Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis

Abstract

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

Figure 1. Spermine increases gap junctional communication between A172 human glioma cells

(A) Examples of intercellular diffusion of Lucifer yellow (LY) in confluent cultures of A172 human glioma cells expressing Cx43. Each image was acquired 20 minutes after loading a single cell (arrow) with LY (0.5 mM) through the patch pipette. In the absence of spermine (SPM) (Control), LY was detected in 8.6±2.5 (B; p<0.02, n=5) neighboring cells (arrowhead). Under presence of 0.5 mM SPM in the patch pipette LY was detected in 17.6±3.5 cells (B; p<0.02, n=5). After knockdown of Cx43 by siRNA treatment, was no detectable Lucifer yellow diffusion to neighboring cells (B; p<0.02, n=5). (B) Summary of the number of fluorescent cells following injection of LY without or with 0.5 mM SPM into control glioma cells or those treated with Cx43 siRNA. Error bars represent standard errors of the mean, and asterisks indicate significant differences (p<0.02, n=5). (C) Western blot analysis of Cx43 in A172 human glioma cells with and without siRNA knock-down of Cx43. India Ink staining used as a loading control for densitometry analysis.

Figure 2. Polyamines prevented uncoupling of Novikoff cells induced by enhanced intracellular calcium or hydrogen concentrations

(A) Examples of junctional currents (I_j) records in response to repeated (0.7 Hz) V_j ramps shown in (B). (C) Changes in the time course of averaged and normalized g_j at different [Ca²⁺]_i and spermine (SPM). An increase of [Ca²⁺]_i from 20 nM to 1 μM accelerated g_j decay (filled circles over filled squares). Addition of SPM at 0.1 mM (up triangles), 1 mM (down triangles) and 10 mM (diamonds) decelerated g_j decay up to full recovery at 10 mM of SPM. (D) A summary of data demonstrating a lessening of Ca²⁺-induced uncoupling by SPM (p<0.05, n=7 in each group). (E) Averaged and normalized g_j show that spermidine (SPD) diminish acidosis-mediated g_j decay in a concentration dependent manner (p<0.05, n=6 in each group). Data shown in grey columns (SPM) demonstrate that SPD effect is lower than that of SPM at all three concentrations. (F) Dose-response relationships of normalized g_j recovery under an influence of SPM and SPD. Curves are fits of a Logistic equation to the experimental data.