Stimulation of protein kinase B and p70 S6 kinase by the histamine H1 receptor in DDT1MF‐2 smooth muscle cells

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Abstract
Previous studies have shown that the histamine H1 receptor activates p42/p44 mitogen‐activated protein kinases (MAPK) in DDT1MF‐2 smooth muscle cells via a phosphatidylinositol 3‐kinase (PI‐3K)‐dependent pathway. In this study the effect of histamine H1 receptor stimulation on protein kinase B (PKB) and p70 S6 kinase, both of which are downstream targets of PI‐3K, has been investigated. Increases in PKB and p70 S6 kinase activation were monitored by Western blotting using phospho‐specific PKB (Ser473) and p70 S6 kinase (Thr421/Ser424) antibodies. Histamine stimulated time and concentration‐dependent increases in the phosphorylation of PKB and p70 S6 kinase in DDT1MF‐2 cells. Both responses were completely inhibited by the histamine H1 receptor antagonist mepyramine and following pre‐treatment with pertussis toxin, to block Gi/Go protein dependent pathways. The PI‐3K inhibitors wortmannin (IC50 5.9±0.5 nM) and LY 294002 (IC50 6.9±0.8 μM) attenuated the increase in PKB phosphorylation induced by histamine (100 μM) in a concentration‐dependent manner. Histamine‐induced increases in p70 S6 kinase phosphorylation were partially sensitive to rapamycin (20 nM; 68% inhibition) but completely blocked by wortmannin (100 nM), LY 294002 (30 μM) and the MAPK kinase inhibitor PD 98059 (50 μM). In summary, these data demonstrate that the histamine H1 receptor stimulates PKB and p70 S6 kinase phosphorylation in DDT1MF‐2 smooth muscle cells. However, functional studies revealed that histamine does not stimulate DDT1MF‐2 cell proliferation or attenuate staurosporine‐induced caspase‐3 activity. The challenge for future research will be to link the stimulation of these kinase pathways with the physiological and pathophysiological roles of the histamine H1 receptor. British Journal of Pharmacology (2002) 135, 1967–1976; doi:10.1038/sj.bjp.0704664