A COMPARISON BETWEEN MUSCARINIC RECEPTOR OCCUPANCY, INOSITOL 1,4,5-TRISPHOSPHATE ACCUMULATION AND CA2+ MOBILIZATION IN PERMEABILIZED SH-SY5Y NEUROBLASTOMA-CELLS

Electrically permeabilized SH-SY5Y neuroblastoma cells have been used to examine the relationship between receptor occupation by muscarinic agonists, inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) accumulation and Ca2+ mobilization from intracellular stores. The kinetics, concentration-dependence and guanine nucleotide-sensitivity of these responses have been characterized for the agonists, carbachol, arecoline and oxotremorine. Carbachol stimulated Ins(1,4,5)P-3 accumulation and Ca2+ mobiIization with an EC(50) value similar to 50 pM, only slightly lower than the apparent affinity of this agonist for the ''free'' receptor (100 mu M). Arecoline and oxotremorine were partial agonists, mobilizing 45 and 21% of the Ca2+ mobilized by carbachol, and yielded EC(50) values for both Ins(1,4,5)P-3 and Ca2+ responses, similiar to their binding affinity. Guanosine 5'-O-3 thio-triphosphate (GTP gamma S) markedly enhanced the responses elicited by all three agonists. Carbachol became significantly more potent for both Ins(1,4,5)P-3 accumulation (EC(50) = 4.1 mu M) and Ca2+ mobilization (EC(50) = 0.25 mu M), revealing a separation of the dose-response relationships. GTP gamma S caused a smaller separation of the responses elicited by arecoline (Ca2+ mobilization EC(50) = 0.9 mu M; Ins(1,4,5)P-3 accumulation EC(50) = 3.6 mu M), and only enhanced maximal responses for oxotremorine. These data reveal that the functional coupling of muscarinic receptors to activation of phosphoinositidase C and subsequent Ca2+ mobilization from intracellular stores is maintained after electrical permeabilization. Furthermore, this model has been used to reveal differences in the relative activities of muscarinic agonists and how they are influenced by a hydrolysis-resistant guanine nucleotide.