An intracellular adenine nucleotide binding site inhibits guanyly cyclase C by a guanine nucleotide-dependent mechanism.
Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), is inhibited by 2-substituted adenine nucleotides in an allosteric fashion. In confluent cultures of Caco-2 intestinal epithelial cells, extracellular 2-methylthioadenosine triphosphate (2MeSATP) had no effect on basal or ST-stimulated cyclic GMP (cGMP) accumulation. However, this nucleotide inhibited cGMP accumulation in digitonin-permeabilized Caco-2 human colon carcinoma cells, demonstrating that allosteric inhibition of GCC by adenine nucleotides is mediated by an intracellular adenine nucleotide binding site rather than purinergic receptors. The role of guanine nucleotides in the regulation of GCC by adenine nucleotides was examined. Increasing GTP concentrations from 5 to 100 microM increased the potency of 2MeSATP inhibition of GCC 20-fold, with a shift in the Ki from 447 to 22 microM, respectively. Also, the hydrolysis-resistant analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), supported 2MeSATP inhibition of GCC with a potency which was 10-fold greater than GTP. In addition, GTP alone, in the absence of adenine nucleotides and at concentrations greater than 1 mM, inhibited GCC through a mechanism convergent with 2MeSATP. Guanine nucleotides supported adenine nucleotide inhibition of GCC at low concentrations and directly inhibited this enzyme at high concentrations when these studies were conducted with receptors expressed in Caco-2 cells, native rat intestine, or cloned rat GCC heterologously expressed in 293 monkey kidney cells. These observations demonstrate that adenine nucleotide inhibition of GCC is mediated through an intracellular mechanism which is guanine nucleotide-dependent.
Parkinson
S J
SJ
Department of Medicine, Division of Clinical Pharmacology, Thomas Jefferson University, Phildelphia, Pennsylvania 19107, USA.
Waldman
S A
SA
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006150:Guanine Nucleotides / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism; D051381:Rats; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology
D000227:Adenine Nucleotides; D006150:Guanine Nucleotides; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D013873:Thionucleotides; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D004072:Digitonin; C008501:2-methylthio-ATP