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pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy

Muller-Borer, Barbara J ; Yang, Hua ; Marzouk, Sayed A. M ; Lemasters, John J ; Cascio, Wayne E

American journal of physiology. Heart and circulatory physiology, 1998-12, Vol.275 (6), p.H1937 [Periódico revisado por pares]

United States

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  • Título:
    pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy
  • Autor: Muller-Borer, Barbara J ; Yang, Hua ; Marzouk, Sayed A. M ; Lemasters, John J ; Cascio, Wayne E
  • Assuntos: Acidosis - metabolism ; Ammonium Chloride - pharmacology ; Animals ; Carbon Dioxide - pharmacology ; Extracellular Space - drug effects ; Extracellular Space - metabolism ; Hydrogen - metabolism ; Hydrogen-Ion Concentration ; Hypercapnia - metabolism ; In Vitro Techniques ; Intracellular Membranes - drug effects ; Intracellular Membranes - metabolism ; Methods ; Microscopy, Confocal ; Microscopy, Fluorescence ; Myocardium - metabolism ; Perfusion ; Rabbits
  • É parte de: American journal of physiology. Heart and circulatory physiology, 1998-12, Vol.275 (6), p.H1937
  • Descrição: Departments of 1  Medicine and 2  Cell Biology and Anatomy, University of North Carolina School of Medicine; and 3  Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-7075 Confocal microscopy and the H + -sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pH i ) or extracellular pH (pH o ) 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 µm 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, pH o was measured during continuous perfusion of SNARF-1 free acid. pH i 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 pH i and pH o were altered by applying CO 2 externally, exchanging HEPES and bicarbonate buffers, and changing pH i with NH 4 Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pH i and pH o changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pH i regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pH i . carboxyseminaphthorhodafluor-1; ventricular myocardium; carbon dioxide; acidosis; alkalosis
  • Editor: United States
  • Idioma: Inglês
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