Protection of ischaemic-reperfused rat heart by dimethylamiloride is associated with inhibition of mitochondrial permeability transition.
1. The aim of the present study was to assess whether protection afforded by the Na(+)/H(+) exchanger blocker dimethylamiloride (DMA) is associated with inhibition of mitochondrial permeability transition (MPT). The effects of DMA were compared with those of cyclosporine (Cs) A, an inhibitor of MPT. 2. Rat hearts were Langendorff perfused with Krebs'-bicarbonate medium containing 10 mmol/L glucose and were subjected to 25 min no-flow global ischaemia and 30 min reperfusion in the presence or absence of 10 micromol/L DMA or 0.2 micromol/L CsA. Cell viability was measured using tetrazolium stain. The MPT was determined by loading hearts with 2-deoxy-[(3)H]-glucose (2DG), which enters mitochondria only during MPT. Total heart 2DG content as an estimation of the extent of tissue damage was also measured. To assess whether DMA has any direct effect on glycolysis, a cell-free heart extract containing all the glycolytic enzymes was used. 3. Dimethylamiloride improved functional recovery (rate-pressure product) from 24 +/- 7 to 68 +/- 11% (P < 0.01) at reperfusion end, attenuated the increase in left ventricular end-diastolic pressure (from 29 +/- 7 to 6 +/- 3% 10 min after reperfusion onset; P < 0.01), improved cell viability (from 21.2 +/- 6.6 to 69.6 +/- 7.1% at reperfusion end; P < 0.05) and lessened lactate accumulation at the end of ischaemia (119 +/- 15 vs 163 +/- 14 micromol/g dry weight; P < 0.05). Dimethylamiloride limited MPT: 2DG mitochondrial entrapment, being 33.1 +/- 14.2 and 96.3 +/- 14.0 at reperfusion end in the treated and control hearts, respectively (P < 0.05), and concomitantly raised total 2DG content (51.3 +/- 4.4 vs 86.8 +/- 1.7 x 10(3) d.p.m./g wet weight in control and treated groups, respectively; P < 0.05). Cyclosporine A improved functional recovery and attenuated the amplitude of ventricular diastolic pressure in ischaemic-reperfused hearts. It also reduced mitochondrial entrapment (67.3 +/- 7.7%; P < 0.05 vs control) and increased total cell 2DG content (162.3 +/- 1.3 x 10(3) d.p.m./g wet weight; P < 0.01 vs control) at the end of reperfusion. Dimethylamiloride did not affect glucose consumption and lactate production in the cell-free heart extract. 4. In conclusion, DMA protects against the noxious effects of ischaemia-reperfusion and inhibits MPT, coinciding with present and previous findings concerning the effects of CsA. Dimethylamiloride also diminished lactate accumulation, although it did not exhibit any direct effect on glycolysis. These data suggest that blockade of Na(+)/H(+) exchange by DMA attenuates the extent of MPT in ischaemic-reperfused rat heart.
Prendes
María G Marina
MG
Department of Biological Sciences, Faculty of Pharmacy and Biochemistry, University of Buenos Aires and IQUIMEFA-CONICET, Buenos Aires, Argentina. mgabymp@yahoo.com.ar
Torresín
Emilia
E
González
Marcela
M
Fernández
María A
MA
Perazzo
Juan C
JC
Savino
Enrique A
EA
Varela
Alicia
A
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D002317:Cardiovascular Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002470:Cell Survival / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology* / Q000627:therapeutic use; D005260:Female; D006019:Glycolysis / Q000187:drug effects; D066298:In Vitro Techniques; D019344:Lactic Acid / Q000378:metabolism; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000503:physiopathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D010477:Perfusion; D010539:Permeability; D051381:Rats; D017208:Rats, Wistar; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors; D016277:Ventricular Function, Left / Q000187:drug effects; D017725:Ventricular Pressure / Q000187:drug effects
D002317:Cardiovascular Agents; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017923:Sodium-Hydrogen Exchangers; D019344:Lactic Acid; C039465:5-dimethylamiloride; D000584:Amiloride; D016572:Cyclosporine