Inositol 1,4,5-trisphosphate formation and ryanodine-sensitive oscillations of cytosolic free Ca2+ concentrations in neuroblastoma x fibroblast hybrid NL308 cells expressing m2 and m4 muscarinic acetylcholine receptor subtypes.
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 microM 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 ethylene-glycol-bis-(beta- aminoethyl)-N,N,N',N'-tetraacetate (EGTA) did not affect the initial [Ca2+]i increases, but reduced the late phases of delta [Ca2+]i in ml- 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 delta [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 (cAD-PR) produced a rise in [Ca2+]i. ACh-induced [Ca2+]i oscillations were not observed in ryanodine-treated m2-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Ishizaka
N
N
Department of Biophysics, Kanazawa University School of Medicine, Japan.
Noda
M
M
Kimura
Y
Y
Hashii
M
M
Fukuda
K
K
Katayama
M
M
Brown
D A
DA
Higashida
H
H
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005470:Fluorometry; D016257:Fura-2 / Q000031:analogs & derivatives; D006822:Hybrid Cells; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism; D037342:Pertussis Toxin; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000187:drug effects; D012433:Ryanodine / Q000494:pharmacology*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; C049925:fura-2-am; D012433:Ryanodine; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D000109:Acetylcholine; D002118:Calcium; D016257:Fura-2