Orlov S N, Tremblay J, Hamet P
Céntre de Recherche Hôtel-Dieu de Montréal, Université de Montréal, Québec, Canada.
J Membr Biol. 1996 Sep;153(2):125-35. doi: 10.1007/s002329900116.
To examine the involvement of Na+,K+,2Cl- cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on 86Rb, 36Cl and 22Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward 86Rb fluxes were not different. Bumetanide decreased basal 86Rb uptake by 70-75% with a Ki of approximately 0.2-0.3 microM. At concentrations ranging up to 1 microM, bumetanide did not affect 36Cl influx and reduced it by 20-30% in the range from 3 to 100 microM. In contrast to 86Rb and 36Cl influx, bumetanide did not inhibit 22Na uptake by VSMC. BS 86Rb uptake was completely abolished in Na(+)- or Cl(-)-free media. In contrast to 86Rb, basal BS 36Cl influx was not affected by Nao+ and Ko+. Hyperosmotic and isosmotic shrinkage of VSMC increased 86Rb and 36Cl influx to the same extent. Shrinkage-induced increments of 86Rb and 36Cl uptake were completely abolished by bumetanide with a Ki or approximately 0.3 microM. Shrinkage did not induce BS 86Rb and 36Cl influx in (Na+ or Cl-)- and (Na+ or K+)-depleted media, respectively. In the presence of an inhibitor of Na+/H+ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated 22Na influx. Bumetanide (1 microM) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na+,K+,2Cl- cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K+ influx is mediated by (Nao+ + Clo-)-dependent K+/K+ exchange and Nao(+)-dependent K+,Cl- cotransport, respectively.
为研究钠钾氯协同转运体在血管平滑肌细胞（VSMC）单价离子通量中的作用，我们比较了布美他尼对大鼠主动脉VSMC静息培养物摄取⁸⁶Rb、³⁶Cl和²²Na的影响。在基础条件下，布美他尼敏感（BS）的内向和外向⁸⁶Rb通量值无差异。布美他尼使基础⁸⁶Rb摄取降低70 - 75%，其抑制常数（Ki）约为0.2 - 0.3微摩尔。在浓度高达1微摩尔时，布美他尼不影响³⁶Cl内流，而在3至100微摩尔范围内使其降低20 - 30%。与⁸⁶Rb和³⁶Cl内流不同，布美他尼不抑制VSMC对²²Na的摄取。在无钠或无氯培养基中，BS⁸⁶Rb摄取完全被消除。与⁸⁶Rb不同，基础BS³⁶Cl内流不受Na⁺和K⁺影响。VSMC的高渗和等渗收缩使⁸⁶Rb和³⁶Cl内流增加程度相同。布美他尼（Ki约为0.3微摩尔）可完全消除收缩诱导的⁸⁶Rb和³⁶Cl摄取增加。在分别缺乏（Na⁺或Cl⁻）和（Na⁺或K⁺）的培养基中，收缩未诱导BS⁸⁶Rb和³⁶Cl内流。在存在钠氢交换抑制剂（EIPA）的情况下，高渗或等渗收缩均未激活²²Na内流。布美他尼（1微摩尔）不改变基础VSMC体积以及钠、钾和氯的细胞内含量，但消除了等渗收缩VSMC的调节性容积增加。这些数据表明VSMC中不存在功能性钠钾氯协同转运体，并提示在这些细胞中，基础和收缩诱导的BS钾内流分别由（Na⁺ + Cl⁻）依赖性钾/钾交换和Na⁺依赖性钾氯协同转运介导。
