Thrombospondin-1 and angiotensin II inhibit soluble guanylyl cyclase through an increase in intracellular calcium concentration

Saumya Ramanathan, Stacy Mazzalupo, Scott Boitano, William R. Montfort

Research output: Contribution to journalArticle

30 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)7787-7799
Number of pages13
JournalBiochemistry
Volume50
Issue number36
DOIs
StatePublished - Sep 13 2011

ASJC Scopus subject areas

  • Biochemistry

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