In vitro ischaemic preconditioning of isolated rabbit cardiomyocytes: effects of selective adenosine receptor blockade and calphostin C.
OBJECTIVE
The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during "metabolic" preconditioning with glucose deprivation or adenosine agonists.


METHODS
Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U.ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling.


RESULTS
Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 microM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 microM), while the A1/A3 antagonist BW 1433U (1 microM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX.


CONCLUSIONS
Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.
Armstrong
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S
Department of Pathology, East Tennessee State University, James H Quillen College of Medicine, Johnson City 37614, USA.
Ganote
C E
CE
D000818:Animals; D066298:In Vitro Techniques; D017202:Myocardial Ischemia / Q000378:metabolism* / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000473:pathology / Q000517:prevention & control*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D009281:Naphthalenes / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D058915:Purinergic P1 Receptor Antagonists; D011817:Rabbits; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
D009281:Naphthalenes; D058915:Purinergic P1 Receptor Antagonists; D014970:Xanthines; C031433:8-(4-sulfophenyl)theophylline; C053414:BW A1433U; C051360:1,3-dipropyl-8-cyclopentylxanthine; D013806:Theophylline; D011493:Protein Kinase C; C058819:calphostin C