Changes in extracellular calcium within the physiological range influence receptor-mediated inositol phosphate responses in brain and tracheal smooth muscle slices

Summary

The effect of changes in extracellular calcium concentration ([Ca²⁺]_e) on the incorporation of myo-[2-³H]-inositol into phosphoinositides and agonist-stimulated ³H-inositol phosphates (³H-InsPs) was examined in rat cerebral cortex and bovine tracheal smooth muscle slices. In brain slices, reduction in [Ca²⁺]_e from 2.4 to 1.2 mmol/l resulted in an approximate doubling of the carbachol and noradrenaline-stimulated ³H-InsP response with no effect on the EC₅₀ values. An identical effect of varying [Ca²⁺]_e was observed for carbachol-stimulated ³H-InsP formation in tracheal smooth muscle with a further increase in ³H-InsPs evident at [Ca²⁺]_e 0.6 mmol/l. In this tissue the effect of changes in [Ca ²⁺]_e on the incorporation of myo-[2-³H]-inositol into the total phosphoinositide pool directly paralleled the changes in ³H-InsPs except in conditions of no added calcium when ³H-InsP responses were markedly impaired. Additional studies in brain slices using buffer where the added calcium varied between 0 and 2.4 mmol/l, showed that both the carbachol stimulated formation of separate inositol phosphates during short incubation periods and incorporation of myo-[2-³H]-inositol into PtdInsP and PtdInsP₂ under basal conditions was maximal at [Ca²⁺]_e 0.3 mmol/l. Omitting Ca²⁺]_e from the buffer resulted in maximal labelling of PtdIns but a decrease in PtdInsP and PtdInsP₂ labelling (compared with the level at [Ca²⁺]_e 0.3 mmol/l) and a markedly impaired inositol polyphosphate response. Alterations in [Ca²⁺]_e following ³H-inositol labelling but immediately prior to carbachol stimulation did not influence ³H-inositol polyphosphate responses. It is therefore clear that even relatively small changes in [Ca²⁺]_e markedly influence agonist-stimulated ³H-InsP responses in brain and tracheal smooth muscle slices and that these reflect changes in the labelling of substrate inositol lipids. These findings have important practical implications for studies examining ³H-InsP responses in central and peripheral tissues and the differential effect of very low [Ca²⁺]_e on PtdIns and PtdlnsP/PtdInsP₂ labelling may explain in part the severe decrease in ³H-InsPs seen under these conditions despite apparent maximal total phosphoinositide labelling.