The indolocarbazole, Gö6976, inhibits guanylyl cyclase-A and -B

Jerid W Robinson, Xiaoying Lou, Lincoln R Potter

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

BACKGROUND AND PURPOSE Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) decrease vascular volume and pressure by activating guanylyl cyclase-A (GC-A). C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) stimulates long bone growth. This study investigated the effects of the indolocarbazole, Gö6976, on the guanylyl cyclase activity of GC-A and GC-B as a first step towards developing small molecule regulators of these enzymes. EXPERIMENTAL APPROACH Whole cell cGMP concentrations or 32P-cGMP accumulation in membrane preparations measured the effects of indolocarbazoles on the enzymatic activity GC-A and GC-B from transfected 293T or endogenously expressing 3T3-L1 cells. KEY RESULTS Gö6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t1/2 for Gö6976 inhibition was 7 s and IC 50 was 380 nM. Gö6976 increased the EC 50 for CNP 4.5-fold, but increasing the CNP concentration did not overcome the inhibition. Half of the inhibition was lost 1 h after removal of Gö6976 from the medium. Cellular exposure to Gö6976 reduced basal and natriuretic peptide-dependent, but not detergent-dependent, GC-A and GC-B activity. Inhibition was also observed when Gö6976 was added directly to the cyclase assay. A constitutively phosphorylated form of GC-B was similarly inhibited. CONCLUSIONS AND IMPLICATIONS These data demonstrate that Gö6976 potently, rapidly and reversibly inhibited GC-A and GC-B via a process that did not require intact cells, known phosphorylation sites or inactivation of all catalytic sites. This is the first report of an intracellular inhibitor of a transmembrane guanylyl cyclase and the first report of a non-kinase target for Gö6976.

Original languageEnglish (US)
Pages (from-to)499-506
Number of pages8
JournalBritish Journal of Pharmacology
Volume164
Issue number2 B
DOIs
StatePublished - Sep 1 2011

Fingerprint

Guanylate Cyclase
C-Type Natriuretic Peptide
3T3-L1 Cells
atrial natriuretic factor receptor A
Natriuretic Peptides
Brain Natriuretic Peptide
Bone Development
Atrial Natriuretic Factor
Detergents
Blood Vessels
Catalytic Domain
B-Lymphocytes
Phosphorylation
Pressure
Membranes
Enzymes

Keywords

  • Gö6850
  • cGMP
  • guanylate cyclase
  • indolocarbazole
  • protein kinase C

Cite this

The indolocarbazole, Gö6976, inhibits guanylyl cyclase-A and -B. / Robinson, Jerid W; Lou, Xiaoying; Potter, Lincoln R.

In: British Journal of Pharmacology, Vol. 164, No. 2 B, 01.09.2011, p. 499-506.

Research output: Contribution to journalArticle

Robinson, Jerid W ; Lou, Xiaoying ; Potter, Lincoln R. / The indolocarbazole, Gö6976, inhibits guanylyl cyclase-A and -B. In: British Journal of Pharmacology. 2011 ; Vol. 164, No. 2 B. pp. 499-506.
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abstract = "BACKGROUND AND PURPOSE Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) decrease vascular volume and pressure by activating guanylyl cyclase-A (GC-A). C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) stimulates long bone growth. This study investigated the effects of the indolocarbazole, G{\"o}6976, on the guanylyl cyclase activity of GC-A and GC-B as a first step towards developing small molecule regulators of these enzymes. EXPERIMENTAL APPROACH Whole cell cGMP concentrations or 32P-cGMP accumulation in membrane preparations measured the effects of indolocarbazoles on the enzymatic activity GC-A and GC-B from transfected 293T or endogenously expressing 3T3-L1 cells. KEY RESULTS G{\"o}6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t1/2 for G{\"o}6976 inhibition was 7 s and IC 50 was 380 nM. G{\"o}6976 increased the EC 50 for CNP 4.5-fold, but increasing the CNP concentration did not overcome the inhibition. Half of the inhibition was lost 1 h after removal of G{\"o}6976 from the medium. Cellular exposure to G{\"o}6976 reduced basal and natriuretic peptide-dependent, but not detergent-dependent, GC-A and GC-B activity. Inhibition was also observed when G{\"o}6976 was added directly to the cyclase assay. A constitutively phosphorylated form of GC-B was similarly inhibited. CONCLUSIONS AND IMPLICATIONS These data demonstrate that G{\"o}6976 potently, rapidly and reversibly inhibited GC-A and GC-B via a process that did not require intact cells, known phosphorylation sites or inactivation of all catalytic sites. This is the first report of an intracellular inhibitor of a transmembrane guanylyl cyclase and the first report of a non-kinase target for G{\"o}6976.",
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T1 - The indolocarbazole, Gö6976, inhibits guanylyl cyclase-A and -B

AU - Robinson, Jerid W

AU - Lou, Xiaoying

AU - Potter, Lincoln R

PY - 2011/9/1

Y1 - 2011/9/1

N2 - BACKGROUND AND PURPOSE Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) decrease vascular volume and pressure by activating guanylyl cyclase-A (GC-A). C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) stimulates long bone growth. This study investigated the effects of the indolocarbazole, Gö6976, on the guanylyl cyclase activity of GC-A and GC-B as a first step towards developing small molecule regulators of these enzymes. EXPERIMENTAL APPROACH Whole cell cGMP concentrations or 32P-cGMP accumulation in membrane preparations measured the effects of indolocarbazoles on the enzymatic activity GC-A and GC-B from transfected 293T or endogenously expressing 3T3-L1 cells. KEY RESULTS Gö6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t1/2 for Gö6976 inhibition was 7 s and IC 50 was 380 nM. Gö6976 increased the EC 50 for CNP 4.5-fold, but increasing the CNP concentration did not overcome the inhibition. Half of the inhibition was lost 1 h after removal of Gö6976 from the medium. Cellular exposure to Gö6976 reduced basal and natriuretic peptide-dependent, but not detergent-dependent, GC-A and GC-B activity. Inhibition was also observed when Gö6976 was added directly to the cyclase assay. A constitutively phosphorylated form of GC-B was similarly inhibited. CONCLUSIONS AND IMPLICATIONS These data demonstrate that Gö6976 potently, rapidly and reversibly inhibited GC-A and GC-B via a process that did not require intact cells, known phosphorylation sites or inactivation of all catalytic sites. This is the first report of an intracellular inhibitor of a transmembrane guanylyl cyclase and the first report of a non-kinase target for Gö6976.

AB - BACKGROUND AND PURPOSE Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) decrease vascular volume and pressure by activating guanylyl cyclase-A (GC-A). C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) stimulates long bone growth. This study investigated the effects of the indolocarbazole, Gö6976, on the guanylyl cyclase activity of GC-A and GC-B as a first step towards developing small molecule regulators of these enzymes. EXPERIMENTAL APPROACH Whole cell cGMP concentrations or 32P-cGMP accumulation in membrane preparations measured the effects of indolocarbazoles on the enzymatic activity GC-A and GC-B from transfected 293T or endogenously expressing 3T3-L1 cells. KEY RESULTS Gö6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t1/2 for Gö6976 inhibition was 7 s and IC 50 was 380 nM. Gö6976 increased the EC 50 for CNP 4.5-fold, but increasing the CNP concentration did not overcome the inhibition. Half of the inhibition was lost 1 h after removal of Gö6976 from the medium. Cellular exposure to Gö6976 reduced basal and natriuretic peptide-dependent, but not detergent-dependent, GC-A and GC-B activity. Inhibition was also observed when Gö6976 was added directly to the cyclase assay. A constitutively phosphorylated form of GC-B was similarly inhibited. CONCLUSIONS AND IMPLICATIONS These data demonstrate that Gö6976 potently, rapidly and reversibly inhibited GC-A and GC-B via a process that did not require intact cells, known phosphorylation sites or inactivation of all catalytic sites. This is the first report of an intracellular inhibitor of a transmembrane guanylyl cyclase and the first report of a non-kinase target for Gö6976.

KW - Gö6850

KW - cGMP

KW - guanylate cyclase

KW - indolocarbazole

KW - protein kinase C

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