Abstract：
OBJECTIVE: To evaluate the basal release of 6-nitrodopamine (6-ND) from human isolated seminal vesicles (HISV) and to characterize its action and origin.
METHODS: Left HISV obtained from patients undergoing prostatectomy surgery was suspended in a 3-mL organ bath containing warmed (37 °C) and gassed (95%O2:5%CO2) Krebs-Henseleit\'s solution (KHS) with ascorbic acid. An aliquot of 2 mL of the supernatant was used to quantify catecholamines by LC-MS/MS. For functional studies, concentration-responses curves to catecholamines were obtained, and pEC50 and Emax values were calculated. Detection of tyrosine hydroxylase and S100 protein were also carried out by both immunohistochemistry and fluorescence in-situ hybridization assays (FISH).
RESULTS: Basal release of 6-ND was higher than the other catecholamines (14.76 ± 14.54, 4.99 ± 6.92, 3.72 ± 4.35 and 5.13 ± 5.76 nM for 6-ND, noradrenaline, adrenaline, and dopamine, respectively). In contrast to the other catecholamines, the basal release of 6-ND was not affected by the sodium current (Nav) channel inhibitor tetrodotoxin (1 μM; 10.4 ± 8.9 and 10.4 ± 7.9 nM, before and after tetrodotoxin, respectively). All the catecholamines produced concentration-dependent HISV contractions (pEC50 4.1 ± 0.2, 4.9 ± 0.3, 5.0 ± 0.3, and 3.9 ± 0.8 for 6-ND, noradrenaline, adrenaline, and dopamine, respectively), but 6-ND was 10-times less potent than noradrenaline and adrenaline. However, preincubation with very low concentration of 6-ND (10-8 M, 30 min) produced significant leftward shifts of the concentration-response curves to noradrenaline. Immunohistochemical and FISH assays identified tyrosine hydroxylase in tissue epithelium of HISV strips.
CONCLUSIONS: Epithelium-derived 6-ND is the major catecholamine released from human isolated seminal vesicles and that modulates smooth muscle contractility by potentiating noradrenaline-induced contractions.
METHODS: Left HISV obtained from patients undergoing prostatectomy surgery was suspended in a 3-mL organ bath containing warmed (37 °C) and gassed (95%O2:5%CO2) Krebs-Henseleit\'s solution (KHS) with ascorbic acid. An aliquot of 2 mL of the supernatant was used to quantify catecholamines by LC-MS/MS. For functional studies, concentration-responses curves to catecholamines were obtained, and pEC50 and Emax values were calculated. Detection of tyrosine hydroxylase and S100 protein were also carried out by both immunohistochemistry and fluorescence in-situ hybridization assays (FISH).
RESULTS: Basal release of 6-ND was higher than the other catecholamines (14.76 ± 14.54, 4.99 ± 6.92, 3.72 ± 4.35 and 5.13 ± 5.76 nM for 6-ND, noradrenaline, adrenaline, and dopamine, respectively). In contrast to the other catecholamines, the basal release of 6-ND was not affected by the sodium current (Nav) channel inhibitor tetrodotoxin (1 μM; 10.4 ± 8.9 and 10.4 ± 7.9 nM, before and after tetrodotoxin, respectively). All the catecholamines produced concentration-dependent HISV contractions (pEC50 4.1 ± 0.2, 4.9 ± 0.3, 5.0 ± 0.3, and 3.9 ± 0.8 for 6-ND, noradrenaline, adrenaline, and dopamine, respectively), but 6-ND was 10-times less potent than noradrenaline and adrenaline. However, preincubation with very low concentration of 6-ND (10-8 M, 30 min) produced significant leftward shifts of the concentration-response curves to noradrenaline. Immunohistochemical and FISH assays identified tyrosine hydroxylase in tissue epithelium of HISV strips.
CONCLUSIONS: Epithelium-derived 6-ND is the major catecholamine released from human isolated seminal vesicles and that modulates smooth muscle contractility by potentiating noradrenaline-induced contractions.
摘要：
目的：评估人离体精囊（HISV）中6-硝基多巴胺（6-ND）的基础释放，并表征其作用和起源。
方法：将从接受前列腺切除术的患者中获得的左HISV悬浮在3-mL器官浴中，其中包含温热（37°C）和充气（95％O2：5％CO2）的Krebs-Henseleit溶液（KHS）和抗坏血酸。2毫升上清液的等分试样用于通过LC-MS/MS定量儿茶酚胺。对于功能研究，获得了对儿茶酚胺的浓度响应曲线，并计算pEC50和Emax值。还通过免疫组织化学和荧光原位杂交测定(FISH)进行酪氨酸羟化酶和S100蛋白的检测。
结果：6-ND的基础释放高于其他儿茶酚胺（6-ND为14.76±14.54、4.99±6.92、3.72±4.35和5.13±5.76nM，去甲肾上腺素,肾上腺素,还有多巴胺,分别)。与其他儿茶酚胺相比，6-ND的基础释放不受钠电流（Nav）通道抑制剂河豚毒素（1μM;10.4±8.9和10.4±7.9nM，河豚毒素之前和之后，分别)。所有儿茶酚胺均产生浓度依赖性HISV收缩（6-ND的pEC50为4.1±0.2、4.9±0.3、5.0±0.3和3.9±0.8，去甲肾上腺素,肾上腺素,还有多巴胺,分别)，但6-ND的效力比去甲肾上腺素和肾上腺素低10倍。然而,用非常低的浓度的6-ND（10-8M，30分钟）对去甲肾上腺素的浓度-反应曲线产生了显着的向左移动。免疫组织化学和FISH测定鉴定了HISV条带的组织上皮中的酪氨酸羟化酶。
结论：上皮衍生的6-ND是从人分离的精囊中释放的主要儿茶酚胺，通过增强去甲肾上腺素诱导的收缩来调节平滑肌收缩性。
方法：将从接受前列腺切除术的患者中获得的左HISV悬浮在3-mL器官浴中，其中包含温热（37°C）和充气（95％O2：5％CO2）的Krebs-Henseleit溶液（KHS）和抗坏血酸。2毫升上清液的等分试样用于通过LC-MS/MS定量儿茶酚胺。对于功能研究，获得了对儿茶酚胺的浓度响应曲线，并计算pEC50和Emax值。还通过免疫组织化学和荧光原位杂交测定(FISH)进行酪氨酸羟化酶和S100蛋白的检测。
结果：6-ND的基础释放高于其他儿茶酚胺（6-ND为14.76±14.54、4.99±6.92、3.72±4.35和5.13±5.76nM，去甲肾上腺素,肾上腺素,还有多巴胺,分别)。与其他儿茶酚胺相比，6-ND的基础释放不受钠电流（Nav）通道抑制剂河豚毒素（1μM;10.4±8.9和10.4±7.9nM，河豚毒素之前和之后，分别)。所有儿茶酚胺均产生浓度依赖性HISV收缩（6-ND的pEC50为4.1±0.2、4.9±0.3、5.0±0.3和3.9±0.8，去甲肾上腺素,肾上腺素,还有多巴胺,分别)，但6-ND的效力比去甲肾上腺素和肾上腺素低10倍。然而,用非常低的浓度的6-ND（10-8M，30分钟）对去甲肾上腺素的浓度-反应曲线产生了显着的向左移动。免疫组织化学和FISH测定鉴定了HISV条带的组织上皮中的酪氨酸羟化酶。
结论：上皮衍生的6-ND是从人分离的精囊中释放的主要儿茶酚胺，通过增强去甲肾上腺素诱导的收缩来调节平滑肌收缩性。
