Endocrine Abstracts

Early events in activated by adrenaline (10⁻⁶ M) and phenylephrine (10⁻⁵M) single hepatocytes of rat have been investigated by quantitative image microfluorometry and microspectrofluorometry. Membrane potential-sensitive cationic DiOC₂(3) and anionic SqSC4 probes have been used for image analysis and transmenbrane potential (Δψ_p) estimation in real-time studies. Fluorescence spectra resulting from the accumulation of dyes in single cells were recorded. A special data acquisition program allowed measuring fluorescent cell parameters. Based on the mean fluorescence intensity the magnitude of Δψ_p was calculated by Nernst equation adapted for lipophilic cationic probes.

DiOC₂(3) has revealed that both hormones induced biphasic hyperpolarization of hepatocytes membrane with alpha-agonist phenylephrine causing a higher amplitude of Δψ_p changes.

The first increase of Δψ_p between 2 and 5 min (ΔΔψ_p=−8.6±4.2 mV) after the Ca²⁺-mobilizing hormone addition evidently depends on Na⁺/K⁺-ATPase activation. 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) at 15 min of experiment, probably is mediated by phenylephrine stimulating action on K⁺-chennels.

Ca²⁺ chelating with EDTA didn’t influence the curve character; however it slightly decreased the hormonal effect on the second peak amplitude.

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. It points to PLD involvement in the mediated by K⁺-channels Δψ_p fluctuations in response to phenylephrine.

Based on SqSC4 fluorescent parameters estimation of Δψ_p relative changes showed the same character of time dependence curve with two phase of hyperpolarization. Synchronic fluctuation of this parameter determined by oppositely charged probes demonstrates that quantitative microfluorimetry allows estimation of slight Δψ_p changes in non-excitable individual cells at short-term hormone action.