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  • 1
    ISSN: 1432-2013
    Keywords: Malpighian tubule ; Formica ; Electrophysiology ; Cable analysis ; Dinitrophenol ; Protonophore
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In Formica Malpighian tubules KCl secretion is driven by a V-type H+ ATPase in the luminal membrane in parallel with a H+/K+ antiporter. The effect of the protonophore dinitrophenol (DNP) was investigated on the isolated, symmetrically perfused tubule. DNP was applied in two different concentrations: 0.2 mmol/l and 1 mmol/l. The effects were fast and rapidly reversible. The equivalent short-circuit current (I sc) was reduced significantly to respectively 25±3% Cn=4) and −3±7% (n=11) of the control value when 0.2 mmol/ l or 1 mmol/l was added to the bath. When 1 mmol/l DNP was applied the transepithelial resistance (R te) decreased significantly to 74±11% of the control value (n=11), and the luminal over basolateral voltage divider ratio (VDR), providing an estimate of luminal over basolateral membrane resistance, decreased to 37±12% of the control (n=6). A concentration of 1 mmol/l DNP was also applied from the lumen. The decrease in I sc was significant, but much less pronounced (74±5% of control; n=6) and no significant changes in R te and VDR were observed. It is argued that, when the concentration in the bath is high enough, DNP may cross the cell and have a protonophoric effect not only on the mitochondria but also across the luminal cell membrane explaining the drop in transepithelial and in relative luminal membrane resistance. The diminished effectiveness of DNP, when applied from the luminal side, suggests that the luminal membrane is somehow less permeable to toxic substances, but that DNP very rapidly enters the cell via the basolateral membrane and may bring about an initial protonophoric effect across this membrane.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-4994
    Keywords: Ratiometric ion indicators ; Fura-2 ; calibration equations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract The absolute values of intracellular ion concentrations as monitored by specific fluorescent indicators are determined by using calibration curves obtained underin vitro andin vivo conditions. In the derivation of the calibration curve by Grynkiewicz et al [(1985)J. Biol. Chem 260, 3440] it is implicitly assumed that the observed fluorescence signal is directly related to the concentrations of the free dye and the dye-ion complex in the ground state. We modified the calibration equation so that ion binding and dissociation in the excited state are taken into account. The extended calibration equation assumes the knowledge of the rate constants in the excited state. Expressions for the calibration curve assuming the absence or presence of an excited-state reaction are compared for the Ca2+ indicator Fura-2. The excited-state rate constants are determined by global compartmental analysis of time-resolved fluorescence decays of Fura-2 collected at various excitation and emission wavelengths using different Ca2+ concentrations. It is found that for Fura-2 there is negligible interference of the excited-state reaction so that the original calibration can used.
    Type of Medium: Electronic Resource
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