Electrical conduction along endothelial cell tubes from mouse feed arteries: confounding actions of glycyrrhetinic acid derivatives

Abstract

BACKGROUND AND PURPOSEElectrical conduction along endothelium of resistance vessels has not been determined independently of the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (i) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; and (ii) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca 2+ signalling. EXPERIMENTAL APPROACHIntact EC tubes were isolated from abdominal muscle feed (superior epigastric) arteries of C57BL/6 mice. Hyperpolarization was initiated with indirect (ACh) and direct (NS309) stimulation of intermediate-and small-conductance Ca 2+ -activated K + channels (IKCa/SKCa). Remote membrane potential (Vm) responses to intracellular current injection defined the length constant (l) for electrical conduction. Dye coupling was evaluated following intracellular microinjection of propidium iodide. Intracellular Ca 2+ dynamics were determined using Fura-2 photometry. Carbenoxolone (CBX) or b-glycyrrhetinic acid (bGA) was used to investigate the role of GJCs. KEY RESULTSSteady-state Vm of ECs was -25 mV. ACh and NS309 hyperpolarized ECs by -40 and -60 mV respectively. Electrical conduction decayed monoexponentially with distance (l~1.4 mm). Propidium iodide injected into one EC spread into surrounding ECs. CBX or bGA inhibited dye transfer, electrical conduction and EC hyperpolarization reversibly. Both agents elevated resting Ca 2+ while bGA inhibited responses to ACh. CONCLUSIONS AND IMPLICATIONSIndividual cells were effectively coupled to each other within EC tubes. Inhibiting GJCs with glycyrrhetinic acid derivatives blocked hyperpolarization mediated by IKCa/SKCa channels, regardless of Ca 2+ signalling, obviating use of these agents in distinguishing key determinants of electrical conduction along the endothelium. AbbreviationsAFA, abdominal muscle feed artery; bGA, b-glycyrrhetinic acid; [Ca 2+ ]i,

Global changes of phospholipids identified by MALDI imaging mass spectrometry in a mouse model of Alzheimer's disease

“…Previous reports showed that glycerophosphatidylcholine concentration typically increases with age; moreover, this increase is more pronounced in aged AD patients and those with enhanced A ␤ plaque formation (29)(30)(31)(32). Breakdown of APP and aggregation of amyloid plaques are the major causes of neuronal deterioration in AD.…”

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

“…Indeed, a number of inhibitors of gap junctions are widely used but are generally considered to be nonspecific, and their mechanism of inhibition have not been clearly defined (Evans and Boitano, 2001;Evans et al, 2012). For example, studies of the functional role of connexins in arterioles have extensively used inhibitors such as glycyrrhetinic acid and carbenoxolone, but recent studies have identified nonspecific effects of these blockers, including inhibition of calcium-activated potassium channels (Behringer et al, 2012). The development of transgenic animals was first thought to be a solution to cope with the lack of specific pharmacological inhibitors, but it is now clear that a number of compensations and/or redistribution of other connexin isoforms occur in Cx KO animals, which could further hinder interpretation of connexins on physiologic function (e.g., Kruger et al, 2002;Simon and McWhorter, 2003;Isakson et al, 2006a).…”

“…b Evidence that although connexin expression and/or gap junction communication levels correlated to disease they do not appear to be directly linked to phenotypic modulation (Matsushita et al, 2007;Behringer et al, 2012).…”

“…Thompson et al, 2006;Iglesias et al, 2008Iglesias et al, , 2009aChekeni et al, 2010;Behringer et al, 2012;Gairhe et al, 2012;Boedtkjer et al, 2013;Zhang et al, 2013 40 Gap27, 37,43 Gap27 Used experimentally to inhibit connexin (Cx37, Cx43) and has been showed to affect pannexin channels. Weingart, 1977;Martin et al, 2004;Turner et al, 2004;Aperia, 2007;Boedtkjer et al, 2013 Heptanol Aliphatic alcohol, gap junction uncoupler/inhibitor 200 mM-3 mM 2/+ ++ Takens- Kwak et al, 1992;Garcia-Dorado et al, 1997;Pelegrin and Surprenant, 2006;Nishida et al, 2008;Li et al, 2010;Li et al, 2012 b +, Partial inhibition; 2/+, conflicting reports; ++, greater than 50% inhibition; 2, no effect; +++, complete inhibition; n/a, no information available a R and S represent the two stereoisomers of mefloquine (Iglesias et al, 2009b) b…”

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

“…The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions 6,7 . In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.…”