Signaling pathways downstream of P2 receptors in human neutrophils.
Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y(2) receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y(2) receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only approximately 75% loss of nucleotide-induced Ca(2+) mobilization indicating that nucleotides cause Ca(2+) mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca(2+) mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca(2+) increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca(2+) mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca(2+) increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca(2+) chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y(2) receptor can cause Ca(2+) mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.
Meshki
John
J
Department of Physiology, Temple University Medical School, 3420 N. Broad Street, Philadelphia, PA, 19140, USA.
Tuluc
Florin
F
Bredetean
Ovidiu
O
Garcia
Analia
A
Kunapuli
Satya P
SP