Proton-sensing G protein-coupled receptor mobilizes calcium in human synovial cells

Abstract

-Lowered extracellular pH in a variety of tissues is associated with increased tissue destruction and initiation of inflammatory processes. Although the acid-sensing receptors described previously are ion channels, we describe a G protein-coupled proton-sensitive receptor that stimulates Ca 2ϩ release from intracellular stores in a tumor-derived synoviocyte cell line (SW982) and in primary cultures of human synovial cells from patients with inflammatory arthropathies. We established a link between proton-dependent receptor activation and intracellular Ca 2ϩ mobilization by demonstrating 1) dependence on the integrity of the intracellular Ca 2ϩ store, 2) independence from extracellular Ca 2ϩ , and 3) proton-induced production of inositol phosphate and 4) by abolishing the effect with GTPase inhibitors. We propose that this G protein-coupled acid-sensing receptor linked to intracellular Ca 2ϩ mobilization in synoviocytes can contribute to downstream inflammatory and cellular proliferative processes in synovial fibroblasts. The acid-sensing receptor has distinct characteristics as a metabotropic G protein-coupled receptor on human synoviocytes in this emerging new class of receptors. calcium imaging; synoviocytes; arthritis; acid-sensing receptor INFLAMMATORY PROCESSES are often accompanied by a decrease in extracellular pH. Protons can modulate cell signaling through acid-sensing ion channels, screening surface charge, or by protonation of residues on integral membrane proteins. Tissue acidosis is an important component of ischemic and inflammatory sequelae. Inflammatory arthropathies such as rheumatoid arthritis (RA) and septic arthritis are often characterized by acidic synovial fluids (SFs) and increased hydrogen ion levels, secondary to increased cellular proliferation, metabolic activation, and subsequent lactic acid production (6,10,20,27,31,33). The effects of lowered extracellular pH on the tissue have been associated with increased destruction and worsening of the inflammatory parameters (8,28,29). In RA, synovial fibroblasts mediate the inflammatory process by releasing a number of factors, including proteases that destroy the surrounding supporting matrix. One of these, cathepsin, requires a lowered pH for activity in the degradation of collagen (28 -30 release from intracellular stores in a tumor-derived synoviocyte cell line (SW982) and in human synovial cells cultured from patients with inflammatory arthropathies. EXPERIMENTAL PROCEDURESCell lines and cell culture. The SW982 cells derived from a human synovial sarcoma are fibroblast-like synovial cells (7) obtained through the American Type Culture Collection (Bethesda, MD). The cells were maintained in Leibovitz's L-15 medium, 10% heated fetal bovine serum (FBS), 2 mM L-glutamine, 1,000 U/ml penicillin G, and 1,000 pg/ml streptomycin. Cells were incubated in a humidified cell incubator (37°C, ambient CO 2) and used in passages 4 -21. Primary lines of surface-adherent synoviocytes were derived from the SFs from therapeutic arthrocenteses of two patie...

Thermosensitive TRP ion channels mediate cytosolic calcium response in human synoviocytes

“…The TRP channel-mediated calcium response is independent of intracellular stores or concurrent activation of voltage-dependent calcium channels. In a previous study we demonstrated (6) that SW982 synoviocytes do not have ryanodine-sensitive calcium stores. Therefore, calcium-induced calcium release also does not contribute to calcium signaling in synovial cells.…”

“…An acidsensing G protein-coupled receptor has been identified on synovial cells that are responsive to low pH (pH 5.5-7.0) in calcium-free media except in the presence of Cu 2ϩ (6). A response profile in low pH (pH 7.0 -7.2) in the presence of Cu 2ϩ was also noted, consistent with transient receptor potential (TRP)V1 activation.…”

“…Increasing the capsaicin concentration to 5 or 10 M at pH 7.2 or 7.0 did not significantly affect the responsive-to-nonresponsive cell ratio and had no significant effect on calcium spike amplitude. Further decreases in extracellular pH were avoided because extracellular acidification below pH 7.0 itself produced significant changes in [Ca 2ϩ ] cyt in synoviocytes due to the activation of an acid-sensitive G protein-coupled receptor we characterized previously (6). The changes in [Ca 2ϩ ] cyt evoked by capsaicin differed from the heat-induced calcium response in that they produced a delayed, transient response of 15 s to 1 min.…”

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

“…Measurement of intracellular Ca 2+ levels helps to elucidate functional changes of Ca 2+ -permeable channels in the plasma membrane or intracellular organelles and the contribution of Ca 2+ to the above processes (9-13). The assay can also be used to investigate the function of G-protein coupled receptors, many of which, upon activation, lead to increased intracellular free Ca 2+ concentrations by releasing Ca 2+ from intracellular Ca 2+ stores or activating other Ca 2+ permeable ion channels (14)(15)(16). FlexStation 3 makes use of Ca 2+ sensitive dyes, which give rise to increased fluorescence intensity, or ratiometric changes, with the increase of intracellular Ca 2+ concentration.…”

An environmental sensor, TRPV4 is a novel regulator of intracellular Ca2+ in human synoviocytes

“…These synoviocytes express many biological sensors including receptors and ion channels (1,2,9,10). In the chronic inflammation and pain associated with rheumatoid arthritis and injury, some of these sensors are active, implying that they are potential targets for treatment.…”