Muller-Borer B J, Yang H, Marzouk S A, Lemasters J J, Cascio W E
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7075, USA.
Am J Physiol. 1998 Dec;275(6):H1937-47. doi: 10.1152/ajpheart.1998.275.6.H1937.
Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 micron were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pHi measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.
共聚焦显微镜和对H⁺敏感的荧光团羧基半萘罗丹明氟-1（SNARF-1）用于测量离体动脉灌注兔乳头肌的细胞内pH（pHi）或细胞外pH（pHo）。从悬浮在与焦平面倾斜的可控气氛中的灌注圆柱形肌肉中，同时记录心内膜表面和下方心肌至300微米深度的单激发、双发射荧光图像。加入丁二酮肟可抑制收缩。在单独的肌肉中，在连续灌注SNARF-1游离酸期间测量pHo。在肌肉加载SNARF-1/AM且细胞外空间清除残留荧光团后进行pHi测量。初步实验证明了作为pH、图像深度和荧光团浓度函数的比率测量的一致性，从而确立了该方法用于定量壁内pH测量的潜在可行性。在随后的实验中，该方法在离体动脉灌注兔乳头肌中，在正常动脉灌注期间以及通过外部施加CO₂、交换HEPES和碳酸氢盐缓冲液以及用NH₄Cl洗脱改变pHi和pHo时得到了验证。我们得出结论，原位共聚焦荧光显微镜可以测量动脉灌注心室心肌的心内膜表面和更深心内膜层的pHi和pHo变化。该方法有可能研究灌注心肌中pHi的调节，在气体、代谢物和肽的扩散预计会改变调节pHi的过程的边界处进行研究。
