Evidence for autocrine activation of the calcium-sensing receptor by glutathione — ASN Events
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  2. CaSR-5 Session 12: The CaSR and glutathione
  3. Evidence for autocrine activation of the calcium-sensing receptor by glutathione

Evidence for autocrine activation of the calcium-sensing receptor by glutathione (#271)

Mahvash A Goolam 1 , Wenhan Chang 2 , Donald T Ward 3 , Stephen J Simpson 1 , Arthur D Conigrave 1 4
  1. School of Life & Environmental Sciences, Charles Perkins Centre (D17), University of Sydney, NSW 2006, Australia
  2. Endocrine Research Unit, San Francisco VA Medical Center and Department of Medicine, University of California, San Francisco, CA 94158, USA
  3. Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, Oxford Road, UK M13 9PT
  4. Department of Endocrinology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia

In addition to its role in calcium homeostasis, the calcium-sensing receptor (CaSR) mediates L-amino acid (AA) dependent control of hormone release via its canonical VenusFlyTrap binding site i.e., distinct from Ca2+ binding sites. With respect to macronutrient metabolism, CaSR-mediated AA-dependent hormone release includes the enteroendocrine hormones gastrin, cholecystokinin, GLP-1 and PYY, and the pancreatic islet hormone glucagon.

Strikingly, in vitro studies have demonstrated robust AA-induced receptor activation in infinite dilution and perifusion assays with much smaller effects in static culture-based assays. This occurs despite receptor sensitivity to extracellular Ca2+ (Ca2+o ) in all these settings. In considering possible explanations for the reduced receptor response to AAs we hypothesized that cultured cells export an endogenous activator(s) that competes with AAs for the canonical binding site.

We investigated our hypothesis in HEK-293 cells stably expressing the CaSR (HEK-CaSR) and assayed IP1 accumulation and pERK in static, plate-based assays. Various Ca2+o (0.5-10 mM) in the absence or presence of AA, L-Phe (10 mM) were used to activate the receptor. Consistent with the hypothesis, stepwise washing of cultured cells reduced Ca2+o sensitivity and revealed positive modulation by AAs.

One candidate autocrine activator of the CaSR’s AA site is the tripeptide, glutathione (GSH). To test whether GSH is responsible for these effects, HEK-CaSR cells were first treated overnight with an inhibitor of GSH synthesis, buthionine sulfoximine (BSO; 100 µM). Interestingly, BSO-treated cells did not require washing to demonstrate AA-dependent activation. Comparable results were obtained upon acute exposure of HEK-CaSR cells to optimized concentrations of bovine kidney g-glutamyl transpeptidase and di-glycine to drive GSH breakdown.

Our findings support a role for AAs/GSH as CaSR co-activators with wide application to downstream signalling pathways, and suggest that receptor sensitivity to AAs in different tissues is modulated by the autocrine release of GSH and/or its regulated breakdown.