David A. Liem, Maaike te Lintel Hekkert, Olivier C. Manintveld, Frans Boomsma, Pieter D. Verdouw, Dirk J. Duncker. Myocardium tolerant to an adenosine-dependent ischemic preconditioning stimulus can still be protected by stimuli that employ alternative signaling pathways. Am J Physiol Heart Circ Physiol 000:H0000-H0000, 0000. Clinical studies on cardioprotection by pre-infarct angina are ambiguous, which may involve development of tolerance to repeated episodes of ischemia. However, not all preconditioning stimuli use identical signaling pathways, and since patients likely experience varying numbers of episodes of different degree and duration of pre-infarct angina, it is important to know whether myocardium tolerant to a particular preconditioning stimulus can still be protected by stimuli employing alternative signaling pathways. Consequently, we tested the hypothesis that development of tolerance to a particular stimulus does not affect cardioprotection by stimuli that employ different signaling pathways. Anesthetized rats underwent classical, remote or pharmacological preconditioning. Infarct size (IS), produced by a 60-min coronary artery occlusion (CAO), was determined after 120 min of reperfusion. Preconditioning by two 15-min periods of CAO, (2CAO15, an adenosine-dependent stimulus) limited IS from 69±2% to 37±6%, but when 2CAO15 was preceded by 4CAO15, protection by 2CAO15 was absent (IS=68±1%). This development of tolerance coincided with a loss of cardiac interstitial adenosine release, whereas two 15-min episodes of 200 µg/min intravenous adenosine infusion still elicited cardioprotection (IS=40±4%). Furthermore, cardioprotection was still produced when 4CAO15 was followed by either the adenosine-independent stimulus 3CAO3 (IS=50±8%), or the remote preconditioning stimulus of two 15-min periods of mesenteric artery occlusion (IS=49±6%). In conclusion, the development of tolerance to cardioprotection by an adenosine-dependent preconditioning stimulus still allows protection by ischemic stimuli or pharmacological intervention employing different signaling pathways.