Figure 8.

GSNO-evoked potentiation of muscarinic signaling is preserved under heterologus expression but the effect diminishes with increasing constitutive activity. The human M4 (hM4) receptor was stably transfected into CHO cells and wild type (WT) or mutant (C133S) cell lines with differential G protein activation capacity were compared with that in rat forebrain membranes. CCh-stimulated G protein activity was determined in control conditions and in membranes pretreated for 30 min with 0.5 mM GSNO, as detailed in the Methods section. Note that cell lines WT-C2, WT-E5 and C133S-H2 have maximal responses comparable to that in native brain tissue. Note also robust G protein activation in WT-A1 cell line, as well as its constitutive activity, as evidenced by the ability of the inverse agonist atropine to inhibit basal G protein activity in this cell line. Potency and efficacy values are summarized in Tables 1 (rat brain) and 2 (hM4 clones). Values are mean ± SE from at least three independent experiments performed in duplicate.