Inositol 1,2-cyclic 4,5-trisphosphate is not a product of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis in rat parotid glands

Abstract

We have employed a neutral-pH extraction technique to look for inositol 1,2-cyclic phosphate derivatives in [3H]inositol-labelled parotid gland slices stimulated with carbachol. The incubations were terminated by adding cold chloroform/methanol (1:2, v/v), the samples were dried under vacuum and inositol phosphates were extracted from the dried residues by phenol/chloroform/water partitioning. Water-soluble inositol metabolites were separated by h.p.l.c. at pH 3.7. 32P-labelled inositol phosphate standards (inositol 1-phosphate, inositol 1,2-cyclic phosphate, inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate) were quantitively recovered through both extraction and chromatography steps. Treatment of inositol cyclic phosphate standards with 5% (w/v) HClO4 for 10 min prior to chromatography resulted in formation of the expected non-cyclic compounds. [3H]Inositol 1-phosphate and [3H]inositol 1,4,5-trisphosphate were both present in parotid gland slices and both increased during stimulation with 1 mM-carbachol. There was no evidence for significant quantities of [3H]inositol 1,2-cyclic phosphate or [3H]inositol 1,2-cyclic 4,5-trisphosphate in control or carbachol-stimulated glands. Parotid gland homogenates rapidly converted inositol 1,4,5-trisphosphate to inositol bisphosphate and inositol tetrakisphosphate, but metabolism of the inositol cyclic trisphosphate was much slower. The results suggest that inositol 1,4,5-trisphosphate, but not inositol 1,2-cyclic 4,5-trisphosphate, is the water-soluble product of muscarinic receptor-stimulated phospholipase C in rat parotid glands.