Department of Physiology and Biophysics, SUNY at Stony Brook, NY 11794-8661, USA.
J Membr Biol. 2010 Feb;233(1-3):51-62. doi: 10.1007/s00232-010-9224-y. Epub 2010 Feb 4.
We previously demonstrated a transmural gradient in Na/K pump current (I (P)) and Na(+), with the highest maximum I (P) and lowest Na(+) in epicardium. The present study examines the relationship between the transmural gradient in I (P) and Na/Ca exchange (NCX). Myocytes were isolated from canine left ventricle. Whole-cell patch clamp was used to measure current generated by NCX (I (NCX)) and inward background calcium current (I (ibCa)), defined as inward current through Ca(2+) channels less outward current through Ca(2+)-ATPase. When resting myocytes from endocardium (Endo), midmyocardium (Mid) or epicardium (Epi) were studied in the same conditions, I (NCX) was the same and I (ibCa) was zero. Moreover, Western blots were consistent with NCX protein being uniform across the wall. However, the gradient in Na(+), with I (ibCa) = 0, should create a gradient in Ca(2+). To test this hypothesis, we measured resting Ca(2+) using two methods, based on either transport or the Ca(2+)-sensitive dye Fura2. Both methods demonstrated a significant transmural gradient in resting Ca(2+), with Endo > Mid > Epi. This gradient was eliminated by exposing Epi to sufficient ouabain to partially inhibit Na/K pumps, thus increasing Na(+) to values similar to those in Endo. These data support the existence of a transmural gradient for Ca(2+) removal by NCX. This gradient is not due to differences in expression of NCX; rather, it is generated by a transmural gradient in Na(+), which is due to a transmural gradient in plasma membrane expression of the Na/K pump.
我们之前已经证明，钠钾泵电流 (I (P)) 和 Na(+) 存在跨壁梯度，心外膜的最大 I (P) 和最低 Na(+)。本研究检查了 I (P) 的跨壁梯度与钠钙交换 (NCX) 之间的关系。从犬左心室分离心肌细胞。使用全细胞膜片钳技术测量 NCX 产生的电流 (I (NCX)) 和内向背景钙电流 (I (ibCa))，定义为通过 Ca(2+) 通道的内向电流减去通过 Ca(2+)-ATP 酶的外向电流。当在相同条件下研究心内膜 (Endo)、心肌中层 (Mid) 或心外膜 (Epi) 的心肌细胞时，I (NCX) 相同，I (ibCa) 为零。此外，Western 印迹与整个壁上均匀的 NCX 蛋白一致。然而，Na(+) 的梯度，I (ibCa) = 0，应该会产生 Ca(2+) 的梯度。为了验证这一假设，我们使用两种基于转运或 Ca(2+) 敏感染料 Fura2 的方法测量了静息 Ca(2+)。这两种方法都显示静息 Ca(2+) 存在明显的跨壁梯度，Endo > Mid > Epi。通过将 Epi 暴露于足够的哇巴因以部分抑制钠钾泵，从而将 Na(+) 增加到与 Endo 相似的值，消除了这种梯度。这些数据支持 NCX 存在钙去除的跨壁梯度。该梯度不是由于 NCX 表达的差异造成的；相反，它是由 Na(+) 的跨壁梯度引起的，这是由于钠钾泵质膜表达的跨壁梯度引起的。
