Kowarski D, Shuman H, Somlyo A P, Somlyo A V
J Physiol. 1985 Sep;366:153-75. doi: 10.1113/jphysiol.1985.sp015790.
The subcellular composition of relaxed and noradrenaline-contracted rabbit main pulmonary artery smooth muscle cells was measured by electron probe X-ray microanalysis of cryosections of rapidly frozen tissue. Some of the preparations were made permeable with saponin and exposed to a known free Ca ion concentration, rapidly frozen, freeze-substituted, and also analysed by electron probe X-ray microanalysis. 98% of intracellular K could be replaced by Rb. This was done to remove the K peak that partially overlaps the Ca peak in the X-ray spectra. The final [Rb]i plus residual [K]i was not significantly different from the [K]i of normal tissue. The [Ca]i in Rb-containing tissue was not significantly different from the [Ca]i in normal, K-containing tissue. Non-mitochondrial micro-regions containing high [Ca] (up to 33 mmol/kg dry wt.) were found at sites 200 nm or more away from the plasma membrane. These micro-regions also contained high [P]. We consider the identification of these regions containing high [Ca] as sarcoplasmic reticulum (s.r.), validated by: (a) conventional electron micrographs that show no other structures in main pulmonary artery smooth muscle in sufficient quantity and location to account for the frequency of these regions, (b) the previous localization of strontium, a functional calcium analogue, in the central s.r. in these smooth muscles (Somlyo & Somlyo, 1971 a), (c) the present demonstration that the central s.r. in this tissue can accumulate large amounts of calcium oxalate. The proportion of regions containing high [Ca] (greater than 12.0 mmol/kg dry wt.) was significantly higher in relaxed (35 of 330 measurements) than in the contracted (14 of 337) tissues (P less than 0.005), or 26 of 34 vs. 6 of 31 high [Ca] measurements in regions identified as s.r. through their high phosphorus content (P less than 0.006). This difference is thought to represent Ca release from the central s.r. There was no significant difference (P greater than 0.05) between the distributions of P in relaxed and contracted smooth muscle. The total cell [Ca]i in relaxed Rb-containing tissue, measured with randomly positioned small probes (3.6 mmol/kg dry wt.), was the same as that measured with large defocused probes, indicating the validity of random sampling. A mathematical model was used to estimate the frequency of including s.r. (35 nm diameter and 5% of cell volume) by a randomly positioned electron probe (50 nm), because we could not visualize s.r. in the cryosections.(ABSTRACT TRUNCATED AT 400 WORDS)
通过对快速冷冻组织的冷冻切片进行电子探针X射线微分析，测量了松弛和去甲肾上腺素收缩的兔主肺动脉平滑肌细胞的亚细胞组成。一些标本用皂角苷使其通透，并暴露于已知的游离钙离子浓度下，快速冷冻，冷冻替代，然后也通过电子探针X射线微分析进行分析。细胞内98%的钾可以被铷替代。这样做是为了去除在X射线光谱中部分与钙峰重叠的钾峰。最终的细胞内铷浓度加上残余的细胞内钾浓度与正常组织的细胞内钾浓度没有显著差异。含铷组织中的细胞内钙浓度与正常含钾组织中的细胞内钙浓度没有显著差异。在距离质膜200纳米或更远的部位发现了含有高钙（高达33 mmol/kg干重）的非线粒体微区。这些微区也含有高磷。我们认为这些高钙区域的鉴定为肌浆网（s.r.），这通过以下几点得到验证：（a）传统电子显微镜照片显示，在主肺动脉平滑肌中没有其他结构在数量和位置上足以解释这些区域的出现频率；（b）之前在这些平滑肌的中央肌浆网中锶（一种功能性钙类似物）的定位（Somlyo和Somlyo，1971a）；（c）目前证明该组织的中央肌浆网可以积累大量草酸钙。在松弛组织（330次测量中有35次）中，含有高钙（大于12.0 mmol/kg干重）的区域比例显著高于收缩组织（337次测量中有14次）（P小于0.005），或者在通过高磷含量鉴定为肌浆网的区域中，高钙测量值为34次中有26次，而收缩组织中31次中有6次（P小于0.006）。这种差异被认为代表了中央肌浆网释放钙。在松弛和平滑肌收缩状态下，磷的分布没有显著差异（P大于0.05）。用随机定位的小探针（3.6 mmol/kg干重）测量的含铷松弛组织中的总细胞内钙浓度与用大散焦探针测量的结果相同，表明随机采样的有效性。由于我们无法在冷冻切片中观察到肌浆网，因此使用数学模型来估计随机定位的电子探针（50纳米）包含肌浆网（直径35纳米，占细胞体积的5%）的频率。（摘要截取自400字）
