Bukoski R D, Bergmann C, Gairard A, Stoclet J C
Laboratoire de Physiologie et Pharmacodynamie, Faculte de Pharmacie, Universite Louis Pasteur, Strasbourg, France.
Am J Physiol. 1989 Nov;257(5 Pt 2):H1728-35. doi: 10.1152/ajpheart.1989.257.5.H1728.
A method is described for the simultaneous measurement of intracellular Ca2+ ([Ca2+]i) and force generation in isolated resistance arteries using the fluorescent Ca2+ indicator fura-2. Branch II mesenteric resistance arteries were isolated from 12-wk-old Wistar-Kyoto rats and mounted in a wire myograph. The myograph was placed on the stage of a compound microscope interfaced with a dual excitation wavelength fluorometer, and the tissue was loaded with fura-2 by incubation over a 30-min period with the cell-permeable form of the dye. When stimulated with physiological salt solution containing 100 mM KCl and 10 microM norepinephrine, a rapid and transient increase in [Ca2+], was observed to precede active force development and plateau at approximately 60% of the maximal level after 50 s. Washout of the agonists induced relaxation of these small arteries, consisting of an 85% decline in active tension over 100 s and a fall in [Ca2+]i to 50% of prerelaxation level over the same time period. Forskolin (1 microM), which increases intracellular adenosine 3',5'-cyclic monophosphate, induced a 50% relaxation over a 150-s period that was preceded by a fall in [Ca2+]i. Nearly identical results were obtained with 100 microM sodium nitroprusside, which stimulates intracellular guanosine 3',5'-cyclic monophosphate production. These findings indicate that the initiating event of forskolin- and sodium nitroprusside-induced relaxation may be a reduction of [Ca2+]i. The method described is useful for examining basic physiological events and Ca2+-related mechanisms of action of vasoactive compounds in isolated resistance arteries.
本文描述了一种使用荧光钙指示剂fura-2同时测量离体阻力动脉细胞内Ca2+（[Ca2+]i）和力产生的方法。从12周龄的Wistar-Kyoto大鼠分离出肠系膜阻力动脉分支II，并将其安装在钢丝肌动描记器中。将肌动描记器放置在与双激发波长荧光计相连的复式显微镜载物台上，通过用细胞可渗透形式的染料孵育30分钟，使组织加载fura-2。当用含有100 mM KCl和10 μM去甲肾上腺素的生理盐溶液刺激时，观察到[Ca2+]i迅速短暂增加，先于主动力产生，并在50秒后达到最大水平的约60%时趋于平稳。冲洗激动剂可诱导这些小动脉舒张，包括在100秒内主动张力下降85%，以及在同一时间段内[Ca2+]i降至舒张前水平的50%。可增加细胞内3',5'-环磷酸腺苷的福斯可林（1 μM）在150秒内诱导50%的舒张，此前[Ca2+]i下降。用100 μM硝普钠刺激细胞内鸟苷3',5'-环磷酸产生，也得到了几乎相同的结果。这些发现表明，福斯可林和硝普钠诱导舒张的起始事件可能是[Ca2+]i的降低。所描述的方法对于研究离体阻力动脉中血管活性化合物的基本生理事件和Ca2+相关作用机制很有用。
