Intracellular free Ca2+ concentrations ([Ca2+]i) were measured in subclones of NL308 neuroblastoma x fibroblast hybrid cells expressing each of the individual muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, m3 and m4. Application of 100 μM acetylcholine (ACh) increased [Ca2+]i in all four subclones. The increased [Ca2+]i levels were significantly higher in m1- and m3-transformed cells than those in m2- and m4-transformed cells. In more than 95% of m2- and m4-transformed cells, [Ca2+]i showed sinusoidal oscillations. ACh-induced increases in [Ca2+]i were not observed in cells treated with an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N∼'-tetraacetic acid (BAPTA). Removal of extracellular Ca2+ with ethyleneglycol-bis-(β-aminoethyl)-N,N,N′,N∼'-tetraacetate (EGTA) did not affect the initial [Ca2+]i increases, but reduced the late phases of Δ[Ca2+]i in m1- and m3-transformed cells by 20-30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slowed in frequency), suggesting that they were of intracellular origin. ACh-induced Δ [Ca2+]i and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50-100 ng ml-1 Pertussis 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 adenosine diphosphate ribose (cADPR) produced a rise in [Ca2+]i. ACh-induced [Ca2+]i oscillations were not observed in ryanodine-treated m2-transformed cells. These results show that, while all four mAChRs utilize Ca2+ as a common second messenger, m2 and m4 receptors use a different signalling pathway to that used by m1 and m3 receptors.
All Science Journal Classification (ASJC) codes
- Clinical Biochemistry
- Physiology (medical)