title: The indolocarbazole, Gö6976, inhibits guanylyl cyclase-A and -B.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  Gö6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t(½) for Gö6976 inhibition was 7 s and IC₅₀ was 380 nM. Gö6976 increased the EC₅₀ 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
  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.
authors.affiliation: Department of Pharmacology, University of Minnesota-Twin Cities, Minneapolis, MN, USA.
authors.forename: Jerid W
authors.initials: JW
authors.lastname: Robinson
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Lou
authors.forename: Lincoln R
authors.initials: LR
authors.lastname: Potter
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology*; D002460:Cell Line; D002461:Cell Line, Transformed; D006152:Cyclic GMP / Q000037:antagonists & inhibitors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D020097:Natriuretic Peptide, Brain / Q000378:metabolism; D020098:Natriuretic Peptide, C-Type / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000099242:Atrial Natriuretic Factor Receptor B
chemical_list: D002227:Carbazoles; D006152:Cyclic GMP; D020097:Natriuretic Peptide, Brain; D020098:Natriuretic Peptide, C-Type; D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B; C081021:Go 6976; C091715:atrial natriuretic factor receptor A