The intracellular signals generated by
carbachol activation of the
muscarinic receptor [release of
inositol phosphates as a consequence of
phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by
quin2)] were studied in intact PC12
pheochromocytoma cells that had been differentiated by treatment with
nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of
inositol trisphosphate and of its possible metabolites,
inositol bis- and mono-
phosphate, and the [Ca2+]i rise were found to occur with almost superimposable
carbachol concentration curves. At the same time
carbachol caused a decrease in the radioactivity of preloaded
phosphatidylinositol 4,5-bisphosphate, the precursor of
inositol trisphosphate. Neither the
inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or
forskolin, the direct activator of
adenylate cyclase. Both signals were partially inhibited by
dibutyryl cyclic AMP, especially when the
nucleotide analogue was applied in combination with the
phosphodiesterase inhibitors RO 201724 and
theophylline. The latter
drug alone profoundly inhibited the
carbachol-induced [Ca2+]i rise, with only minimal effect on
phosphoinositide hydrolysis. Because of the diverging results obtained with
forskolin on the one hand,
dibutyryl cyclic AMP and
phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular
cyclic AMP concentration. Two further drugs tested,
mepacrine and MY5445, inhibited
phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by
carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate
phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the
carbachol concentration curves for
inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by
theophylline of the Ca2$ signal without major effects on
inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.