Dataset Information

Blockade of gap junction coupling by glycyrrhetinic acids in guinea pig cochlear artery: a whole-cell voltage- and current-clamp study.

ABSTRACT:

Background and purpose

Glycyrrhetinic acids (GAs) are widely used as gap junction blockers, but their efficacy and side effects have not been well determined.

Experimental approach

Whole-cell electrical recordings were made from vascular smooth muscle cells (VSMCs) embedded in or dissociated from, guinea pig cochlear artery segments.

Key results

18beta- & 18alpha-GA concentration-dependently increased membrane input resistance (R(in)) of in situ VSMCs, with a maximal input conductance (G(in)=1/R(in)) reduction of 92% & 77% and IC(50) of 2.0 & 4.4 microm, respectively. 18betaGA (30 microM) resulted in a R(in) of 2.2 GOmega and C(in) of 12 pF, comparable to those of freshly dissociated VSMCs (3.1 GOmega & 6.1 pF). The GAs (> or =30 microM) caused a depolarization in VSMCs in situ. In dispersed VSMCs, they both inhibited delayed rectifiers; 18betaGA also activated a non-selective cation conductance while 18alphaGA inactivated a voltage-independent K+-conductance. ACh induced an outward current in VSMCs in situ at -40 mV, with a positive slope I/V relation and a reversal potential near E(K). The ACh-induced current was attenuated by 18beta- & 18alphaGA with an IC(50) of 4.3 & 7.8 microM, respectively.

Conclusions and implications

18betaGA blocked the vascular gap junctions, achieving a complete electrical isolation of the recorded VSMC at > or =30 microM while causing a mild depolarization by a complex conductance alteration. 18betaGA suppressed the ACh-induced current in VSMC by blocking the myoendothelial gap junction and by a non-junctional action. 18alphaGA at 30-100 microM failed to fully block the gap junctions while exerting side actions.

SUBMITTER: Guan BC

PROVIDER: S-EPMC2042944 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:A variety of mechanisms that govern connexin channel gating and permeability regulate coupling in gap junction networks. Mutations in connexin genes have been linked to several pathologies, including cardiovascular anomalies, peripheral neuropathy, skin disorders, cataracts and deafness. Gap junction coupling and its patho-physiological alterations are commonly assayed by microinjection experiments with fluorescent tracers, which typically require several minutes to allow dye transfer to a limited number of cells. Comparable or longer time intervals are required by fluorescence recovery after photobleaching experiments. Paired electrophysiological recordings have excellent time resolution but provide extremely limited spatial information regarding network connectivity.Here, we developed a rapid and sensitive method to assay gap junction communication using a combination of single cell electrophysiology, large-scale optical recordings and a digital phase-sensitive detector to extract signals with a known frequency from Vf2.1.Cl, a novel fluorescent sensor of plasma membrane potential. Tests performed in HeLa cell cultures confirmed that suitably encoded Vf2.1.Cl signals remained confined within the network of cells visibly interconnected by fluorescently tagged gap junction channels. We used this method to visualize instantly intercellular connectivity over the whole field of view (hundreds of cells) in cochlear organotypic cultures from postnatal mice. A simple resistive network model reproduced accurately the spatial dependence of the electrical signals throughout the cellular network. Our data suggest that each pair of cochlear non-sensory cells of the lesser epithelial ridge is coupled by ~1500 gap junction channels, on average. Junctional conductance was reduced by 14% in cochlear cultures harboring the T5M mutation of connexin30, which induces a moderate hearing loss in connexin30T5M/T5M knock-in mice, and by 91% in cultures from connexin30-/- mice, which are profoundly deaf.Our methodology allows greater sensitivity (defined as the minimum magnitude of input signal required to produce a specified output signal having a specified signal-to-noise ratio) and better time resolution compared to classical tracer-based techniques. It permitted us to dynamically visualize intercellular connectivity down to the 10th order in non-sensory cell networks of the developing cochlea. We believe that our approach is of general interest and can be seamlessly extended to a variety of biological systems, as well as to other connexin-related disease conditions.

Project description:Fibroblasts play a significant role in the development of electrical and mechanical dysfunction of the heart; however, the underlying mechanisms are only partially understood. One widely studied mechanism suggests that fibroblasts produce excess extracellular matrix, resulting in collagenous septa that slow propagation, cause zig-zag conduction paths, and decouple cardiomyocytes, resulting in a substrate for cardiac arrhythmia. An emerging mechanism suggests that fibroblasts promote arrhythmogenesis through direct electrical interactions with cardiomyocytes via gap junction (GJ) channels. In the heart, three major connexin (Cx) isoforms, Cx40, Cx43, and Cx45, form GJ channels in cell-type-specific combinations. Because each Cx is characterized by a unique time- and transjunctional voltage-dependent profile, we investigated whether the electrophysiological contributions of fibroblasts would vary with the specific composition of the myocyte-fibroblast (M-F) GJ channel. Due to the challenges of systematically modifying Cxs in vitro, we coupled native cardiomyocytes with in silico fibroblast and GJ channel electrophysiology models using the dynamic-clamp technique. We found that there is a reduction in the early peak of the junctional current during the upstroke of the action potential (AP) due to GJ channel gating. However, effects on the cardiomyocyte AP morphology were similar regardless of the specific type of GJ channel (homotypic Cx43 and Cx45, and heterotypic Cx43/Cx45 and Cx45/Cx43). To illuminate effects at the tissue level, we performed multiscale simulations of M-F coupling. First, we developed a cell-specific model of our dynamic-clamp experiments and investigated changes in the underlying membrane currents during M-F coupling. Second, we performed two-dimensional tissue sheet simulations of cardiac fibrosis and incorporated GJ channels in a cell type-specific manner. We determined that although GJ channel gating reduces junctional current, it does not significantly alter conduction velocity during cardiac fibrosis relative to static GJ coupling. These findings shed more light on the complex electrophysiological interplay between cardiac fibroblasts and myocytes.