Histamine activation of the ERK signalling pathway in U373 MG astrocytoma cells: comparison with an NK1 receptor agonist

Emma McWilliams, Lynda A. Sellers & J. Michael Young, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB5 9DU.

Both histamine and substance P (SP) stimulate mitogenesis in human U373 MG cells (Hernández-Angeles et al., 2001). However, whereas the MAP kinase signalling pathways mediating responses to SP have been studied in some detail (Castagliuolo et al., 2000), there is little information on the pathways activated by histamine. Both H1 and NK1 receptors are Gq-coupled, but there is a marked difference in agonist-stimulated levels of inositol 1,4,5-trisphosphate (Young et al., 1998). It was thus also of interest to test whether this apparent difference in receptor-coupled events is reflected in differences between histamine and an NK1 agonist in MAP kinase signalling.

U373 MG cells were grown for 3 days in 12-well plates in DMEM/F-12 mixture, before incubation for 3 h at 37°C in serum-free medium and then addition of histamine or sar9met(O2)11-substance P (sar-metSP), usually for 10 min. Proteins were separated by SDS-PAGE and ERK-P detected by Western blotting. Responses were quantitated by densitometry or using a GeneGnome system (Syngene). Inhibitors were added 10 min before the agonist and responses expressed as a percentage of that to the agonist alone.

Histamine stimulated the formation of activated ERK (ERK-P), with a pEC50 6.48 ± 0.09 (EC50 0.33 µM). In comparison, the EC50 values for histamine-induced increases in total inositol phosphates and Ca2+i were 3.2 ± 0.6 and 5.9 ± 0.6, respectively. Histamine (10 µM) activation of ERK was blocked by the H1 antagonist mepyramine (1 µM) and strongly inhibited, 87 ± 6% (7), by the MEK inhibitor PD98059 (40 µM). The increase in ERK-P stimulated by 10 m M histamine was maximal at 5-10 min; levels declined rapidly thereafter. Sar-metSP stimulated ERK-P formation with an pEC50 of 8.86 ± 0.22 (EC50 1.4 nM) and with a similar time-course. The responses to both 10 m M histamine and 30 nM sar-metSP were reduced by chelation of extracellular Ca2+ with EGTA, but the response to histamine was inhibited to a significantly greater extent than that to sar-metSP (52 ± 8%, n=13, and 29 ± 7%, n=7, inhibition respectively). Ionomycin (1 µM) also stimulated a PD98059-sensitive increase in the level in ERK-P. The responses to both histamine and ionomycin were reduced by the CaMKinase II inhibitor KN-62 (20 µM) (47 ± 3%, n=3, and 42 ± 11%, n=3, inhibition, respectively). ERK-P formation induced by SP has been reported to require transactivation of the EGF receptor tyrosine kinase (Castagliuolo et al., 2000) and in accord with this the response to sar-metSP was markedly reduced by the EGF tyrosine kinase inhibitor AG1478 (10 m M), 77 ± 6% inhibition (9). However, ERK-P formation induced by histamine was significantly less sensitive to AG1478, 35 ± 8% inhibition (9). The response to ionomycin was also inhibited by AG1478, 55 ± 11% inhibition (11), implying that Ca2+ can also activate the pathway via EGF. These findings indicate that there are differences between histamine and sar-metSP in the pathways of activation of ERK. Whereas the action of sar-metSP is largely via EGF transactivation, an additional pathway, probably involving induced Ca2+ entry, has a major role in the response to histamine.

Castagliuolo, I. et al. (2000) J. Biol. Chem., 275, 26545-26550.
Hernández-Angeles, A. et al.(2001) J. Neuro-Oncol., 55, 81-89.
Young, K.W. et al. (1998) Cell Calcium, 24, 59-70.