Synergistic activation by serotonin and GTP analogue and inhibition by phorbol ester of cyclic Ca2+ rises in hamster eggs.
1. Synergistic activation of a GTP-binding protein (G protein) by external serotonin (5-hydroxytryptamine, 5-HT) and internally applied guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) in hamster eggs was demonstrated by the facilitation of repetitive increases in cytoplasmic Ca2+ as measured by their associated hyperpolarizing responses (HRs) and by aequorin luminescence. 2. Rapid application of 70 nM-5-HT caused a single HR of 10-12 s duration and with a delay of 80 s. The critical concentration of 5-HT to cause an HR was 50 nM. 3. With 10 microM-5-HT four to six HRs were often elicited with a delay to the first HR of 8-30 s. HRs disappeared after prolonged or repeated application of 5-HT, indicating an apparent desensitization. 4. 5-HT-induced HRs were completely inhibited by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (TPA) (100 nM). Conversely, the PKC inhibitor sphingosine (2 microM) enhanced the series of HRs by shortening the delay to the first HR (3-9 s) and by causing more HRs. 5. Ionophoretic injection of GTP gamma S into the egg usually produced a large HR with a delay of 120-240 s followed by a series of much smaller HRs. When 5-HT was applied within 1 min of injection of GTP gamma S. 70 nM-5-HT induced a number of large HRs and even 1 nM-5-HT could induce HR(s). In contrast, when 5-HT was applied after the size of GTP gamma S-induced HRs had declined, as much as 10 microM-5-HT could only elicit a single large HR. Thus, GTP gamma S apparently caused a sensitization and then a desensitization of the action of 5-HT. 6. GTP gamma S-induced Ca2+ transients were facilitated when injected in the presence of 5-HT concentrations as low as 0.1 nM. The time delay to the first HR was 65 s in 0.1 nM-5-HT or 4 s in 100 nM-5-HT whereas it was 170 s without 5-HT (mean values). The magnitude as well as frequency of HRs succeeding the first HR was enhanced by 5-HT at concentrations above 0.01 nM. 7. TPA (100 nM) blocked the GTP gamma S-plus-5-HT-potentiated HRs after the first four to five HRs. Sphingosine (2 microM) augmented the series of HRs.(ABSTRACT TRUNCATED AT 400 WORDS)
Miyazaki
S
S
Department of Physiology, Tokyo Women's Medical College, Japan.
Katayama
Y
Y
Swann
K
K
D000331:Aequorin / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D004357:Drug Synergism; D005260:Female; D019204:GTP-Binding Proteins / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008647:Mesocricetus; D010063:Ovum / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011985:Receptors, Serotonin / Q000187:drug effects; D012701:Serotonin / Q000494:pharmacology*; D012702:Serotonin Antagonists / Q000494:pharmacology; D013110:Sphingosine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000331:Aequorin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium