Pittner J, Liu R, Brown R, Wolgast M, Persson A E G
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Acta Physiol Scand. 2003 Nov;179(3):309-17. doi: 10.1046/j.1365-201X.2003.01137.x.
Nitric oxide (NO) is an important signal transmitter with multiple haemodynamic functions in the kidney. Study of these is complicated by the difficulty in measuring NO directly or visualizing its production. Recently the synthesis of a group of new NO-sensitive fluorescent dyes, diaminofluoresceins (DAF), suitable for imaging applications has been reported. We attempted to use one DAF (DAF-2 DA) to investigate the relationship between endothelial calcium, NO production and afferent arteriolar reactivity.
We used the isolated, perfused juxtamedullary nephron preparation (JMN) and loaded the afferent arteriolar endothelium with Fura-2 AM and DAF-2 DA (4,5-diaminofluorescein-2-diacetyl). After in vitro calibration of the imaging system, we measured Fura-2 and DAF-2 fluorescence in single endothelial cells of afferent arterioles (AA) perfused at a pressure of 100 mmHg.
Carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO) (10-3 m), a specific NO scavenger, decreased DAF-2 fluorescence in the endothelium by 16.1% and the mid-afferent arteriolar diameter by 10.2%, and increased endothelial calcium by 17.8%. Nomega-nitro-l-arginine methyl ester (l-NAME) (10-4 m) decreased fluorescence intensity of DAF-2 by 18.6%, increased cellular calcium level by 19.7% and constricted the vessels by 11.6%. Addition of carbachol (10-4 m) increased average DAF-2 fluorescence by 22.8% and endothelial calcium concentration by 28.9%, whereas the arteriolar diameter remained essentially unchanged. Carbachol failed to increase DAF-2 fluorescence when administered after l-NAME pre-treatment.
We conclude that endothelial NO homeostasis is an important determinant of AA reactivity and suggest that DAF are suitable for real-time imaging of afferent arteriolar NO production in the isolated, perfused JMN and may be used in combination with calcium-sensitive fluorophores. We have found that NO reduction by carboxy-PTIO or l-NAME increases endothelial calcium, suggesting involvement of calcium signalling in an autocrine NO production feedback in the endothelium. This method should help to further clarify the role of endothelial NO in renal haemodynamics.
一氧化氮（NO）是一种重要的信号传递分子，在肾脏中具有多种血液动力学功能。由于难以直接测量NO或观察其生成过程，对这些功能的研究变得复杂。最近，有报道称合成了一组适用于成像应用的新型NO敏感荧光染料——二氨基荧光素（DAF）。我们试图使用一种DAF（DAF-2 DA）来研究内皮细胞钙、NO生成与传入小动脉反应性之间的关系。
我们使用分离的、灌注的近髓肾单位制备物（JMN），并用Fura-2 AM和DAF-2 DA（4,5-二氨基荧光素-2-二乙酰）加载传入小动脉内皮细胞。在对成像系统进行体外校准后，我们测量了在100 mmHg压力下灌注的传入小动脉（AA）单个内皮细胞中的Fura-2和DAF-2荧光。
特异性NO清除剂羧基-2-苯基-4,4,5,5-四甲基-咪唑啉-1-氧基-3-氧化物（羧基-PTIO）（10⁻³ m）使内皮细胞中的DAF-2荧光降低了16.1%，使传入小动脉中段直径减小了10.2%，并使内皮细胞钙增加了17.8%。Nω-硝基-L-精氨酸甲酯（L-NAME）（10⁻⁴ m）使DAF-2的荧光强度降低了18.6%，使细胞钙水平升高了19.7%，并使血管收缩了11.6%。加入卡巴胆碱（10⁻⁴ m）使平均DAF-2荧光增加了22.8%，使内皮细胞钙浓度增加了28.9%，而小动脉直径基本保持不变。在L-NAME预处理后给予卡巴胆碱未能增加DAF-2荧光。
我们得出结论，内皮细胞NO稳态是AA反应性的重要决定因素，并表明DAF适用于在分离的、灌注的JMN中对传入小动脉NO生成进行实时成像，并且可与钙敏感荧光团联合使用。我们发现羧基-PTIO或L-NAME降低NO会增加内皮细胞钙，这表明钙信号参与了内皮细胞中自分泌NO生成的反馈。该方法应有助于进一步阐明内皮细胞NO在肾血流动力学中的作用。
