INOSITOL 1,4,5-TRISPHOSPHATE FORMATION AND RYANODINE-SENSITIVE OSCILLATIONS OF CYTOSOLIC-FREE CA2-RECEPTOR SUBTYPES( CONCENTRATIONS IN NEUROBLASTOMA X FIBROBLAST HYBRID NL308 CELLS EXPRESSING M2 AND M4 MUSCARINIC ACETYLCHOLINE)

Intracellular free Ca2+ concentrations ([Ca2+](i)) were measured in su bclones of NL308 neuroblastoma x fibroblast hybrid cells expressing ea ch of the individual muscarinic acetylcholine receptor (mAChR) subtype s m1, m2, m3 and m4. Application of 100 mu M ace tylcholine (ACh) incr eased [Ca2+](i) in all four subclones. The increased [Ca2+](i) levels were significantly higher in m1- and m3-transformed cells than those i n m2- and m4-transformed cells. In more than 95% of m2- and m4-transfo rmed cells, [Ca2+](i) showed sinusoidal oscillations. ACh-induced incr eases in [Ca2+](i) were not observed in cells treated with an intracel lular Ca2+ chelator, ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceti c acid (BAPTA). Removal of extracellular Ca2+ with ethylene-glycol-bis -(beta-aminoethyl) -N,N,N',N'-tetraacetate (EGTA) did not affect the i nitial [Ca2+](i) increases, but reduced the late phases of Delta[Ca2+] (i) in m1- and m3-transformed cells by 20-30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slo wed in frequency), suggesting that they were of intracellular origin. ACh-induced Delta[Ca2+](i) and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50-100 ng ml(-1) Pertu ssis toxin (PTX) for 12 h in m2- and m4-transformed cells, but not in m1- and m3-transformed cells. In all cells, extracellular application of caffeine and ryanodine, or intracellular application of cyclic aden osine diphosphate ribose (cAD-PR) produced a rise in [Ca2+](i). ACh-in duced [Ca2+](i) oscillations were not observed in ryanodine-treated m2 -transformed cells. These results show that, while all four mAChRs uti lize Ca2+ as a common second messenger, m2 and m4 receptors use a diff erent signalling pathway to that used by m1 and m3 receptors.