No abstract

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and< or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form.

The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gamma-thio]-triphosphate, the latter indicating that some PDBu-sensitive elements were downstream of the receptor. The PKC inhibitor, Ro-318220, protected against PDBu but did not enhance responses to maximal concentrations of carbachol, indicating no feedback inhibition by agonist-activated PKC. Muscarinic antagonist activity of Ro-318220 complicated such assessment at low agonist concentrations. Carbachol or PDBu induced cytosol to membrane translocation of PKC alpha. This was faster and possibly greater with PDBu, which may explain the lack of feedback by agonist-activated PKC. These results indicate that, in SH-SY5Y cells, PDBu activation of PKC preferentially inhibits rapid muscarinic receptor-mediated phosphoinositide and Ca2+ responses via suppression of PtdIns(4,5)P2 hydrolysis. This is at least partially through inhibition of Gq-protein/phosphoinositidase C coupling. However, at least at high agonist concentrations, a major agonist-mediated PKC feedback is not present in these cells.

Inositol lipids and phosphates play vital roles in the control of cell function through their participation in signalling pathways downstream of receptor-regulated enzymes such as phospholipase C and phosphatidylinositol 3-kinase, which generate established (inositol 1,4,5-trisphosphate and diacylglycerol) or putative (phosphatidylinositol 3,4,S-trisphosphate) intracellular signalling molecules respectively (1 -3). Further progress in our understanding of the functions of these second messengers in complex cellular processes may come by studying signalling pathways in organisms with easily controlled genetics.The fission yeast, Schizosaccharomycespombe, in which mating is controlled by the reciprocal release of the pheromones M-factor and P-factor (4), may provide a genetically tractable organism in which to study these events. The pheromone response pathway appears to include certain features similar to well characterised signal transduction events in mammalian systems. The receptors for both pheromones (encoded by themam2 and map3 genes) have seven transmembrane spanning domains and are members of the receptor superfamily whose prototype is mammalian rhodopsin. Like other receptors of this family, the pheromone receptors are coupled to a G protein (encoded bygpal). The Gcr sub-unit is responsible for transducing the pheromone signal (5). S. Pombe also contain homologues of the mammalian MAP kinase cascade (byr2, byrl, ste8) and of ras (Rasl) and these proteins are also believed to be components of the pheromone signalling pathway, although their precise functions have yet to be defined (4) The direct target for the Gpal protein is unknown and has been the focus of our investigations. Preliminary experiments analysing the effect of pheromone stimulation of S. pombe cells have implicated the involvement of certain inositol lipids and phosphates in the signalling pathway. The most consistent response to pheromone was a triphasic accumulation of diacylglycerol (DAG), shown in Figure 1; this was measured by a mass assay involving radioenzymatic conversion of DAG to phosphatidic acid (PtdOH) by E.coli DAG kinase (6). In addition, increases in compounds having the chromatographic characteristics of glycerophosphoinositol and InsP, although not consistently in the levels of Ins( 1,4,5)P3, were observed upon pheromone stimulation in [3H]inositol-labelled S. pombe cells. Such observations implicate the activation of specific phospholipases in response to pheromone addition. Extracts from S. pombe contain a number of the inositol phosphates commonly found in mammalian cells, alongside some of the enzymes which metabolise them. Exogenously added [3H]Ins( 1,4,5)P3, [3H]Ins(1,4)P2 and [3H]Ins1P can be metabolised by activities which appear to share some of the characteristics of their mammalian counterparts (7). For example [3H]Ins( 1,4,5)P3 was metabolised by a 5-phosphatase, a 3-kinase and also by a minor pathway involving a putative 6-kinase. [3H]Ins( I,3,4)P3 appears to be dephosphorylated to [3H]Ins( 1,4)P2 and [3H]Ins(...

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...