Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium.
The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.
Koch
B D
BD
Dorflinger
L J
LJ
Schonbrunn
A
A
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D037342:Pertussis Toxin; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D011956:Receptors, Cell Surface / Q000502:physiology; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000378:metabolism / Q000494:pharmacology*; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D011956:Receptors, Cell Surface; D017481:Receptors, Somatostatin; D010566:Virulence Factors, Bordetella; D006165:Guanylyl Imidodiphosphate; C014109:thyrotropin-releasing hormone, 3-Me-; D014660:Vasoactive Intestinal Peptide; D013004:Somatostatin; D013973:Thyrotropin-Releasing Hormone; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D011188:Potassium; D002118:Calcium; D011761:Pyrrolidonecarboxylic Acid