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

Abstract

We had reported that activation of presynaptic histamine H 3 -receptors inhibits norepinephrine exocytosis from depolarized cardiac sympathetic nerve endings, an action associated with a marked decrease in intraneuronal Ca 2ϩ that we ascribed to a decreased Ca 2ϩ influx. An H 3 -receptor-mediated inhibition of cAMP-dependent phosphorylation of Ca 2ϩ channels could cause a sequential attenuation of Ca 2ϩ influx, intraneuronal Ca 2ϩ and norepinephrine exocytosis. We tested this hypothesis in sympathetic nerve endings (cardiac synaptosomes) expressing native H 3 -receptors and in human neuroblastoma SH-SY5Y cells transfected with H 3 -receptors. Norepinephrine exocytosis was elicited by K ϩ or by stimulation of adenylyl cyclase with forskolin. H 3 -receptor activation markedly attenuated the K ϩ -and forskolin-induced norepinephrine exocytosis; pretreatment with pertussis toxin prevented this effect. Similar to forskolin, 8-bromo-cAMP elicited norepinephrine exocytosis but, unlike forskolin, it was unaffected by H 3 -receptor activation, demonstrating that inhibition of adenylyl cyclase is a pivotal step in the H 3 -receptor transductional cascade. Indeed, we found that H 3 -receptor activation attenuated norepinephrine exocytosis concomitantly with a decrease in intracellular cAMP and PKA activity in SH-SY5Y-H 3 cells. Moreover, pharmacological PKA inhibition acted synergistically with H 3 -receptor activation to reduce K ϩ -induced peak intracellular Ca 2ϩ in SH-SY5Y-H 3 cells and norepinephrine exocytosis in cardiac synaptosomes. Furthermore, H 3 -receptor activation synergized with N-and L-type Ca 2ϩ channel blockers to reduce norepinephrine exocytosis in cardiac synaptosomes. Our findings suggest that the H 3 -receptor-mediated inhibition of norepinephrine exocytosis from cardiac sympathetic nerves results sequentially from H 3 -receptor-G i /G o coupling, inhibition of adenylyl cyclase activity, and decreased cAMP formation, leading to diminished PKA activity, and thus, decreased Ca 2ϩ influx through voltage-operated Ca 2ϩ channels.

Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway

“…In the presence of imetit (100 nM), K + -induced NE release was inhibited by ~40-60% ( Fig. 2A-C), an effect which we have previously shown to be prevented by clobenpropit [5], indicating that the inhibition of the K + -induced NE exocytosis is mediated by H 3 R activation. As shown in Figure 2A, the MEK/ERK inhibitor PD98059 [15] diminished the anti-exocytotic effect of imetit by ~30% at 3 μM and abolished it at 10 μM.…”

“…Although the N-type Ca 2+ -channels are the dominant Ca 2+ entry pathway triggering sympathetic transmitter release [27;28], it is possible that L-type Ca 2+ -channels may also participate in NE exocytosis and be inhibited by H 3 R and EP 3 R activation [5]. H 3 R and EP 3 R are both coupled to a G i protein [12;21].…”

“…Further, Gβγ i is known to decrease adenylyl cyclase activity [32]. Thus, it is possible that in addition to the attenuation of adenylyl cyclase by Gα i [5], Gβγ i will also play a role in the H 3 R-mediated decrease in cAMP. A decrease in intracellular cAMP would diminish PKA activity and phosphorylation of L-type Ca 2+ channels resulting in a decreased Ca 2+ influx.…”

“…3A). Pretreatment with the selective H 3 R antagonist clobenpropit (50 nM) [5] prevented the imetit-induced increase of cPLA 2 activity, indicating that this increase was dependent on H 3 R activation. Similar to imetit, the Ca 2+ -ionophore A23187 (10 μM) also enhanced cPLA 2 activity (~20%), however this effect was not inhibited by clobenpropit (50 nM; Fig.…”

“…H 3 R activation reduces norepinephrine (NE) exocytosis and is associated with a marked decrease in the peak intraneuronal Ca 2+ ([Ca 2+ ] i ) response [4]. We recently reported that the H 3 R-mediated attenuation of NE exocytosis involves an H 3 R-G i /G o coupling, adenylyl cyclase inhibition by Gα i , decreased cAMP formation and diminished PKA activity [5]. Diminished PKA activity is likely to result in reduced phosphorylation of voltage-operated Ca 2+ -channels (VOCC), which would be reflected in a decrease in Ca 2+ current (I Ca ).…”