Bretylium abolishes neurotransmitter release without necessarily abolishing the nerve terminal action potential in sympathetic terminals

BACKGROUND AND PURPOSE: The antidysrhythmic bretylium is useful experimentally because it selectively abolishes neurotransmitter release from sympathetic peripheral nerve terminals. Its mechanism of action seemed settled, but recent results from optical monitoring of single terminals now suggests a new interpretation.EXPERIMENTAL APPROACH: Orthograde transport of a dextran-conjugated Ca(2+) indicator to monitor Ca(2+) in nerve terminals of mouse isolated vas deferens with a confocal microscope. In some experiments, local neurotransmitter release was detected by monitoring neuroeffector Ca(2+) transients (NCTs) in adjacent smooth muscles, a local measure of purinergic transmission. Sympathetic terminals were identified with catecholamine fluorescence (UV excitation) or post-experiment immunohistochemistry.KEY RESULTS: Bretylium (10 microM) abolished NCTs at 60/61 junctions over the course of 2 h, indicating effective abolition of neurotransmitter release. However, bretylium did not abolish the field stimulus-induced Ca(2+) transient in most nerve terminals, but did increase both action potential delay (by 2+/-0.4 ms) and absolute refractory period (by 4+/-2 ms). Immunohistochemistry demonstrated that 85-96% of terminals orthogradely filled with a dextran-conjugated fluorescent probe contained Neuropeptide Y (NPY). A formaldehyde-glutaraldehyde-induced catecholamine fluorescence (FAGLU) technique was modified to allow sympathetic terminals to be identified with a Ca(2+) indicator present. Most terminals contained catecholamines (based on FAGLU) or secrete ATP (as NCTs in adjacent smooth muscle cells are abolished).CONCLUSIONS AND IMPLICATIONS: Bretylium can inhibit neurotransmitter release downstream of Ca(2+) influx without abolishing the nerve terminal action potential. Bretylium-induced increases in the absolute refractory period permit living sympathetic terminals to be identified.     

Caffeine-inducible ATP release is mediated by Ca2+-signal transducing system from the endoplasmic reticulum to mitochondria

Adenosine triphosphate (ATP) is released as an autocrine/paracrine signal from a variety of cells. The present study was undertaken to clarify the Ca(2+)-signal pathway involved in the caffeine-inducible release of ATP from cultured smooth muscle cells (SMC). The release of ATP induced by caffeine (3 mM) was almost completely inhibited by ryanodine and tetracaine, but not by 2-APB, thus being mediated by ryanodine receptors (RyR). The expression of messenger RNA from only RyR-2 was detected in the cells. Furthermore, the induced release was attenuated by mitochondrial inhibitors, rotenone and oligomycin and by Cl(-) channel blockers, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Increase in Ca(2+)-signals with fluo 4 and rhod-2 caused by caffeine were reduced by tetracaine and oligomycin plus carbonyl cyanide m-chlorophenylhydrazone, respectively. A close spatial relation between the endoplasmic reticulum (ER) and mitochondria was electromicroscopically observed in the SMC, supporting the existence of a Ca(2+)-signaling bridge on both the organelli. These results suggest that caffeine stimulates ryanodine receptor (RyR-2) and facilitates a Ca(2+)-signal transducing system from ER to mitochondria, and then, the signal appears to accelerate the ATP synthesis in mitochondria. In addition, the mitochondrial event may lead further cell signaling to the cell membrane and activates Cl(-) channels, resulting in the extracellular release of cytosolic ATP.     

Antioxidant treatment ameliorates diabetes‐induced dysfunction of the vas deferens in a rat model

Diabetes mellitus (DM) affects the male ejaculatory function. This study was designed to evaluate the role of oxidative stress in the development of diabetes‐induced dysfunction of vas deferens (VD) in the rat. DM was induced by streptozotocin in 40 male Wistar rats. Subsequently, the diabetic animals were divided into three groups: DM group, DM + Eda group and DM + Tau group. These groups were administered saline, edaravone and taurine, respectively, daily for 4 weeks. Another group of ten rats served as a control group. DM was diagnosed in the 40 streptozotocin‐injected rats. DM significantly reduced the VD weight. Additionally, DM induced in vitro VD hypercontractility, VD histological abnormalities and increased the serum and VD tissue concentration of malondialdehyde. VD immunohistochemistry revealed overexpression of three markers of oxidative stress. DM significantly reduced serum testosterone levels. No live birth was documented in all DM rats in mating experiments. Antioxidants significantly improved all the aforementioned parameters, except the testosterone levels. This study indicates a deleterious impact of DM‐induced oxidative stress on VD histological and functional features. Antioxidant treatment may provide an adjunct tool to alleviate ejaculatory disorders for male patients with type 1 diabetes.     

The CFTR gene mild variants poly‐T,TG repeats and M470V detection in Indian men with congenital bilateral absence of vas deferens

The aim of the study was to detect the frequency of the CFTR gene variants poly‐T, TG repeats and c.1408A>G p.Met470Val (M470V) in Indian men with congenital bilateral absence of the vas deferens (CBAVD). Men diagnosed with CBAVD (n = 76), their female partners (n = 76) and healthy men from general population (n = 50) were recruited. Genomic DNA was isolated and the polymorphic regions of IVS9‐ c.1210‐12T [5] and M470V were amplified using specific primers followed by Sanger's DNA sequencing. A statistically significant increase in the frequency of heterozygous IVS9‐ c.1210‐12T [5] (39.4%) was observed in CBAVD men as compared to controls (14%). The allelic distribution of c.1210‐12T [5], c.1210‐12T [7] and c.1210‐12T [9] in CBAVD men was 21%, 64.4% and 13% and that in healthy controls was 7%, 73% and 20% respectively. Longest TG repeat c.1210‐34TG [13] was found in association with c.1210‐12T [5] with an allelic frequency of 5.9% in CBAVD men. We found a significant association of c.1210‐34TG [12]/c.1210‐34TG [13] ‐ c.1210‐12[5] –V470 allele in CBAVD men. Twelve female partners harboured a heterozygous c.1210‐12T [5] allele. The study emphasises the need to screen both partners for the polymorphisms M470V, poly‐T, TG tract repeats in addition to population‐specific known CFTR gene mutations.     

先天性精囊缺如的临床特点和诊断分析

目的 探讨先天性精囊缺如的临床特点，提高其诊断水平。方法 分析21例患者的临床资料。经超声检查、盆腔CT或精道造影检查确诊。6例行睾丸穿刺活检或附睾穿刺检查。结果 双侧精囊缺如14例，单侧精囊缺如7例。合并先天性双侧输精管缺如（CBAVD）者13例，合并单侧输精管缺如（CUA-VD）者5例，合并双侧输精管异位开口于苗勒管囊肿者1例，同时合并单侧输精管缺如及对侧输精管异位开口于苗勒管囊肿者2例。6例行睾丸或附睾穿刺者均生精功能正常。结论 先天性精囊缺如不会单独出现，常合并输精管缺如或输精管末端异位开口。双侧精囊缺如者无生育能力，单侧精囊缺如者临床表现复杂多变。仔细检查阴囊内容物多可提供重要信息；超声、盆腔CT和精道造影检查可确诊。     

Vas Deferens Abscess Rupture: A Case Report

A vas deferens abscess is a very rare complication of acute vasitis and lower urinary tract infection. A case of vas deferens rupture due to an abscess with severe pelvic inflammation requiring surgical drainage is reported.Teaching Point: Vas deferens abscess rupture is an example of a very rare complication of severe inflammation of the vas deferens.     

Relaxation of hormonally stimulated smooth muscular tissues by the 8-bromo derivative of cyclic GMP

Summary Substances that cause contraction or relaxation of smooth muscle have been shown to increase intracellular levels of cyclic GMP. Because of the unclear role of cyclic GMP in the control of smooth muscle tone, cyclic GMP derivatives were exogenously applied to various smooth muscle preparations and their effects on tissue tone were studied.Whereas the basal tone of the rat ductus deferens was not affected by exogenous cyclic GMP or its dibutyryl or 8-bromo derivatives, the contractile responses of this tissue to noradrenaline and acetylcholine were depressed by preincubation with 10 M 8-bromo cyclic GMP (Br-cGMP). The 8-bromo derivatives of 2:3-cyclic GMP, 5-GMP and guanosine were without effects. Cyclic AMP levels were not changed by Br-cGMP. The frequency of oxytocin-stimulated rat uteri was also depressed by Br-cGMP (10 M). In helical strips of rat and rabbit aortae, Br-cGMP (1–100 M) caused a concentration-dependent, rapid decrease in noradrenaline-stimulated tissue tension. Br-2:3-cyclic GMP was ineffective. Noradrenaline-stimulated strips from hog spleen arteries were less sensitive to Br-cGMP than aortic tissue. In ductus deferentes and aortic strips stimulated by K⁺ at a depolarizing concentration, Br-cGMP caused less relaxation than under hormonal stimulation.These findings support the concept that cyclic GMP is involved in the control of smooth muscle tone and that hormone- and drug-induced elevations of the cyclic GMP level can reduce contractile responses to neurotransmitters and hormones.Abbreviations cGMP Guanosine 3:5-monophosphate, cyclic GMP - dibutyryl cGMP N², 2-O-dibutyryl guanosine 3:5-monophosphate - Br-cGMP 8-bromo guanosine 3:5-monophosphate - Br-2:3-cGMP 8-bromo guanosine 2:3-monophosphate - Br-GMP 8-bromo guanosine 5-monophosphate - Br-Guo 8-bromo guanosine, Br-guanosine - cAMP adenosine 3:5-monophosphate, cyclic AMP - dibutyryl cAMP N⁶, 2-O-dibutyryl adenosine 3:5-monophosphate - Br-cAMP 8-bromo adenosine 3:5-monophosphate This work was supported by grants from the Deutsche Forschungsgemeinschaft. Preliminary reports were presented (Schultz, 1977b; Schultz et al., 1978).     

爱普列特对原代培养大鼠输精管上皮细胞及bcl-2表达的影响

目的　探讨爱普列特对原代培养SD大鼠输精管上皮细胞及原癌基因bcl 2表达的影响。方法　应用HE染色、电镜、流式细胞术和免疫组化方法研究爱普列特对输精管上皮细胞形态改变、细胞凋亡率以及bcl 2表达的影响。结果　 0 1 μmol·L- 1 爱普列特对细胞形态无显著影响 ;0 3和 1 0 μmol·L- 1 爱普列特作用 72h后 ,细胞呈核固缩、深染、碎裂、染色质边集等多种形态改变 ,电镜下可见典型的凋亡细胞特征 ;流式细胞仪分析结果显示 ,溶剂对照组细胞凋亡率为 3 42 %± 1 49% ,0 1、0 3和 1 0 μmol·L- 1 爱普列特作用 72h后 ,细胞凋亡率分别为 4 66 %± 2 2 3 % ,39 0 4 %± 1 0 69%和 52 74%± 8 91 % ;免疫组化结果显示输精管上皮细胞bcl 2阳性率为 76 96 %± 9 2 5 % ,0 1、0 3和 1 0 μmol·L- 1 爱普列特作用 72h后 ,Bcl 2阳性表达的百分率分别为 63 93 %± 3 40 %、52 82 %± 8 66 %和 2 9 64 %± 8 74%。结论　 0 1 μmol·L- 1 爱普列特对大鼠输精管上皮细胞无影响 ,0 3和 1 0 μmol·L- 1 可诱导大鼠输精管上皮细胞发生凋亡 ,其作用机制可能与降低bcl 2表达有关