HL-1 myocytes exhibit PKC and K(ATP) channel-dependent delta opioid preconditioning.
BACKGROUND
Opioid preconditioning protects the myocardium against ischemia/reperfusion (IR) injury. By enhancing cardiomyocyte viability, opioids can enhance cardiac function and recovery from IR injury during acute cardiac care. The myocyte model HL-1 is an immortalized, mouse atrial cell line that expresses functional delta-opioid receptors. The HL-1 myocyte may be useful for IR injury research exploring opioid cardioprotection.


METHODS
In study I, microplates of HL-1 were subjected to 10 min pre-treatment with either basal media, delta-opioid agonist DADLE(10uM), or DADLE(10uM) + delta-antagonist naltrindole (10uM). Study II treatment groups included PKC inhibitor chelerythrine (2uM), K(ATP) channel closer glybenclamide (100uM), or mitochondrial K(ATP) channel opener diazoxide (100uM) administered in various combinations followed by DADLE (10uM) or control. Microplates were subjected to normal oxygen/substrate conditions or ischemic (<1% 0(2)) and substrate deficient (10 uM 2-Deoxyglucose versus 10 mM glucose) conditions, then reperfused with normal oxygen and glucose-containing media. Microplate supernatants were subjected to lactate dehydrogenase (LDH) assay.


RESULTS
Compared to untreated control, the LDH assay showed significant reduction in opioid-only pretreated groups at all time points. These effects were attenuated with delta-opioid antagonist co-administration. Co-administration of non-selective K(ATP) channel closer glybenclamide and DADLE abolished DADLE cytoprotection, while selective mitochondrial K(ATP) opener diazoxide mimicked DADLE cytoprotection Co-administration of chelerythrine and DADLE significantly reduced chelerythrine cytotoxicity.


CONCLUSIONS
Delta-opioid preconditioning of HL-1 myocytes significantly decreased necrosis from in vitro simulated ischemia/reperfusion as measured by LDH release; this effect was reversed by delta-antagonist naltrindole. Cytoprotection was PKC and K(ATP) channel-dependent. HL-1 myocytes exhibit opioid-induced cytoprotection from IR injury, and present a novel model of pharmacologic preconditioning.
Seymour
Elisabeth M
EM
Department of Cardiac Surgery, B558 MSRBII 0686, University of Michigan Medical School, Ann Arbor, MI, USA.
Wu
Shu-Yung James
SY
Kovach
Melissa A
MA
Romano
Matthew A
MA
Traynor
Jonathan R
JR
Claycomb
William C
WC
Bolling
Steven F
SF
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002460:Cell Line; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D007700:Kinetics; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D032342:Muscle Cells / Q000502:physiology*; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000166:cytology; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D015221:Potassium Channels; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000187:drug effects / Q000502:physiology*
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D010617:Phenanthridines; D015221:Potassium Channels; D017465:Receptors, Opioid, delta; C117780:mitochondrial K(ATP) channel; D009271:Naltrexone; D016308:Enkephalin, Leucine-2-Alanine; C016299:chelerythrine; D011493:Protein Kinase C; C055382:naltrindole