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 (Δψp) 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 Δψp 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 Δψp changes.
The first increase of Δψp between 2 and 5 min (ΔΔψp=−8.6±4.2 mV) after the Ca2+-mobilizing hormone addition evidently depends on Na+/K+-ATPase activation. The second peak of hyperpolarization (ΔΔψp=−13.2±3.2 mV) between 25 and 30 min, after a transient decrease of Δψp (ΔΔψp=10.9±4.3 mV) at 15 min of experiment, probably is mediated by phenylephrine stimulating action on K+-chennels.
Ca2+ chelating with EDTA didnt 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 Δψp but it abolished the second phase of hyperpolarization. It points to PLD involvement in the mediated by K+-channels Δψp fluctuations in response to phenylephrine.
Based on SqSC4 fluorescent parameters estimation of Δψp 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 Δψp changes in non-excitable individual cells at short-term hormone action.
30 Apr - 04 May 2011
European Society of Endocrinology