Interaction of polyanions with the recognition sites for inositol 1,4,5-trisphosphate in the bovine adrenal cortex

Abstract

Inositol 1,4,5-trisphosphate (InsP 3) serves as a second messenger for Ca 2+ mobilization in a wide variety of cells. InsP 3 activates a specific receptor/channel located on an internal Ca 2+ store. Because heparin has already been shown to block the action of InsP 3, we have looked at the influence of other polyanions (ddextran sulfate and polyvinyl sulfate) on the action and metabolism of InsP 3 in the bovine adrenal cortex. Polyvinyl sulfate blocked InsP 3 binding to adrenal cortex microsomes with a half-maximal efficiency of 250 nM. Scatchard analyses revealed that this effect was not competitive. The Ca 2+ releasing activity of InsP 3 on the same microsomal preparation was monitored with the fluorescent indicator, fura-2. Polyvinyl sulfate blocked this activity with a half-maximal efficiency of 80 nM. The effect of polyvinyl sulfate could not be overcome by supramaximal doses of InsP 3, suggesting a non-competitive inhibitory effect. The activity of InsP 3 phosphatase from bovine adrenal cortex microsomes was also studied. Polyvinyl sulfate inhibited the activity of the phosphatase with a half-maximal efficiency of 5 μM. Lineweaver-Burk plots revealed that this effect was not competitive. Polyvinyl sulfate was also able to inhibit the activity of InsP 3 kinase from bovine adrenal cortex cytosol. The half-maximal dose was 15 nM and the Lineweaver-Burk analysis showed that the inhibition was not competitive. The effect of dextran sulfate 5000 (DS-5000) on these activities was also studied. DS-5000 inhibited in a competitive manner the binding of InsP 3 to its receptor (IC 50 of 34 μM), the release of Ca 2+ induced by InsP 3 (IC 50 of 6.5 μM) and the activity of InsP 3 phosphatase (IC 50 of 57 μM). DS-5000 strongly inhibited in a non-competitive manner the activity of InsP 3 kinase (IC 50 of 8 nM). These results show that polyanions share similar structural properties with InsP 3. Their inhibitory effects demonstrated some interesting selectivity, specially that of DS-5000 on InsP 3 kinase activity.