NaF and guanine nucleotides modulate adenylate cyclase activity in NG108-15 cells by interacting with both Gs and Gi.
1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.
Department of Pharmacology, Medical School, Univesity of Birmingham.
E
E
Kelly
M
M
Keen
P
P
Nobbs
J
J
MacDermot
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D005576:Colforsin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D011464:Epoprostenol / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D016285:Iloprost; D051379:Mice; D009020:Morphine / Q000494:pharmacology; D009423:Nervous System Neoplasms / Q000201:enzymology / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism; D037342:Pertussis Toxin; D012969:Sodium Fluoride / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D006150:Guanine Nucleotides; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D019830:Adenosine-5'-(N-ethylcarboxamide); D009020:Morphine; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D011464:Epoprostenol; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D016285:Iloprost; D000241:Adenosine