Activation of presynatic histamine H3 receptors (H3R) down-regulates norepinephrine exocytosis from cardiac sympathetic nerve terminals, in both normal and ischemic conditions. Analogous to the effects of ␣2-adrenoceptors, which also act prejunctionally to inhibit norepinephrine release, H 3R-mediated antiexocytotic effects could result from a decreased Ca 2؉ influx into nerve endings. We tested this hypothesis in sympathetic nerve terminals isolated from guinea pig heart (cardiac synaptosomes) and in a model human neuronal cell line (SH-SY5Y), which we stably transfected with human H 3R cDNA (SH-SY5Y-H3). We found that reducing Ca 2؉ influx in response to membrane depolarization by inhibiting Ntype Ca 2؉ channels with -conotoxin ( -CTX) greatly attenuated the exocytosis of W e have found that sympathetic nerve endings in the guinea pig (1) and human heart (2) express the H 3 histamine receptor subtype (H 3 R). Activation of these H 3 R down-regulates norepinephrine exocytosis in both normal and ischemic conditions (3, 4).The H 3 R is a G-protein-coupled receptor linked to the inhibition of adenylyl cyclase (5). The H 3 R is modestly related in sequence to most biogenic amine receptors, including ␣ 2 -adrenoceptors (6). Like the H 3 R, cardiac ␣ 2 -adrenoceptors act prejunctionally to inhibit norepinephrine exocytosis (3, 7). Moreover, inhibition of Ca 2ϩ influx into sympathetic nerve endings with the selective N-type Ca 2ϩ channel blocker -conotoxin ( -CTX) (8) potentiates both H 3 R-and ␣ 2 -adrenoreceptor-mediated attenuation of cardiac adrenergic responses (1). Therefore, it is conceivable that, as for ␣ 2 -adrenoceptors (9), H 3 R activation may also inhibit norepinephrine release by diminishing Ca 2ϩ influx into cardiac sympathetic terminals.The human neuroblastoma cell line SH-SY5Y has been used as a model to study mechanisms of neurotransmitter release (10). To determine whether H 3 R activation modulates norepinephrine release by impeding Ca 2ϩ influx, we stably transfected SH-SY5Y cells with the cDNA for the H 3 R (SH-SY5Y-H 3 ). We report that upon K ϩ -induced depolarization, activation of H 3 R is associated with a marked decrease in intracellular Ca 2ϩ (Ca i ) and, thus, with an attenuation of norepinephrine exocytosis. Materials and MethodsPreparation of Cardiac Synaptosomes. Male Hartley guinea pigs (Charles River Breeding Laboratories) weighing 250-300 g were killed by cervical dislocation under light anesthesia with CO 2 vapor. The rib cage was rapidly opened and the heart was dissected away. A cannula was inserted into the aorta, and the heart was perfused for 5 min at constant pressure (40 cm H 2 O) in a Langendorff apparatus (11) with Ringer's solution (154 mM NaCl͞5.61 mM KCl͞2.16 mM CaCl 2 ͞5.95 mM NaHCO 3 ͞5.55 mM glucose) equilibrated with 100% O 2 at 37°C. This procedure ensured that no blood traces remained in the coronary circulation. Hearts were then freed from fat and connective tissue and minced in ice-cold 0.32 M sucrose containing 1 mM EGTA (pH 7.4). Synaptosomes were isolated as...