The intracellular distribution of inositol polyphosphates

Abstract

Cells make a malor energetic investment in the synthesis and turriover of inositol 1,3,4,5,6 pentakisphosphate iInsP,l and inositol hexakisphosphate [lnsP,I [refs 1-41 At concentrations of between 1 0 pM and 1 mM, these are the most abundant inositol derivatives in most eukaryotic cells 15 61There is accumulating evidence to suggest that cellular concentrations of InsP, and InsP, may be capable of influencing various cell functions 17-1 11 These include control of neuronal excitability, heart rate and blood pressure, regulation of pituitary function. modulation of the oxygen affinity of haemogiobin in avian erythrocytes, putative roles as antioxidants or antineoplastic agents, and inhibition of enzymes such as an Ins(l.3.4,5IP4 3phosphatase and alkaline phosphatase Interest has recently focussed on possible interactions with arrestin and the clathrin-assembly protein AP-2 However, It remains impossible to interpret these effects in a cellular context, since we do not know whether InsPS and InsP, are located in the appropriate intracellular compartments to exert these effects m situ If either InsP, or InsP, was to have an extracellular role, it should be packaged within vesicles prior to release into the external medium To look for such packaging. we have used several techniques selectively to permeabilize the plasma membrane of the HL60 cell (a human promyelocytic leukaemia cell line that contains a relatively large amount of InsP, and InsP,l without disruption of intracellular organelles within which InsPS and InsP, might be located The degrees to which inositol polyphosphates and intracellular markers were released was then compared InsP, and InsP, were released from cells permeabilized by digitonin, by osmotic lysis or by electroporation in parallel with cytosolic markers ( 6phosphogluconate dehydrogenase and ATPI, and without significant release of the primary granule enzyme ! 3 galactosidase Electroporation was the technique that achieved the most selective permeabilisation of the plasma membrane, and the results obtained with this technique are shown below (Fig 1) During the course of these experiments it was noted that the inclusion of certain ion chelators had modest effects on InsP, and InsP, release from electroporated cells, without effecting the release of inositol or ATP Release I 2 2 x hL.LI i s mlns apart 39.2 i 2.1 36.1 * S.S 65.2 * 1.5 56.8* 1.2 -56.7 i 2.8 73.9 i 9.4 70.6 i 0.6 56.8 f 0.8 66.8 i 0.7 -44.1 i 3.7 SY.3 i 2.3 -57.0 i 1.5 -of InsP, and InsP, was increased significantly by the addition of BAPTA, EGTA or EDTA, but not by the FeiAl-selective agent desferroxamine (Fig 2) 100 90 80 7 0 U 60 m o m m 50 L a4 40 30 20 10 0 Fig. 1 The release of inositol polyphosphates, inositol and cellular markers from HL60 cells permeabilized by high voltage electroporation. /I , ; I 7 InsP,+, i i n o s i t o l I , ; I 7 InsP,+, i i n o s i t o l I I ~ ~---_----0 125 250 375 500 625 750 875 1000 V o l t a g e d i s c h a r g e d ( V / c m ) Data shown are p e r c e n t a~e r of the f o~a l released when 5...

Involvement of Inositol in Reproduction

“…This information has been reviewed in detail elsewhere. 20,21 Other inositol phosphates have various biologic functions. Inositol(3,4,5,6)P 4 regulates Ca 2 -stimulated Cl secretions-a process that is relevant to salt and uid secretions-and thereby regulates osmolarity, pH, and smooth muscle excitability.…”

“…20 Inositol(1,3,4,5,6)P 5 and InositolP 6 are the two most abundant inositol polyphosphates in eukaryotic cells. 21 It has been suggested that InositolP 5 and InositolP 6 merely serve as stores of inositol and/or phosphate. 22 Their ability to bind to certain proteins, however, might serve several biologic functions.…”