Biomedical subjects

B R Takens-Kwak

Publications and source records attributed to B R Takens-Kwak.

3 recordsLinked to original sources

PubMed1993

Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains.

To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (gj), we compared the effects of 2 mM 1-heptanol and 20 microM 2-(methoxy-ethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (rss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6 +/- 1.1% (mean +/- SEM, n = 5) and 9.8 +/- 0.6% (n = 5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2 mM heptanol uncoupled cell pairs completely (n = 6), whereas 20 microM A2C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n = 5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased rss by 14.9 +/- 3.0% (n = 5), i.e., decreased cholesterol domain fluidity, whereas A2C had no effect on rss (-0.4 +/- 6.7%, n = 5). Following an increase of cellular "cholesterol" content (by loading the cells with DHE), 2 mM heptanol did not uncouple cell pairs completely: gj decreased by 80 +/- 20% (range 41-95%, n = 5). The decrease in gj was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of nonjunctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreases gj by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.

Alcohols↗

PubMed1992

Cardiac gap junctions: three distinct single channel conductances and their modulation by phosphorylating treatments.

The effects of an increase in intracellular cyclic GMP (cGMP)-concentration on gap junctional current (Ij) were studied in cultured neonatal rat heart cells using both the whole-cell and perforated patch voltage-clamp method. In whole-cell measurements, exposure to 8-bromo-cGMP or carbachol reduced Ij. With the perforated patch technique, on the other hand, Ij was not affected by either 8-bromo-cGMP or carbachol. Addition of alkaline phosphatase prevented the carbachol-induced decrease in Ij in whole-cell measurements. Reduction of Ij in well-coupled cell pairs by application of heptanol allowed us to study the effects of these substances on the single gap junction channel level. We found that cGMP-treatment shifts the single channel conductance (gamma j) from 43 to 21 pS in whole-cell measurements and that intracellular addition of phosphatase prevents this shift. In contrast, intracellular phosphatase-treatment itself shifts gamma j to 70 pS. Our results indicate that Cx43-gap junction channels may exhibit three conductance levels (21 pS, 40-45 pS and 70 pS), depending on the phosphorylation state of the protein.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Mechanism of heptanol-induced uncoupling of cardiac gap junctions: a perforated patch-clamp study.

The influence of heptanol on gap junctional and non-junctional membrane currents was studied in cultured neonatal rat heart cells using both the whole cell and perforated patch voltage-clamp method. With both methods, exposure to heptanol produced a dose-dependent decrease in the junctional current (dissociation constant = 0.54 and 1.20 mM for whole cell and perforated patch experiments, respectively). Heptanol-induced uncoupling was reversible. In the same concentration range, heptanol reduced all nonjunctional membrane ionic currents examined. This suggests that heptanol does not act specifically on gap junction channels but rather on the structure of the lipid membrane. This hypothesis is strengthened by the observation that in monolayer cultures of neonatal rat heart cells fluorescence steady-state anisotropy decreased proportional with increasing the heptanol concentration in the bath. Single-channel conductances (gamma j) were identical with both recording methods (21 and 40-45 pS); heptanol did not alter gamma j. Under conditions of reduced junctional coupling induced by heptanol, junctional conductance (gj) displayed voltage sensitivity at values of gj at which no voltage sensitivity could be observed under control conditions. These results suggest that heptanol-dependent uncoupling was caused by a decrease in open probability of the gap junction channels.

Alcohols↗