Sphingosylphosphorylcholine-induced contraction of feline ileal smooth muscle cells is mediated by Gα_i3 protein and MAPK

Abstract

We studied the mechanism of sphingosylphosphorylcholine (SPC)-induced contraction in feline ileal smooth muscle cells. Western blotting revealed that G protein subtypes of Gα_i1, Gα_i3 and Gα_o existed in feline ileum. Gα_i3 antibody penetration into permeabilized cells decreased SPC-induced contraction. In addition, incubation of [³⁵S]guanosine 5′-O-(3-thiotriphosphate) ([³⁵S]GTPγS) with membrane fraction increased its binding to Gα_i3 subtype after SPC treatment, suggesting that the signalling pathways invoked by SPC were mediated by Gα_i3 protein. MAPK kinase (MEK) inhibitor PD98059 blocked the contraction significantly, but p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 did not. Chelerythrine and neomycin also inhibited the contraction. However, cotreatment of PD98059 and chelerythrine showed no significant difference. Phosphorylation of p44/42 MAPK was increased by SPC treatment, which was reversed by pretreatment of inhibitors of signalling molecules that decreased SPC-induced contraction previously. The same result was obtained in the assay of MAPK activity.

Introduction

A variety of phosphorylated lipids act as intracellular and extracellular messengers for a diverse array of cellular processes, and numerous studies have provided abundant evidence of their importance in metabolic events [1], [2], [3], [4], [5]. Sphingolipids display a wide spectrum of biological activities, including such effects as protection of cells from apoptosis [6], activation of calcium signalling [7] and stimulation of nitric oxide production [8]. Additional effects include modulation of phosphatidylserine homeostasis [9], cell proliferation [10], wound healing [11] and prevention of nerve excitation and neuronal degeneration [12]. An untimely or improper accumulation of sphingolipids results in pathological changes in brain and other organs [13]. One of the sphingolipids, sphingosylphosphorylcholine (SPC), is generated by N-deacylation of sphingomyelin, which is one of the most abundant lipids in the cell membrane. SPC has been shown to act as an extracellular signal. For example, in common with peptide growth factors, SPC rapidly activates mitogen-activated protein kinase (MAPK) and stimulates DNA synthesis in Swiss 3T3 fibroblasts [1], [14], [15].

It is presumed that SPC interacts with a seven-transmembrane-spanning domain G protein-coupled receptor (GPCR), because all SPC-stimulated signalling events are inhibited by pertussis toxin (PTX) treatment, demonstrating the involvement of a G protein that leads to stimulation of phospholipases A2 and C (PLA2 and PLC) [15], [16], [17].

Activation of MAPK leads to the phosphorylation of various proteins that regulate gene transcription factor phosphorylation and has been implicated in the stimulation of cell proliferation [18], [19]. MAPK is activated by a variety of extracellular stimuli, including peptide growth-promoting factors acting on tyrosine kinase receptors and heterotrimeric G protein-coupled receptor agonists. Although the activation of the MAPK pathway by receptors with tyrosine kinase activity is well defined, the signalling mechanism leading to MAPK activation via G protein-coupled receptors is less clear. Several G protein-coupled receptors, including lysophosphatidic acid (LPA) [20], M₁ and M₃ muscarinic ACh [21], ANG II [22], α_2A-adrenergic [23], D₂ dopamine [24] and bradykinin (BK) [25] receptors, have been shown to mediate the signalling pathway to activate MAPK. The signalling mechanism used by a given G protein-coupled receptor agonist to stimulate MAPK depends on the class of G protein and second messenger in a given cell type.

The best-characterized members of the MAPK superfamily of protein kinases are the extracellular signal-regulated protein kinases (Erk/MAPK or Erks, p42 and p44 kDa) [26]. The Erk/MAPK can be activated by many different stimuli and are involved in smooth muscle cell contraction. These proteins are activated when phosphorylated on both tyrosine and threonine residues. Activation of the MAPK pathway ultimately results in the phosphorylation of target enzyme essential for cellular responses. The protein directly responsible for Erk/MAPK phosphorylation is MAPK kinase (MEK)/Erk kinase [27].

It is also reported that SPC might be able to decrease the length of smooth muscle cells isolated from the rectosigmoid of the rabbit and pig vascular smooth muscle via the activation of MAPK [28] and Ca²⁺ signalling [8], respectively. In this study, we examined whether SPC-induced ileal smooth muscle cell contraction is G protein subtype specific and whether MAPK is related to the contraction and finally what are the components of the linkage of G protein and MAPK.