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G-proteins of the q family have been implicated as mediators of bombesin receptors action. We cloned Xenopus Gαq and Gα11 and specifically disrupted the synthesis of either protein with selective antisense oligonucleotides. Gαq antisense inhibited responses mediated by neuromedin B receptor (NMB-R) by 74%, though not by gastrin-releasing peptide receptor (GRP-R). Gα11 antisense had little effect on either GRP-R- or NMB-R-mediated responses. This suggests that NMB-R couples to Gαq, and that GRP-R and NMB-R show distinct G-protein coupling preferences in the Xenopus oocyte.

We tested the contribution of extracellular calcium (Ca o 2+ ) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Ca o caused a decrease in both the rapid (D 1 ) and the slow (D 2 ) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca o 2+ removal on the muscarinic response was mimicked by the addition of 1 mM Mn 2+ , an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca 2+ -free medium, subsequent addition of 1.8 mM CaCl 2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca o 2+ -dependent component of membrane electrical responses, the Ca 2+ -evoked current was reversibly abolished by Mn 2+ , though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca 2+ -evoked current, implying negative feedback of calcium channels by calcium. Injection of 10–100 fmol ofinositol 1,4,5-trisphosphate (IP 3 ) resulted in a two-component depolarizing current. IP 3 injection promoted the appearance of Ca o 2+ -evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage- insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP 3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.

Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)-sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein-activated K+ channel (GIRK) as a reporter. Low [LPA] induced IK+ in

Shallow injection of inositol 1,4,5-trisphosphate (IP3) near the animal pole of the Xenopus oocyte resulted in a large depolarizing current that decayed rapidly. A similar injection near the vegetal pole produced a much smaller response characterized by a significantly slower rate of decay. Injection of CaCl2 near the animal pole of the oocyte resulted in a large depolarizing current characterized by rapid rise and decay times. Injection near the vegetal pole of the cell produced responses that exhibited similar amplitudes but much longer rise and decay times. The protein kinase C (PK-C) activator, β-phorbol 12-myristate 13-acetate (PMA), significantly enhanced the rapid responses to IP3 injections at either hemisphere but did not affect the amplitudes of the responses to CaCl2. The PK-C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) had no effect on the responses to CaCl2. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.