Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739-744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex. PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha). Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor. These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling.

Nitric oxide (NO) has various physiological functions. However, uncontrolled overproduction of NO can be toxic in many pathologic conditions involving inflammatory tissue damage. In the present study, we examined effects of 23,24-dihydrocucurbitacin D (DHCD) isolated from the root of Bryonia alba L. on macrophage NO generation. DHCD (Ͻ80 M) effectively abolished NO generation from macrophages activated with lipopolysaccharide and interferon-␥. DHCD decreased the levels of protein and mRNA for inducible NO synthase (iNOS). DHCD potently blocked nuclear factor-B (NF-B) activation, a process necessary for transcriptional activation of iNOS. These results suggested that DHCD inhibited NO generation by blocking NF-B activation and iNOS gene transcription. Because NF-B activation is necessary not only for NO generation but also for many inflammatory processes, DHCD and its derivatives could be developed as anti-inflammatory drugs.

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We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the 1 -adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed G h 5 ) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, G h5 was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed highaffinity GTP S binding which is Mg 2 + -dependent and saturable. The relative order of affinity of nucleotide binding by G h 5 was GTP> GDP > ITP >> ATP adenyl-5'-yl imidodiphosphate, which was similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, the G h 5 demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTP S. In support of these observations, the G h 5 was recognized by a specific antibody to G h 7 or TGase II, indicating a homology with G h (TGase II) family. These results demonstrate that 77-kDa G h5 from pig heart is an 1 -adrenoceptor-coupled G h (TGase II) family which has species-specificity in molecular mass.