Direct evidence of chloride ion efflux in ischaemic and pharmacological preconditioning of cultured cardiomyocytes.

Abstract

AIMS:

We have previously shown that reduction of ischaemic cell swelling via enhanced cell volume regulation is a key mechanism of ischaemic preconditioning (IPC) in cardiomyocytes. We have also shown that pharmacological blockade of Cl(-) channels abolishes cardioprotection achieved by IPC in Langendorff-perfused hearts and freshly isolated cardiomyocytes, thus suggesting that Cl(-) plays a key role in IPC cardioprotection. However, direct evidence of Cl(-) channel activation resulting in transsarcolemmal Cl(-) efflux by IPC had been lacking. To address this issue, 24 h cultured rabbit cardiomyocytes were loaded with 5 mM 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), a specific fluorescence indicator that is quenched by Cl(-) so that cellular efflux of Cl(-) results in an increase in SPQ fluorescence.

METHODS AND RESULTS:

After stabilization for 10 min, cardiomyocytes were preconditioned either with 10 min simulated ischaemia/10 min simulated reperfusion or with 10 min treatment with 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA). IPC and APNEA significantly (P < 0.001) reduced the intracellular Cl(-) concentration ([Cl(-)](i)) to 31.9 +/- 3.2 mM (mean +/- SEM) and 32.5 +/- 2.8 mM, respectively, from an initial [Cl(-)](i) (pooled stabilization 61.5 +/- 7.1 mM). [Cl(-)](i) did not change in control (non-preconditioned) cardiomyocytes (control 58.1 +/- 1.9 mM and control + vehicle 62.6 +/- 4.9 mM, P = 0.98 and 0.99 vs. pooled pre-treatment baseline, respectively). Inhibition of Cl(-) channels with 50 microM indanyloxyacetic acid 94 completely blocked preconditioning-induced Cl(-) efflux. Thus, a net Cl(-) efflux of 29.6 and 29.0 mM was triggered by IPC and APNEA.

CONCLUSION:

These findings provide the first direct evidence of activation of sarcolemmal Cl(-) channels by ischaemic and pharmacological preconditioning in cardiomyocytes.

• Authors: Roberto J Diaz, Alina Hinek, Gregory J Wilson
• Journal: Cardiovascular research (Cardiovasc Res) Vol. 87 Issue 3 Pg. 545-51 (Aug 01 2010) ISSN: 1755-3245 [Electronic] England
• PMID: 20228398 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Adenosine A1 Receptor Agonists
• Adenosine A3 Receptor Agonists
• Cardiovascular Agents
• Chloride Channels
• Chlorides
• Fluorescent Dyes
• Glycolates
• N(6)-2-(4-aminophenyl)ethyladenosine
• Quinolinium Compounds
• Receptor, Adenosine A1
• Receptor, Adenosine A3
• MK 473
• 6-methoxy-N-(3-sulfopropyl)quinolinium
• Adenosine

Topics

• Adenosine (analogs & derivatives, pharmacology)
• Adenosine A1 Receptor Agonists
• Adenosine A3 Receptor Agonists
• Animals
• Cardiovascular Agents (pharmacology)
• Cell Size (drug effects)
• Cells, Cultured
• Chloride Channels (drug effects, metabolism)
• Chlorides (metabolism)
• Fluorescent Dyes
• Glycolates (pharmacology)
• Ion Channel Gating (drug effects)
• Ischemic Preconditioning, Myocardial
• Microscopy, Fluorescence
• Myocytes, Cardiac (drug effects, metabolism)
• Quinolinium Compounds
• Rabbits
• Receptor, Adenosine A1 (metabolism)
• Receptor, Adenosine A3 (metabolism)
• Sarcolemma (drug effects, metabolism)
• Time Factors