SYNERGISTIC ACTIVATION BY SEROTONIN AND GTP ANALOG AND INHIBITION BY PHORBOL ESTER OF CYCLIC CA-2+ 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) © 1990 The Physiological Society