Dissertation

Regulation of ERK1/2 and SAPK/JNK phosphorylation by histamine

Dandekar, Radhika D

2009

Overview

Mitogen activated protein kinases (MAPKs) are specific serine/threonine kinases which respond to various stimuli and control various cellular activities including gene expression, mitosis, cell differentiation, and cell survival/apoptosis. Histamine is implicated in allergic disease and asthma and SAPK/JNK and ERK1/2 are involved in certain aspects of allergic inflammation such as TH2 differentiation and proliferation and apoptosis. This study was designed to investigate the effects of histamine on ERK1/2 and SAPK/JNK phosphorylation in splenocytes. C57BL/6 splenocytes were treated with different concentrations of histamine (10-4 M to 10-11 M). Histamine at higher concentration (10-4 M) increased ERK2 phosphorylation. There was, however no significant effect seen at other concentrations (10-6 M - 10-11 M). Surprisingly, H1 receptor agonist betahistine (10-5 M), H2 agonist amthamine (10-5 M), H3 agonist methimepip (10 -6 M) and H4 agonist 4-methyl histamine (10-6 M), all increased ERK2 phosphorylation. H1R antagonist pyrilamine (10-6 M), H2R antagonist ranitidine (10-5 M), H3/H4R antagonist thioperamide (10-6 M), and H3R antagonist clobenpropit (10-5 M) inhibited histamine mediated ERK2 phosphorylation suggesting that all four histamine receptor subtypes played some role in this phosphorylation. Since TNF-alpha causes phosphorylation of ERK1/2, we investigated whether histamine acted via secretion of TNF-alpha to affect ERK1/2 phosphorylation. As a consequence, TNF-alpha knockout mice were used and we found that TNF-alpha was involved in ERK2 phosphorylation. There was complete inhibition of ERK2 phosphorylation by histamine via H2, H3 and H4 agonists, but effects of H1 agonist were inconclusive in TNF-alpha knockout splenocytes. This suggested that histamine indirectly affected the ERK2 phosphorylation via its effects on the secretion of TNF-alpha and H1 receptor played a role in this process. We performed similar experiments with stress activated protein kinases/c-jun N-terminal kinases (SAPK/JNK). C57BL/6 mice splenocytes were treated with different concentrations of histamine (10-4 M to 10 -11 M), phorbol 12 myristate 13-acetate (PMA) was used as a positive control and phosphorylation of SAPK/JNK was determined. Histamine inhibited phosphorylation of SAPK/JNK at high concentrations (10-4 M-10-8 M) and had no effect on SAPK/JNK phosphorylation at lower concentrations (10 -9 M- 10-11 M). Histamine receptor specific agonists were used to identify the histamine receptors involved in the inhibition of SAPK/JNK phosphorylation. H1R agonist betahistine (10-5 M) decreased the phosphorylation of SAPK/JNK. The decrease in SAPK/JNK phosphorylation by histamine was predominantly an H1 receptor effect. H2R agonist amthamine (10-5 M) did not show any significant effect on SAPK/JNK phosphorylation. H3R agonist methimepip (10-6 M) and H4R agonist 4-methyl histamine (10-6 M), increased SAPK/JNK phosphorylation. H1R antagonist pyrilamine (10-6 M), H2R antagonist ranitidine (10-5 M), H3/H4R antagonist thioperamide (10-6 M), and clobenpropit (10-5 M), partially reversed the histamine mediated inhibition of SAPK/JNK phosphorylation. TNF-alpha knockout mice were used to determine if histamine regulated SAPK/JNK phosphorylation via TNF-alpha. In TNF-alpha knockout mice splenocytes, histamine inhibited SAPK/JNK phosphorylation. Activation of H1 receptors inhibited SAPK/JNK phosphorylation in knockout as was the case in wild type mice. Histamine via H2 receptor inhibited SAPK/JNK phosphorylation in knockout mice, but did not show any significant effect in wild type mice. Activation of H3 receptors decreased SAPK/JNK phosphorylation in knockout mice, as opposed to an increase in wild type mice suggesting that another cytokine besides TNF-alpha was involved in SAPK/JNK phosphorylation. H4 receptor did not show any significant effect in knockout mice, but showed an increase in SAPK/JNK phosphorylation in wild type mice suggesting that TNF-alpha is required for histamine mediated effects via H4 receptors. This data showed a role of TNF-alpha in histamine-mediated effects of SAPK/JNK phosphorylation via H4 receptors.

• Publisher: ProQuest Dissertations & Theses
• Subject: Pharmacology
• ISBN: 1109552483, 9781109552485