A Stimulator of Atrial Natriuretic Peptide Secretion and Its Mechanism of Action

Ouabain increases atrial natriuretic peptide (ANP) secretion. When isolated superfused rat left atria were paced at 2 Hz, ouabain at concentrations of 50, 100, and 200 ,M increased ANP secretion by 2.0±0.3-, 3.2+0.5-, and 4.2+0.5-fold, respectively. In this study, we examine the mechanism of ouabain-stimulated ANP secretion using the dose of 100 ,uM. To determine whether calcium played a role, atria were superfused with the calcium antagonist lanthanum. Superfusion with 2 mM LaCl3 completely inhibited ouabain-stimulated secretion, suggesting that calcium influx and/or sarcoplasmic reticulum (SR) calcium release provide essential sources of calcium for the stimulatory pathway. To determine the contribution of calcium from the SR, atria were superfused with ryanodine, an agent that depletes the SR of calcium. Superfusion with 1 luM ryanodine inhibited ouabain-stimulated secretion by 47%. Inhibition of Na+ ,K+ATPase allows sodium to accumulate in the cell. A rise in intracellular sodium alters Na+-Ca2' exchange, leading to an increase in cytosolic calcium. To determine the mechanism of sodium entry, atria were superfused with 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of Na+-H+ exchange, or with bumetanide, an inhibitor of Na+-K+-Clcotransport. Superfusion with 25 ,tM HMA inhibited ouabainstimulated secretion by 71%; however, 100 ,M bumetanide had no significant effect on secretion. Ouabain failed to stimulate ANP secretion by nonpaced (nonbeating) atria. Likewise, superfusion with the combination of ryanodine (1 ,&M) and the calcium channel antagonist isradipine (10 ,uM) totally blocked ouahain-stimulated ANP secretion. We conclude as follows: 1) Calcium influx and SR calcium release contribute equally to the secretory response. 2) Calcium influx through the Na+-Ca2' exchanger is inadequate to stimulate secretion when SR calcium release and calcium channels are blocked. 3) The primary mechanism of sodium entry required for stimulated secretion is through Na+-H+ exchange, with Na+-K+-Clcotransport playing little to no role. 4) By inference, sodium entry by means of the fast sodium channel also plays a role in ouabain-stimulated secretion. These results lend further support to the idea that calcium is an important second messenger in regulated secretion of ANP. (Circulation Research 1993;72:1035-1043)