Two-probe microfluorometry estimation of transmembrane potential (ΔΨ(p)) slight changes in individual hepatocytes...

Early events in individual hepatocytes of rat activated by adrenaline (10(-6)M) and phenylephrine (10(-5)M) have been investigated by quantitative image microfluorometry and microspectrofluorometry. Cationic DiOC2 and anionic SqSC4 probes have been used for image analysis and transmembrane potential (ΔΨ p) estimation in real-time studies. Fluorescence spectra resulting from the accumulation of dyes in single cells were recorded. Based on the mean fluorescence intensity, the magnitude of ΔΨ p was calculated by Nernst equation adapted for lipophilic cationic probes. DiOC2 has revealed that both hormones induce biphasic hyperpolarization of hepatocytes membrane with α-agonist phenylephrine causing ΔΨ p changes at higher amplitude. The first increase of ΔΨ p within 2 and 5 min (ΔΔΨ p = -8.6 ± 4.2 mV) apparently related to Na(+)/K(+)-ATPase activation by the Ca(2+)-mobilizing hormone. The second peak of hyperpolarization (ΔΔΨ p = -13.2 ± 3.2 mV) between 25 and 30 min, after a transient decrease of ΔΨ p (ΔΔΨ p = 10.9 ± 4.3 mV) over 15 min experiment, probably is mediated by phenylephrine stimulating action on K(+)-channels. K(+) channel blocker (Ba(2+) or 4-aminopyridine) as well as elevating of extracellular K(+) prevented the hyperpolarization. Modulation of PLD-dependent signal transduction pathway by 0.4% butanol had a weak influence on the first increase of ΔΨ p but it abolished the second phase of hyperpolarization. That points to PLD involvement in the ΔΨ p fluctuations mediated by K(+)-channels in response to phenylephrine. Based on SqSC4, fluorescent parameters estimation of relative changes of ΔΨ p revealed similar character of time dependence with two phases of hyperpolarization. Synchronic fluctuation of ΔΨ p determined by oppositely charged probes demonstrate that the quantitative microfluorometry allows to evaluate slight ΔΨ p changes separately from ΔΨ m in non-excitable individual cells at the short-term hormone action.