Two-probe microfluorometry estimation of transmembrane potential (ΔΨ(p)) slight changes in individual hepatocytes under short-term hormone action.

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.

Effect of tilorone and its analogues on the change of mitochondrial potential of rat hepatocytes.

The influence of tilorone dihydrochloride and its analogues--diphenyl derivatives on the changes of transmembrane potential of mitochondrial membranes of the isolated rat hepatocytes has been estimated. Authors have shown a significant increase in mitochondrial potential thirty minutes after the introduction of the test compounds to the cells using the fluorescent probe JC-1. These results indicate the rapid activation with tilorone and its analog--dihydrochloryde 4,4'-bis-[2-(diethylamino)ethoxy]diphenyl--of the RLR signaling pathway. The final stage of this pathway is the cell production of IFN type I. The authors concluded that there is an increasing of the organelles resistance to the extra/intracellular damaging agents under the influence of the test compounds.