Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate

Abstract

The stereo specificity of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize Ca2+ from an intracellular store has been examined in permeabilized rat pituitary-tumour GH3 and Swiss 3T3 cells. A comparison of D-Ins(1,4,5)P3 with the synthetic enantiomer L-Ins(1,4,5)P3 and the racemate DL-Ins(1,4,5)P3 clearly demonstrates the marked stereospecificity of the response. Whereas D-Ins(1,4,5)P3 released 30-50% of non-mitochondrially-bound Ca2+ with a EC50 (concentration producing 50% of maximal response) of 200 nM, the L isomer was both substantially less potent and efficacious. A high concentration of the L isomer (10 microM) did not significantly shift the dose-response curve for the D isomer in Swiss 3T3 cells, suggesting that the less active isomer is probably a very weak agonist. Other studies revealed, in contrast with previous work, that the other naturally occurring isomer, D-Ins(1,3,4)P3, was essentially inactive in releasing Ca+, whereas a novel 5-phosphatase-resistant analogue, DL-myo-inositol 1,4,5-trisphosphorothioate, was a relatively potent full agonist in GH3 cells. These data reveal, for the first time, the stereoselectivity of the intracellular receptor associated with Ca2+ release. They also provide evidence for the activity of the novel phosphorothioate analogue of Ins(1,4,5)P3, but suggest that D-Ins(1,3,4)P3 is not involved in cellular Ca2+ mobilization.

myo‐Inositol 1,4,5‐trisphosphorothioate is a potent competitive inhibitor of human erythrocyte 5‐phosphatase

“…The Lenantiomer of IP3 has already been shown to be very weak at binding to the D-IPS-specific cerebellum site [lo] and is very poor at releasing calcium [9]. In the present experiments, no breakdown of L-IP3 by 5phosphatase was discernable, but this enantiomer does inhibit the enzyme.…”

“….k"T-, ; oPo*2- (2) [7], wherein the three phosphate groups have been replaced by phosphorothioate groups ( fig.1) and which is a potent mobiliser of calcium [8,9] but is resistant to 5phosphatase [lo]. We reasoned that, since D-IPS3 is a good structural analogue of D-IP3, it might be expected to be an effective inhibitor of the 5phosphatase.…”

Muscarinic receptors coupled to phosphoinositide hydrolysis and elevated cytosolic calcium in a human neuroblastoma cell line SK‐N‐SH

“…These studies have been repeated with the SH-SY5Y clone in which the time taken to change between wavelengths has been reduced to 3.8 s. Under these conditions we see at least one point on the upstroke of the calcium spike, peaking at 8.7 s and, by use of a single wavelength (340 nm), we have confirmed a similar time to reach a maximum level (unpublished observations). It is interesting to note that the production of total inositol phosphates ( Figure la) (Strupish et al, 1988) that the mobilization of calcium from intracellular pools is stereospecific, with the D-isomer of Ins(1,4,5) -P3 being active whilst the L-isomer and Ins(1,3,4)P3 were inactive. Indeed D-Ins(1,4,5)P3 had 2000 fold greater affinity than L-InsP3 to receptor sites in cerebellum (Willcocks et al, 1987).…”

“…The method used was as described previously (Strupish et al, 1988). Briefly, cells were permeabilised with saponin (100 pg ml -1) for one minute and then suspended in a cytosol-like buffer containing oligomycin (2 yg ml-1) (De Lean et al, 1978).…”

Defining the minimal structural requirements for partial agonism at the type I myo‐inositol 1,4,5‐trisphosphate receptor

“…The interaction of Ins(l,4,5)P 3 with its receptor is highly stereospecific, the vicinal 4,5-bisphosphate and 6-hydroxyl motif being the crucial structural features, and the 1-phosphate of lns (l,4,5)P3 further contributing to receptor binding specificity (reviewed [2]). Partial or total phosphorothioate substitution of the phosphate groups appears to well tolerated, producing high affinity ligands which are resistant to metabolism catalysed by the specific enzymes Ins(l,4,5)P 3 -3-kinase and Ins(l,4,5)P 3 -5-phosphatase [3][4][5]. Whilst Ins(l,4,5)PS 3 has only a slightly lower affinity than Ins(l,4,5)P 3 at binding sites characterised with [ 3 H]Ins(l,4,5)P 3 , inositol l,4,5-[ 35 S(U)]trisphosphorothioate was able to specifically label two specific binding site populations [6].…”