Cardiovascular Drugs and Therapy 1992;6:447-449 © Kluwer Academic Publishers, Boston. Printed in U.S.A. Massive Atrial Natriuretic Ischemia Reperfusion Peptide (ANP) Release in A. Lochner, S. Genade, and R. Mouton MRC Centrefor Molecular and Cellular Biology, Department of Medical Physiology and Biochemistry, Faculty of Medicine, University of Stellenbosch, Tygerberg, Republic of South Africa Key Words. atrial natriuretic peptide, ischemia, reperfusion, sarcolemma Dear Sir, Recent studies reported the stimulatory effects of Ca 2+ channel and al receptor agonists [1,2] on ANP release by atrial tissue. Therefore the increased cytosolic Ca z÷ [3] and release of endoge- nous catecholamines [4] occurring during ischemia-reperfusion may be associated with ANP release. Hypoxia has previously been shown to be a potent stimulus for ANP release by the isolated perfused heart [5,6], as well as in conscious animals [7] and humans [8]. In view of the marked differences in coronary flow rates in ischemia and hypoxia, and the role of coronary hemodynamics in ANP secretion [9], ANP release during reper- fusion was monitored in this study after exposure of the isolated rat heart to either 15 or 25 minutes normothermic ischemic ar- rest (representing reversible and irreversible ischemic injury, respectively [10]). ANP released during ischemia alone was evaluated by determining the amounts released into the recircu- lating buffer of hearts subjected to 35 minutes normothermic cardioplegic arrest (a condition causing overt ischemic damage). To eliminate the possible contribution of a high K+-cardioplegic solution to ANP release, myocardial ANP release was also moni- tored during exposure to 90 minutes of hypothermic arrest, a procedure that has been shown to offer complete protection against ischemic damage [11]. Male Wistar rats (weight +- 200 g), fed ad libitum until exper- imentation were used in all experiments. The hearts were per- fused retrogradely for 20 minutes, during which time the pulmo- nary vein was cannulated. Atrial stretch was then induced by perfusion in the working mode for 10 minutes (preload 15 cm H20; afterload 100 cm H20). The perfusion conditions and buffer, as well as induction of total global ischemia, have been described previously [10]. The hearts were reperfused in the retrograde mode for 10 minutes, followed by 5 minutes perfusion in the working mode. Normothermic (37°C) as well as hypothermic (20°C) cardio- plegic arrests were induced by using 20 ml Plasmaiyte B (Sabax, South Africa) to which CaC12 (1.25 mM) and KC1 (25 mM) were added (final composition in mM: Na ÷ 130; K ÷ , 29; C1- 109; bicar- bonate 28; Ca 2+ 1.25). The cardioplegic solution was gassed with 95% 02/5% CO2 and administered for 1 minute at 10-minute intervals at a pressure of 40 mmHg during the arrest period. Reperfusion of these hearts was done as described above. At least six hearts were studied in each group. Samples for measurement of perfusate ANP were collected over a period of 1 minute at the following time intervals during the control perfusion period: 10 and 20 minutes retrograde perfusion, 5 and 10 minutes working heart perfusion. During reperfusion after normothermic or hypothermic arrest, samples were collected at 1, 2, and 5 minutes of retrograde perfusion and at 1, 2, and 5 minutes of working heart perfusion. In the case of the cardio- plegic hearts, the recirculating cardioplegic fluid was collected at the end of the cardioplegic arrest. Samples were kept frozen at -70°C until assayed. At the end of the experimental period, the hearts were blotted dry, dried in an oven for 24 hours, and weighed. ANP release was expressed as fentomol/g dry weight/min. Perfusate ANP was analyzed using Amersham's ~ANP [12~I] radioreceptor assay system. This system utilizes a high specific activity (3 - [125I] iodotyrasy128) aANP tracer, together with a specific and sensitive ANP binding receptor preparation from bovine adrenal gland. Crossreactivity with human and rat a-ANP is 100% and 73%, respectively. The assay involves a 90-minute room temperature incubation and measures aANP in the range 1-128 fentomol/tube. The ED50 and the intraassay coefficient of variation averaged 25-29 fmol and 6%, respec- tively. Nonspecific binding was 4%. The standard curve and unknowns were evaluated by a RIA-calc computer program. In view of the fact that similar values were obtained in the amounts of ANP released during control perfusion, the values of the four series of experiments were pooled (Table 1). Atrial stretch, induced by switching from retrograde to working heart perfusion, caused an 88-100% increase (p < 0.001) in ANP re- lease. The severity of ischemic damage markedly affected the pattern and amounts of ANP released: (a) Reperfusion in the retrograde mode after induction of 15 minutes ischemia (revers- ible damage) caused a massive ANP release within 2 minutes (62059 -+ 14037 and 49466 + 8604 fmoles/g/min). Although the release tended to decline after 5 minutes of retrograde reperfu- sion, the amounts of ANP released were still significantly higher (25384 fmoles/g/min) than that observed during atrial stretch in the control period. ANP release during reperfusion in the work- ing mode after 15 minutes of ischemia was similar to that oc- curring during the corresponding control period, indicating that the washout of the ischemia-induced ANP release was probably completed. (b) Irreversible damage (25 minutes ischemia) was associated with an initial lesser degree of ANP release, which might be due to lower coronary flow rates during retrograde perfusion (mean coronary flow rates during first 2 minutes of retrograde perfusion after 15 and 25 minutes of ischemia were 16 -+ 1 and 11 -+ 1 ml/min, respectively). However, ANP release remained high throughout the reperfusion period and was not further affected by atrial stretch. (c) The magnitude of ANP released during ischemia is reflected by the massive amounts of ANP discharged into the cardioplegic fluid. Although high concentrations of KCI have been shown to induce ANP release in rat hypothalamus [12], it was unable to do so in Langendorff- 447