title: Thrombospondin-1 and angiotensin II inhibit soluble guanylyl cyclase through an increase in intracellular calcium concentration.
abstract: Nitric oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca(2+)](i)), and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells and that inhibition requires an increase in [Ca(2+)](i). Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca(2+)](i), up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca(2+)](i), also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca(2+) remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca(2+)](i) in response to NO, inducing vasodilation, but also is inhibited by high [Ca(2+)](i), providing a fine balance between signals for vasodilation and vasoconstriction.
authors.affiliation: Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721, United States.
authors.forename: Saumya
authors.initials: S
authors.lastname: Ramanathan
authors.forename: Stacy
authors.initials: S
authors.lastname: Mazzalupo
authors.forename: Scott
authors.initials: S
authors.lastname: Boitano
authors.forename: William R
authors.initials: WR
authors.lastname: Montfort
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D051928:CD47 Antigen; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism; D010766:Phosphorylation; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D019700:Thrombospondin 1 / Q000494:pharmacology*; D014661:Vasoconstriction / Q000187:drug effects; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
chemical_list: D051928:CD47 Antigen; D018160:Receptors, Cytoplasmic and Nuclear; D019700:Thrombospondin 1; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D002118:Calcium