Received for publication June 8, 2004.
Revised August 10, 2004.
Accepted for publication August 11, 2004.

Histamine H₃-Receptor-Induced Attenuation of Norepinephrine Exocytosis: A Decreased PKA Activity Mediates a Reduction in Intracellular Calcium

Abstract

We had reported that activation of pre-synaptic histamine H₃-receptors inhibits norepinephrine exocytosis from depolarized cardiac sympathetic nerve endings, an action associated with a marked decrease in intraneuronal Ca²⁺ which we tentatively ascribed to a decreased Ca²⁺ influx. An H₃-receptor-mediated inhibition of cAMP-dependent phosphorylation of Ca²⁺-channels could cause a sequential attenuation of Ca²⁺ influx, intraneuronal Ca²⁺ and norepinephrine exocytosis. We tested this hypothesis in sympathetic nerve endings (cardiac synaptosomes) expressing native H₃-receptors and in human neuroblastoma SH-SY5Y cells transfected with H₃-receptors. Norepinephrine exocytosis was elicited by K⁺ or by stimulation of adenylyl cyclase with forskolin. H₃-receptor activation markedly attenuated the K⁺- and forskolin-induced norepinephrine exocytosis; pre-treatment with pertussis toxin prevented this effect. Similar to forskolin, 8-bromo cAMP elicited norepinephrine exocytosis but, unlike forskolin, it was unaffected by H₃-receptor activation, demonstrating that inhibition of adenylyl cyclase is a pivotal step in the H₃-receptor transductional cascade. Indeed, we found that H₃-receptor activation attenuated norepinephrine exocytosis concomitantly with a decrease in intracellular cAMP and PKA activity in SH-SY5Y-H₃ cells. Moreover, pharmacological PKA inhibition acted synergistically with H₃-receptor activation to reduce K⁺-induced peak intracellular Ca²⁺ in SH-SY5Y-H₃ cells and norepinephrine exocytosis in cardiac synaptosomes. Further, H₃-receptor activation synergized with N- and L-type Ca²⁺-channel blockers to reduce norepinephrine exocytosis in cardiac synaptosomes. Our findings suggest that the H₃-receptor-mediated inhibition of norepinephrine exocytosis from cardiac sympathetic nerves results sequentially from H₃-receptor-G_i/G_o coupling, inhibition of adenylyl cyclase activity and decreased cAMP formation, leading to diminished PKA activity, and thus, decreased Ca²⁺ influx through voltage-operated Ca²⁺-channels.

Key words: PKA, adenylyl cyclase, calcium channels, histamine H3 receptor, intracellular calcium, norepinephrine excocytosis

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