TY - JOUR
T1 - PHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy
AU - Muller-Borer, Barbara J.
AU - Yang, Hua
AU - Marzouk, Sayed A.M.
AU - Lemasters, John J.
AU - Cascio, Wayne E.
PY - 1998
Y1 - 1998
N2 - Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHj) or extracellular pH (pH0) 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 ; /m 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. pH; 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 pH0 were altered by applying CC> 2 externally, exchanging HEPES and bicarbonate buffers, and changing pH; with NH4C1 washout. We conclude that in situ confocal fluorescent microscopy can measure pH; and pH0 changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pH; regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.
AB - Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHj) or extracellular pH (pH0) 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 ; /m 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. pH; 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 pH0 were altered by applying CC> 2 externally, exchanging HEPES and bicarbonate buffers, and changing pH; with NH4C1 washout. We conclude that in situ confocal fluorescent microscopy can measure pH; and pH0 changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pH; regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.
KW - Acidosis
KW - Alkalosis
KW - Carbon dioxide
KW - Carboxyseminaphthorhodafluor-1
KW - Ventricular myocardium
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M3 - Article
C2 - 9843791
AN - SCOPUS:0032416118
SN - 0002-9513
VL - 275
SP - H1937-H1947
JO - American Journal of Physiology
JF - American Journal of Physiology
IS - 6 PART 2
ER -