with reserpine or 6-OH-DA induced a supersensitivity of this effect. The second action is a presycaptic effect (M₂), which is manifested by a rapid increase in the muscular twitch, is dose-dependent to Ach, and is potentiated significantly by reserpine, neostigmine and clonidine. This latter effect was not modified by atropine, hexamethonium, yohimbine, prazosin or theophylline. The normal efficacy of ACh is 185 times greater in presynaptic over postsynaptic receptors. The increase in the twitch is not considered to be by the release of endogenous ACh or ATP. The evidence presented here indicates that the presynaptic effect of ACh (M₂) is due to participation of α₂ - adrenoceptors in enhancement of the release of norepinephrine (NE) from the nerve terminals.     

The Vas Deferens of the Spanish Lobster,Scyllarus chacei

Summary

The male reproductive tract of Scyllarus chacei consists of paired testes and vasa deferentia that conduct sperm containing spermatophores to the genital pores at the base of each fifth walking leg. The testis is joined to the vas deferens which can be divided into four regions: (1) the anterior vas deferens can be further divided into three regions. It is highly convoluted and is the region in which the sperm become encapsulated in ovoid spermatophores of approximately 100 sperm as well as produces seminal fluids. (2) The middle vas deferens is the primary site of sperm storage and adds to seminal fluids which formed in the anterior region. (3) The posterior region is highly muscularized and may serve for limited sperm storage. (4) The most distal portion is the ejaculatory duct which is highly muscularized for extruding the spermatophoric mass for transfer to the female. A final seminar product is added here.     

Noradrenaline-Containing Cells in the Epididymis and Vas Deferens of the Tortoise Testudo hermanni

The vas deferens and epididymis of the tortoise, Testudo hermanni, contained very large amounts of noradrenaline but no certain amounts of adrenaline and dopamine. Small polymorf cells exhibiting a fluorescence typical of catecholamines were present in the tissues. It is concluded that most of the noradrenaline present in these organs is confined to the small cells. Their origin and possible function is discussed, and it is suggested that they may have the same function as the short adrenergic neurons present in the male genital tract of mammals.     

An Improved Method for the Purification of "Light" Noradrenaline Vesicles from Rat Vas Deferens: Some Biochemical Characteristics

"Light" noradrenaline storage vesicles from nerve endings have been isolated by differential centrifugation and differential gradient centrifugation. They have been further purified by isopycnic sucrose/D2O centrifugation. By using these centrifugation techniques, we obtained an isopycnic gradient fraction in which noradrenaline was enriched about 41 times versus a total homogenate. This factor could be raised to 61 by using seminal ducts of castrated rats. Comparison of the distribution patterns in sucrose/D2O isopycnic gradients indicated that light noradrenaline vesicles of nerve endings contain Mg2+-stimulated ATPase and ATP, but that only a minor part of the dopamine beta-hydroxylase can be associated with these vesicles.     

Characterization of [3H]Ro 5-4864 Binding Sites in Rat Vas Deferens

The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5-4864 as a radioligand. The binding of [3H]Ro 5-4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 +/- 0.7 x 10(7) M-1 min-1, and the dissociation rate constant (k-1) was 0.031 +/- 0.003 min-1. The dissociation constant (KD) determined by saturation binding was 5.22 +/- 0.56 nM. The density of binding was 4,926 +/- 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 +/- 0.01, an indication that [3H]Ro 5-4864 binds to a single site. The [3H]Ro 5-4864 binding was inhibited competitively by Ro 5-4864 and 2-hydroxy-5-nitrobenzyl-6-thioguanosine and noncompetitively by PK 11195, nitrendipine, alpha,beta-methylene-ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5-4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5-4864 binding sites in rat vas deferens is suggested.     

Comparative Assessment of the Functional Activity of Rat Epididymal Mitochondria in Oxidative Stress Induced by Hyperhomocysteinemia and L-NAME Administration

Journal of Evolutionary Biochemistry and Physiology - The state of protein carbonylation with the determination of the adaptive reserve potential and analysis of the proportion of primary and...     

CFTR Haplotype Analysis Reveals Genetic Heterogeneity in the Etiology of Congenital Bilateral Aplasia of the Vas Deferens

Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations.     

Immunolocalization of Aquaporin-1, -5, and -7 in the Avian Testis and Vas Deferens

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been found in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs have been poorly investigated in the male reproductive system of birds. The localization of AQP subtypes (AQP1, 2, 3, 4, 5, 7, 8, 9, and 11) in the goose testis and vas deferens has been studied through immunohistochemistry and immunobloting. Interestingly, the testicular and deferential tissues were positive for AQP1, -5, and -7 but not the others. AQP1 immunoreactivity was detected in the capillary endothelial cells of testis and vas deferens. AQP5 was localized in the interstitial tissue of the testis, including Leydig cells, as well as in the basal cells of vas deferens. Double-labeling confocal microscopy revealed coexpression of AQP5 with capillary AQP1 in the testis. AQP7 was expressed in elongated spermatid and spermatozoa tails in the testis, as well as spermatozoa tails in the vas deferens. These results suggest that several subtypes of AQPs are involved in the regulation of water homeostasis in the goose male reproductive system. (J Histochem Cytochem 57:915–922, 2009)     

Modulation of A-type potassium current in smooth-muscle cells of the rat Vas Deferens by Ca2+/calmodulin-dependent protein kinase II

Using a standard patch-clamp technique in the perforated patch configuration, we studied the effect of a highly specific membrane-permeable inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII), KN-93, on fast outward A-type potassium current in isolated smooth-muscle cells (SMCs) of an epididymal region of the rat vas deferens. This inhibitor significantly changed the dynamics of the studied current; in particular, it increased the rate of inactivation and considerably slowed down the recovery after inactivation. In the presence of 5 μM KN-93, we observed a moderate (nearly by 20%) decrease in the peak amplitude of fast A-type current. Based on the data obtained, we conclude that voltage-sensitive fast A-type potassium current in SMCs of the epididymal part of the rat vas deferens can be significantly regulated by the activity of CaM-KII. Therefore, by influencing the kinetic characteristics of the above current, this enzyme can be indirectly involved in the control of electrical activity in SMCs. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 419–422, July–October, 2007.     

The Effect of Intracellular Current Pulses in Smooth Muscle Cells of the Guinea Pig Vas Deferens at Rest and during Transmission

The effect of intracellular current pulses on the membrane of smooth muscle cells of the guinea pig vas deferens at rest and during transmission was studied. Two main response types were identified: active response cells, in which a spike was initiated in response to depolarizing currents of sufficient strength and duration; passive response cells, in which depolarizing currents gave only electrotonic potential changes. These cells were three times more numerous than the active response cells. During the crest of the active response the input resistance fell by about 25% of the resting value. Comparison of the active response with the action potential due to stimulating the hypogastric nerve showed that the former was smaller in amplitude and had a slower rate of rise and higher threshold. Electrical coupling occurred between the smooth muscle cells during the propagation of the action potential. Depolarizing current pulses had no effect on the amplitude of the excitatory junction potential (E.J.P.) in passive response cells, but in general did decrease its amplitude in active response cells. These results are discussed with respect to the mechanism of autonomic neuroeffector transmission.     

Analysis of Epithelial Protein Profiles of Prostatic Glands Induced Heterotypically in the Bladder Epithelium of the Rat

When urinary bladder epithelia of rats were grown in association with fetal urogenital sinus mesenchyme, prostatic morphogenesis was induced. The epithelial proteins were examined by HPLC fractionation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). More than 500 bands of silver-stained epithelial proteins were analyzed. The glandular epithelia induced from both adult and fetal bladder epithelia lost all of the 7 bladder-specific bands (BE 1–7) in most recombinants and expressed a number of prostate-specific bands. Among the 18 bands commonly found in all prostatic lobes, 13 (PE 4, 7–18) were constantly and 3 (PE 1–3) were sporadically detected, while the other 2 (PE 5 and 6) bands were not detected when the adult epithelium was used in recombination. Among the 7 prostatic lobe-specific bands (vPE 14, dPE 1–3), most of them were detected when the fetal epithelium was used, while few of them when the adult epithelium was used. These results demonstrate that prostatic morphogenesis induced in the bladder epithelium was associated with most of biochemical features of prostate. In addition to the biochemical study, histological examination revealed that the prostatic differentiation was more complete in the fetal bladder epithelium than the adult one.     

大鼠输精管平滑肌细胞的体外培养及形态学特征(英文）

建立大鼠输精管平滑肌细胞的培养方法。取大鼠输精管,剥离外膜和内膜,用组织块法进行体外培养。用抗α-SMA(anti α-smooth muscle actin)免疫组化染色的方法鉴定培养的细胞。结果显示,在倒置显微镜下观察细胞形态多样,表现为长梭形或星形,细胞伸出突起互相接触,彼此融合,部分区域细胞多层重叠,部分区域细胞单层高低起伏,呈"峰-谷"状生长。免疫组化染色鉴定呈阳性反应,用该方法所分离、培养的输精管平滑肌细胞纯度达99%以上。应用组织块法培养大鼠输精管平滑肌细胞,操作简单,结果稳定。     

Methylprednisolone Attenuates Airway and Vascular Responses Induced by Reactive Oxygen Species in Isolated, Plasma-Perfused Rat Lungs

The effects of methylprednisolone (MP) on the acute airway and pulmonary vascular responses induced by reactive oxygen species (ROS) were investigated in isolated, plasma-perfused rat lungs. ROS were generated by adding xanthine oxidase and hypoxan-thine to the perfusate. MP was administered in 3 different ways: 1. Added to the perfusate (1 mg*ml^-1) 5 min prior to xanthine oxidase and hypoxanthine, 2. Given as intraperitoneal injections (40 mg*kg^-1) to lung donor rats 12 and 2 hours prior to the experiments, or 3. Combining 1 and 2. The lungs were perfused at constant volume inflow (15 ml*min^-1). Pulmonary arterial pressure and transpulmonary pressure were followed for 30 min after addition of xanthine oxidase and hypoxanthine. ROS induced a powerful, acute broncho- and vasoconstriction, which was inhibited by addition of MP to the perfusate. Pretreatment with MP also inhibited the vascular and airway responses. Adding MP to the perfusate of pretreated lungs further reduced the ROS-induced smooth muscle constriction. In conclusion, MP inhibits vasoconstriction and bronchocon-striction induced by ROS in isolated rat lungs.     

Lack of Rapid Desensitization of Ca2+ Responses in Transfected CHO Cells Expressing the Rat Neurotensin Receptor Despite Agonist-Induced Internalization

Abstract: Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca²⁺]_i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca²⁺]_i resulted from release of Ca²⁺ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca²⁺]_i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for ～70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells. A possible absence of desensitization of the neurotensin receptor itself is also discussed.     

Antibody-Induced Lysis of Isolated Rat Epididymal Adipocytes and Complement Activation In Vivo

Objective: To identify human monoclonal antibodies selectively binding to human adipocytes and to evaluate their ability to induce lysis of isolated rat adipocytes in vitro and to reduce rat complement levels in vivo. Research Methods and Procedures: Using phage display technology, human monoclonal antibodies binding to human adipocyte plasma membranes were identified. Three antibodies (Fat 13, Fat 37, and Fat 41) were selected based on their additional cross-reaction with rat adipocytes and reformatted as a rat chimeric IgG2bs. The ability of these antibodies, both singly and in combination, to induce lysis of rat epididymal adipocytes in vitro and the reduction of serum complement levels in vivo in the rat was evaluated. Results: All antibodies caused similar time- and dose-dependent lysis of isolated rat adipocytes. Calculated mean EC₅₀ values (maximum percentage of lysis in parentheses) were 0.680 μg/mL (63.2%), 0.546 μg/mL (72.4%), and 0.391 μg/mL (73.7%) for Fat 13, Fat 37, and Fat 41, respectively. Combinations were no more effective than individual antibodies in inducing lysis. Anti-adipocyte antibodies (both singly and in combination) were also similarly effective in vivo. In rats, doses of monoclonal antibody up to 10 mg/kg intraperitoneal generally caused almost complete depletion of serum complement up to 24 hours after dosing recovering to baseline values by day 5. Discussion: Individual and combinations of monoclonal anti-adipocyte antibodies produced a complement-dependent and concentration-dependent activity to lyse adipocytes in vitro and in vivo as measured by a dramatic depletion in serum complement.     

Testosterone Replacement Therapy Prevents Alterations of Coronary Vascular Reactivity Caused by Hormone Deficiency Induced by Castration

The present study aimed to determine the effects of chronic treatment with different doses of testosterone on endothelium–dependent coronary vascular reactivity in male rats. Adult male rats were divided into four experimental groups: control (SHAM), castrated (CAST), castrated and immediately treated subcutaneously with a physiological dose (0.5 mg/kg/day, PHYSIO group) or supraphysiological dose (2.5 mg/kg/day, SUPRA group) of testosterone for 15 days. Systolic blood pressure (SBP) was assessed at the end of treatment through tail plethysmography. After euthanasia, the heart was removed and coronary vascular reactivity was assessed using the Langendorff retrograde perfusion technique. A dose–response curve for bradykinin (BK) was constructed, followed by inhibition with 100 μM L-NAME, 2.8 μM indomethacin (INDO), L-NAME + INDO, or L-NAME + INDO + 0.75 μM clotrimazole (CLOT). We observed significant endothelium–dependent, BK–induced coronary vasodilation, which was abolished in the castrated group and restored in the PHYSIO and SUPRA groups. Furthermore, castration modulated the lipid and hormonal profiles and decreased body weight, and testosterone therapy restored all of these parameters. Our results revealed an increase in SBP in the SUPRA group. In addition, our data led us to conclude that physiological concentrations of testosterone may play a beneficial role in the cardiovascular system by maintaining an environment that is favourable for the activity of an endothelium–dependent vasodilator without increasing SBP.