ATP triggers a robust intracellular [Ca²⁺]‐mediated signalling pathway in human synovial fibroblasts

Abstract

In human articular joints, synovial fibroblasts (HSFs) have essential physiological functions that include synthesis and secretion of components of the extracellular matrix and essential articular joint lubricants, as well as release of paracrine substances such as ATP. Although the molecular and cellular processes that lead to a rheumatoid arthritis (RA) phenotype are not fully understood, HSF cells exhibit significant changes during this disease progression. The effects of ATP on HSFs were studied by monitoring changes in intracellular Ca ([Ca ] ), and measuring electrophysiological properties. ATP application to HSF cell populations that had been enzymatically released from 2-D cell culture revealed that ATP (10-100 μm), or its analogues UTP or ADP, consistently produced a large transient increase in [Ca ] . These changes (i) were initiated by activation of the P Y purinergic receptor family, (ii) required G -mediated signal transduction, (iii) did not involve a transmembrane Ca influx, but instead (iv) arose almost entirely from activation of endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (IP ) receptors that triggered Ca release from the ER. Corresponding single cell electrophysiological studies revealed that these ATP effects (i) were insensitive to [Ca ] removal, (ii) involved an IP -mediated intracellular Ca release process, and (iii) strongly turned on Ca -activated K current(s) that significantly hyperpolarized these cells. Application of histamine produced very similar effects in these HSF cells. Since ATP is a known paracrine agonist and histamine is released early in the inflammatory response, these findings may contribute to identification of early steps/defects in the initiation and progression of RA.

The emerging fibroblast‐like synoviocyte channelome

“…In this issue of Experimental Physiology , the latest study by Kondo et al. () goes some way to address this by developing a fundamentally important working hypothesis for control of synoviocytes that links purinergic and histamine receptor activation to intracellular calcium mobilization and cell membrane hyperpolarization.…”

“…Kondo et al. () use an unusually well‐laden tool bag of cell physiological techniques to put this schema together, with a panel of 19 qPCR primer pairs, intracellular Ca 2+ imaging, flash photolysis of caged IP 3 and, of course, patch‐clamp electrophysiology. Two separate inflammatory mediators, ATP and histamine, both act in parallel to trigger phospholipase C activation, IP 3 elevation, thence Ca 2+ elevation and activation of a potassium conductance.…”

“…Well, the study by Kondo et al. () documents the all‐important control of synoviocytes rather than how that system will have a practical influence on joint function or even joint pain, but it does not take much extrapolation to contextualize this. Activation of Ca 2+ ‐activated potassium channels somehow drives invasiveness of synoviocytes and progression of arthritis in both human and rodent rheumatoid arthritis models by increasing invasiveness, proliferation and production of both inflammatory mediators and catabolic enzymes (Friebel et al., ; Hu et al., ; Tanner et al., ).…”

“…Investigation of all these questions will now be a far more realistic possibility in the light of the new ‘working hypothesis’ model of Kondo et al. ().…”

“…Sadly, whilst we do know a fair amount about the composition and function of the synovium, we still have a lot to learn about how to control the important fibroblast-like synoviocytes within. In this issue of Experimental Physiology, the latest study by Kondo et al (2018) goes some way to address this by developing a fundamentally important working hypothesis for control of synoviocytes that links purinergic and histamine receptor activation to intracellular calcium mobilization and cell membrane hyperpolarization. Kondo et al (2018) use an unusually well-laden tool bag of cell physiological techniques to put this schema together, with a panel of 19 qPCR primer pairs, intracellular Ca 2+ imaging, flash photolysis of caged IP 3 and, of course, patch-clamp electrophysiology.…”