Stephen T. Safrany

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2+-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase

The ability of D‐6‐deoxy‐myo‐inositol 1,4,5‐trisphosphate [6‐deoxy‐Ins(1,4,5)P3], a synthetic analogue of the second messenger D‐myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH‐SY5Y neuroblastoma cells was investigated. 6‐Deoxy‐Ins(1,4,5)P3 was a full agonist (EC30 = 6.4 μM), but was some 70‐fold less potent than Ins (1,4,5)P3 (EC30 = 0.09 μM), indicating that the 6‐hydroxyl group of Ins(1,4,5)P3 is most important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6‐Deoxy‐Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5‐Phosphatase, but inhibited both the hydrolysis of 5‐[22P] + Ins (1,4,5)P3 (K 1 76 μM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent K 1 5.7 μM) 6‐Deoxy‐Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins(1,4,5)P3 3‐kinase.

3-Position modification of myo-inositol 1,4,5-trisphosphate: consequences for intracellular Ca2+ mobilisation and enzyme recognition

The ability of the novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogues, L-chiro-inositol 2,3,5-trisphosphate (L-ch-Ins(2,3,5)P3) and D-3-deoxy-3-fluoro-myo-inositol 1,4,5-trisphosphate (3F-Ins(1,4,5)P3), to bind to the Ins(1,4,5)P3 receptor, mobilise intracellular Ca2+ stores and interact with metabolic enzymes has been investigated. L-ch-Ins(2,3,5)P3 and 3F-Ins(1,4,5)P3 were bound by the Ins(1,4,5)P3 receptor from bovine adrenal cortex with relatively high affinity (Ki values 60.4 and 8.0 nM respectively) but with lower affinity than Ins(1,4,5)P3 (KD = 5.9 nM). Both analogues were apparent full agonists at the Ca2+ mobilising receptor in SH-SY5Y cells, but were less potent than Ins(1,4,5)P3 (EC50 L-ch-Ins(2,3,5)P3 = 1.4 microM, 3F-Ins(1,4,5)P3 = 0.37 microM and Ins(1,4,5)P3 = 0.12 microM). L-ch-Ins(2,3,5)P3 and 3F-Ins(1,4,5)P3 were resistant to Ins(1,4,5)P3 3-kinase, and were potent inhibitors of the enzyme (Ki values 7.1 and 8.6 microM respectively). 3F-Ins(1,4,5)P3 was hydrolysed by Ins(1,4,5)P3 5-phosphatase at a similar rate to Ins(1,4,5)P3, but inhibited dephosphorylation of [3H]Ins(1,4,5)P3 with high potency (apparent Ki = 3.9 microM) L-ch-Ins(2,3,5)P3 was also recognised by the enzyme with high affinity (Ki = 7.7 microM) but was resistant to hydrolysis. These results suggest that the environment around C-3 is of major importance for recognition not only by Ins(1,4,5)P3 3-kinase but also by Ins(1,4,5)P3 5-phosphatase.

Ca2(+)-mobilising properties of synthetic fluoro-analogues of myo-inositol 1,4,5-trisphosphate and their interaction with myo-inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

The ability of two fluoro‐analogues of D‐myo‐inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) to mobilize intracellular Ca2+ stores in SH‐SY5Y neuroblastoma cells has been investigated. DL‐2‐deoxy‐2‐fluoro‐scyllo‐Ins(1,4,5)P3 (2F‐Ins(1,4,5)P3) and DL‐2,2‐difluoro‐2‐deoxy‐myo‐Ins(1,4,5)P3 (2,2‐F2‐Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 μM respectively) and slightly less potent than D‐Ins(1,4,5)P3 (EC50 0.13 μM), indicating that the axial 2‐hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3‐kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F‐Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5‐phosphatase but 2,2‐F2‐Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 μM).

Muscarinic receptors, phosphoinositide metabolism and intracellular calcium in neuronal cells.

1. We have utilised SH-SY5Y human neuroblastoma cells and primary cultures of rat neonatal cerebellar granule cells, both expressing M3 muscarinic receptors, to examine agonist driven polyphosphoinositide hydrolysis and alterations in intracellular calcium. 2. Stimulation of SH-SY5Y cells leads to a biphasic increase in intracellular calcium, the initial peak being due to the release of calcium from an intracellular store and the second maintained phase being due to calcium entry across the plasma membrane. The channel involved does not appear to be voltage sensitive, to involve a pertussis toxin sensitive G protein, or be opened by inositol polyphosphates. 3. Muscarinic receptor stimulation also leads to increased inositol polyphosphate formation in SH-SY5Y cells. Ins(1,4,5)P3 mass formation was biphasic in profile whereas Ins(1,3,4,5)P4 mass formation was slower and monophasic in profile. These data are consistent with substantial activity of 5-phosphatase (dephosphorylating Ins(1,4,5)P3 to Ins(1,4)P2) and 3-kinase (phosphorylating Ins(1,4,5)P3 to Ins(1,3,4,5)P4) in SH-SY5Y cells. 4. In order to better understand the role of Ins(1,4,5)P3 and its metabolites in calcium homeostasis we have examined the ability of a variety of natural and synthetic analogues to release intracellular sequestered calcium. The Ins(1,4,5)P3 calcium mobilizing receptor displays a remarkable degree of stereo- and positional selectivity with the most potent agonist to date being Ins(1,4,5)P3 (EC50 = 0.09 microM). 5. As an alternative to the continuous SH-SY5Y neuroblastoma (tumour derived) cell line we have used the primary cultured cerebellar granule cell. These cells also display a biphasic increase in Ins(1,4,5)P3 mass and a subsequent release of intracellular stored calcium. In our hands carbachol appears to increase calcium influx, a response which is only visible in the absence of magnesium.

Synthesis of myo-inositol 1,2,4,5-tetrakisphosphate, a Ca2+-mobilising tetrakisphosphate with a potency similar to myo-inositol 1,4,5-trisphosphate

Abstract The synthesis of myo-inositol 1,2,4,5-tetrakisphosphate from inositol is described; this tetrakisphosphate is a highly potent Ca2+-mobilising agonist at the Ins(1,4,5)P3 receptor.

Synthesis of L-chiro-inositol 1,4,6-trisphosphorothioate, a potent and selective inhibitor of myo-inositol 1,4,5-trisphosphate 5-phosphatase

Abstract L- chiro -inositol 1,4,6-trisphosphate and trisphosphorothioate have been synthesized from L-quebrachitol; the trisphosphorothiate is the most potent inhibitor of Ins(1,4,5)P 3 5-phosphatase yet discovered.