NDLI: Two-Probe Microfluorometry Estimation of Transmembrane Potential (ΔΨ $_{p}$) Slight Changes in Individual Hepatocytes Under Short-Term Hormone Action

Content Provider	Springer Nature Link
Author	Kavok, N. S. Malyukina, M. Y. Borovoy, I. A. Obukchova, E. N. Klimov, S. A.
Copyright Year	2013
Abstract	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 DiOC$_{2}$ and anionic SqSC$_{4}$ 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. DiOC$_{2}$ 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 SqSC$_{4}$, 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.
Starting Page	763
Ending Page	771
Page Count	9
File Format	PDF
ISSN	10859195
Journal	Cell Biochemistry and Biophysics
Volume Number	67
Issue Number	2
e-ISSN	15590283
Language	English
Publisher	Springer US
Publisher Date	2013-03-24
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Transmembrane potential changes Microfluorometry Single hepatocytes Hormone effect Probes Biochemistry Pharmacology/Toxicology Biotechnology Cell Biology Biophysics and Biological Physics
Content Type	Text
Resource Type	Article
Subject	Cell Biology Medicine Biochemistry Biophysics

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