Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides

Abstract

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.

Time-dependent potentiation of insulin release induced by alpha-ketoisocaproate and leucine in rats: possible involvement of phosphoinositide hydrolysis

“…In some experiments the nature of the 3H-containing molecules was assessed in pooled perifusate samples collected during the final 10 min of the perifusion. In other experiments, the levels of free 3H-inositol, glycerophosphoinositol (GPI) and labelled inositol phosphates were determined after extraction with 10% perchloric acid by methods previously detailed [13,14]. Briefly, after neutralisation with 0.25-0.28 ml 6 N KOH, the further addition of 5 ml water, and centrifugation, the supernatant was applied to columns.…”

Right atrial dilatation increases inositol‐(1,4,5)trisphosphate accumulation Implications for the control of atrial natriuretic peptide release

“…The stretch signal was terminated by chilling the hearts in ice-cold saline. Right atria were dissected, weighed and inositol phosphates extracted using a chloroform/methanol extraction method as described elsewhere [7].…”

“…Water-soluble [3H]inositol-labelled compounds were separated using anion-exchange high-performance liquid chromatography as described elsewhere [6] or Dowex column chromatography [7]. The column used was a Whatman partisil SAX column packed by Waters (Milford, MA, USA) for use in a radial compression system on a Waters model 441 liquid chromatograph.…”

Human neutrophil cytosolic phospholipase C: partial characterization

“…Hydrolysis was terminated by the addition of chloroform/methanol (2:1, v/v). lnositol phosphates were isolated on Dowex AGI-X8-formate columns eluted with 0.2 M ammonium formate/1.0 M formic acid and their radioactivities were determined in liquid scintillation counter [19].…”