Abstract
Adenophostin A is the most potent known agonist ofd-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors. Equilibrium competition binding studies with 3H-Ins(1,4,5)P3 showed that the interaction of a totally synthetic adenophostin A with both hepatic and cerebellar Ins(1,4,5)P3 receptors was indistinguishable from that of the natural product. At pH 8.3, a synthetic analog of adenophostin A (which we named acyclophostin), in which most elements of the ribose ring have been removed, bound with substantially higher affinity (K d = 2.76 ± 0.26 nM) than Ins(1,4,5)P3 (K d = 7.96 ± 1.02 nM) to the 3H-Ins(1,4,5)P3-binding sites of hepatic membranes. At pH 7, acyclophostin (EC50 = 209 ± 12 nM) and Ins(1,4,5)P3 (EC50 = 153 ± 11 nM) stimulated 45Ca++ release to the same maximal extent and from the same intracellular stores of permeabilized hepatocytes. Comparison of the affinities of a range of Ins(1,4,5)P3 and adenophostin analogs with their abilities to stimulate Ca++ release revealed that although all other agonists had similar EC50/K dratios, that for acyclophostin was significantly higher. Similar results were obtained with cerebellar membranes, which express almost entirely type 1 InsP3 receptors. When the radioligand binding and functional assays of hepatocytes were performed under identical conditions, the higher EC50/K d ratio for acyclophostin was retained at pH 8.3, but it was similar to that for Ins(1,4,5)P3 when the assays were performed at pH 7. To directly assess whether acyclophostin was a partial agonist of hepatic Ins(1,4,5)P3 receptors, the kinetics of45Ca++ efflux from permeabilized hepatocytes was measured with a temporal resolution of 80 ms using rapid superfusion. At pH 7, the kinetics of 45Ca++release, including the maximal rate of release, evoked by maximal concentrations of acyclophostin or Ins(1,4,5)P3 were indistinguishable. At pH 8.3, however, the maximal rate of45Ca++ release evoked by a supramaximal concentration of acyclophostin was only 69 ± 7% of that evoked by Ins(1,4,5)P3. We conclude that acyclophostin is the highest affinity ribose-modified analog of adenophostin so far synthesized, that at high pH it is a partial agonist of inositol trisphosphate receptors, and that it may provide a structure from which to develop high-affinity antagonists of inositol trisphosphate receptors.
Footnotes
- Received July 14, 1998.
- Accepted September 25, 1998.
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Send reprint requests to: Dr. Colin W. Taylor, Department of Pharmacology, Tennis Court Road, University of Cambridge, Cambridge, CB2 1QJ UK. E-mail: cwt1000{at}cam.ac.uk
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This work was supported by grants from the Wellcome Trust to C.W.T. (039662) and B.V.L.P. (045491) and from the Biotechnology and Biological Sciences Research Council to C.W.T. J.S.M. is supported by a Wellcome Prize Fellowship (018484).
- The American Society for Pharmacology and Experimental Therapeutics
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