Measurement of rates of phagocytosis: the use of cellular monolayers.

A method has been developed for measuring the rate of phagocytosis rather than the quantity of particles ingested per cell when the process is virtually complete. The method, which is simpler and more rapid than those described previously, utilizes cellular monolayers, radioactive particles, and short incubation times. Under the conditions described, the rate of uptake of particles by either guinea-pig peritoneal or human blood leukocytes was proportional to both cell concentration and the time of incubation, and was independent of changes in the concentration of particles during the measurement. The particles were retained by the cells for at least 90 min. The most suitable particles so far used have been (32)P-labeled Salmonella typhimurium, and acetyl-(14)C- or methyl-(14)C-labeled starch particles. The oxidation of (14)C-labeled glucose has been studied under the same conditions that were used for the assays of phagocytosis: the greatest increase in formation of (14)CO(2) from glucose-1-(14)C occurred a few minutes after the most rapid period of phagocytosis.

Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate.

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.

Inositol lipids in cellular signalling mechanisms.

At the opening of the 1980s, two camps vigorously contested whether receptor-stimulated inositol lipid hydrolysis was a transmembrane signalling reaction that brought about an elevation of cytosolic [Ca2+] or simply a frequent, but unexplained, response of many stimulated cells to a stimulated elevation of cytosolic [Ca2+]. Since 1984, this discussion has been replaced by intensive work that is well on the way to providing a detailed description of the complex set of signalling pathways initiated by phosphatidylinositol 4,5-bisphosphate hydrolysis to form the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. In addition, it has been realized that cells closely regulate their levels both of a novel family of 3-phosphorylated inositol lipids and of a large number of water-soluble inositol polyphosphates; the definition of the functions of these molecules will be a job for the 1990s.

Diacylglycerols and phosphatidates: which molecular species are intracellular messengers?

In eukaryotes, many receptor agonists use phospholipase-generated lipids as intracellular messengers. Receptor occupation stimulates the production of polyunsaturated 1,2-diacylglycerols by phosphatidylinositol-4,5-bisphosphate specific phospholipases C and/or of mono-unsaturated and saturated phosphatidates by phospholipase-D-catalysed phosphatidylcholine breakdown. The primary phospholipase products are rapidly metabolized: polyunsaturated 1,2-diacylglycerols are converted to polyunsaturated phosphatidates by diacylglycerol kinase; mono-unsaturated and saturated phosphatidates are dephosphorylated to give mono-unsaturated and saturated 1,2-diacylglycerols by phosphatidate phosphohydrolase. The phospholipase-generated polyunsaturated 1,2-diacylglycerols and mono-unsaturated and saturated phosphatidates appear to be intracellular messengers, whereas their immediate metabolites probably do not have signalling functions.

Second messengers. Sphingolipid signalling.

Sphingomyelin metabolites seem increasingly likely to be intracellular messengers controlling a variety of cell activities, but it is not yet clear exactly which metabolites control which functions, or how they do so.

The stress-activated phosphatidylinositol 3-phosphate 5-kinase Fab1p is essential for vacuole function in S. cerevisiae.

Polyphosphoinositides have many roles in cell signalling and vesicle trafficking [1-3]. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a recently discovered PIP2 isomer, is ubiquitous in eukaryotic cells and rapidly accumulates in hyperosmotically stressed yeast. PI(3,5)P2 is synthesised from PI(3)P in both yeast and mammalian cells [4,5]. A search of the Saccharomyces cerevisiae genome database identified FAB1, a gene encoding a PIP kinase homologue and potential PI(3)P 5-kinase. Fab1p shows PI(3)P 5-kinase activity both in vivo and in vitro. A yeast strain in which FAB1 had been deleted was unable to synthesise PI(3,5)P2, either in the presence or absence of osmotic shock. A loss of PI(3,5)P2 was observed also in a temperature-sensitive FAB1 strain at the non-permissive temperature. A recombinant glutathione-S-transferase (GST)-Fab1p fusion protein was shown to have selective PI(3)P 5-kinase activity in vitro. Thus, we have demonstrated that Fab1p is a PI(3)P-specific 5-kinase and represents a third class of PIP kinase activity, which we have termed type III. Deletion of the FAB1 gene produces a loss of vacuolar morphology [6]; it is therefore concluded that PI(3,5)P2, the lipid product of Fab1p, is required for normal vacuolar function.

Novel inositol containing phospholipids and phosphates: their synthesis and possible new roles in cellular signalling.

Details of the widely employed PtdIns(4,5)P2 hydrolysis receptor-stimulated signalling pathway continue to be elucidated rapidly. However, it has recently become apparent that numerous other inositol lipids and phosphates are widespread and are likely to have important cellular functions. In this review, we focus particularly on three rapidly progressing areas: the synthesis and possible functions of 3-phosphorylated inositol lipids, particularly phosphatidylinositol 3,4,5-trisphosphate; the roles of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in coordinating intracellular Ca2+ mobilization and Ca2+ influx in stimulated cells; and the metabolism and possible functions of other inositol polyphosphates and of inositol polyphosphate pyrophosphates.

A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes.

Haemoglobin-free human erythrocyte ghosts that were prepared in the presence of EDTA and were then exposed to Ca2+ showed a substantial loss of phosphatidylinositol phosphate and phosphatidylinositol diphosphate, measured either chemically or by loss of 32P from the lipids of prelabelled membranes. At the same time there was, as reported previously (Allan, D. and Michell, R.H., (1976) Biochim. Biophys. Acta 455, 824--830), and approximately equivalent rise in the diacylglycerol content of the membranes. Analysis of the 32P-labelled water-soluble material released during this process showed that the major products were inositol diphosphate and inositol triphosphate. No change was seen in the phosphatidylinositol or phosphatidate content of the membranes, and there was no Ca2+-activated loss of 32P from the phosphatidate of prelabelled membranes: this suggests that Ca2+ did not activate phosphoinositide phosphomonoesterases or phosphatidate phosphomonoesterase in human erythrocyte membranes. It is concluded that human erythrocyte membranes contain at their cytoplasmic surface a Ca2+-activated phosphodiesterase that is active against both phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Rabbit erythrocytes also contained this enzyme, but in these cells there was also evidence for the presence of a Ca2+-activated phosphatidate phosphomonoesterase.

Production of 1,2-diacylglycerol in human erythrocyte membranes exposed to low concentrations of calcium ions.

A specific increase in the membrane content of 1,2-diacylglycerol occurred when erythrocytes were lysed at 20 degrees C in media which did not inclued a chelator of Ca2+ and also when Ca2+ was added to haemoglobin-free erythrocyte ghosts which had been prepared in the presence of ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA). The maximum increase was about 20-fold. The production of 1,2-diacylglycerol appeared to be caused by an endogenous membrane-bound phospholipase C which was half-maximally activated at less than 1 muM Ca2+ and which had access to only about 0.6-0.8% of the cells' glycerolipids. This activity was optimal at pH 7.0-7.2 in the presence of 0.1 mM Ca2+; under these conditions diacylglycerol production was complete within 5-10 min. Enzyme activity was markedly decreased at low temperatures, and was abolished by heating at 100 degrees C for 1 min.

Inhibition of porcine brain inositol 1,3,4-trisphosphate kinase by inositol polyphosphates, other polyol phosphates, polyanions and polycations.

We have partially purified an enzyme activity that phosphorylates inositol 1,3,4-trisphosphate from porcine brain, rat liver and bovine testis by FPLC chromatography on Q-Sepharose anion-exchange resin and Heparin-agarose. The products of this reaction were inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate. The same enzyme appears to be responsible for both 6-kinase and 5-kinase activities against inositol 1,3,4-trisphosphate (the 6-kinase: 5-kinase activity ratio is approximately 4 to 1), has a pH optimum of approximately 6.8 and requires Mg2+ for activity. The Km values of the enzyme for inositol 1,3,4-trisphosphate and ATP were approximately 0.5 microM and approximately 100 microM, respectively. Inositol 3,4,5,6-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate are all competitive inhibitors with K(i) values of 0.4 microM, 3 microM and 5 microM, respectively, well within their likely intracellular concentration ranges: they inhibited 6-kinase and 5-kinase activities equally. 2,3-Bisphosphoglycerate and spermine were also competitive inhibitors, with K(i) values of 0.8 mM an 12 mM, respectively. Dextran sulphate was a non-competitive inhibitor with a Ki of approximately 15 microM, and poly-L-lysine (IC50 approximately 200 microM), polyvinylsulphate (IC50 approximately 250 microM) and heparin (IC50 approximately 2 mg/ml) also inhibited. Inhibition by these compounds suggests that inositol 3,4,5,6-tetrakisphosphate (and to a lesser extent inositol 1,3,4,5-tetrakisphosphate and other naturally occurring intracellular ions) may restrict the synthesis of inositol 1,3,4,6-tetrakisphosphate and hence regulate the rate of inositol penta- and hexakisphosphate synthesis from receptor-generated inositol phosphates.

Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes.

When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack. Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and de and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.

Preparation of erythrocyte ghosts by a glycol-induced osmotic lysis under isoionic conditions.

A procedure has been developed for obtaining haemoglobin-free, erythrocyte ghosts under ionic conditions approximating that of the cell cytoplasm. Haemolysis was effected by incorporating glycol into cells suspended in the isoionic medium and then diluting with a large volume of glycol-free medium. The ghosts were of uniform spherical shape throughout the preparative procedure and were impermeable to macromolecules. Analysis of polypeptides by sodium dodecyl sulphate-gel electrophoresis at each stage of preparation and comparison with ghosts prepared under hypo-ionic conditions served to distinguish membrane components from those of cytoplasm.

Permeability characteristics of erythrocyte ghosts prepared under isoionic conditions by a glycol-induced osmotic lysis.

A detailed study has been made of the permeability characteristics of human erythrocyte ghosts prepared under isoionic conditions by a glycol-induced lysis (Billah, M.M., Finean, J.B., Coleman, R. and Michell, R.H. (1976) Biochim. Biophys. Acta 433, 45-54). Impermeability to large molecules such as dextran (average molecular weight 70 000) was restored immediately and spontaneously after each of the 5-7 lyses that were required to remove all of the haemoglobin. Permeabilities to smaller molecules such as MgATP2-, [3H]inositol and [14C]choline were initially high but could be greatly reduced by incubation at 37 degrees C for an hour. The extent of such resealing decreased as the number of lyses to which the ghosts had been subjected increased. Both removal of haemoglobin and permeabilities to small molecules were affected significantly by pH, CA3+ concentrations and divalent cation chelators. Maximum resealing was achieved in ghosts prepared in the basic ionic medium (130 mM KCl, 10 nM NaCl, 2 mM MgCl2, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES)) at pH 7.0 (0 degrees C) and with a calcium level around 10(-5) M. Acidic pH facilitated the removal of haemoglobin whilst the presence of divalent cation chelators showed down its release. Retention of K+ by ghosts leaded with K+ during the first lysis and subsequently incubated at 37 degrees C was substantial but lation chelators slowed down its released. Retention of K+ by ghosts loaded with K+ during the first lysis and subsequently incubated at 37 degrees C was substantial but little K+ could be retained within the haemoglobin-free ghosts. Permeability of the ghosts to K+ after one lysis was affected by temperature, pH, Ca2+ concentrations and by the presence of divalent cation chelators.

Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils.

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.

Intracellular concentrations of inositol, glycerophosphoinositol and inositol pentakisphosphate increase during haemopoietic cell differentiation.

We have analysed the levels of soluble inositol metabolites in HL60 cells as they differentiate towards neutrophils in response to a combination of all-trans-retinoic acid and granulocyte colony-stimulating factor and towards monocytes in response to 1 alpha-25-dihydroxyvitamin D3. In both cases, differentiation was accompanied by increases in intracellular inositol (Ins), glycerophosphoinositol (GroPIns) and inositol pentakisphosphate (InsP5) concentrations. [GroPIns] reached a peak early in the differentiation of both neutrophils and monocytes and subsequently fell to about double the starting level as the cells acquired mature characteristics, and [InsP5] rose later. Similarly, neutrophils derived in culture by the spontaneous differentiation of myeloid blast cells contained increased levels of Ins, GroPIns and InsP5 when compared to their parental blast cells. We have also compared the inositol metabolites present in two pairs of cell lines which are representative of immature and mature B and T lymphocytes. The mature cells again contained the higher levels of GroPIns and InsP5. We have previously demonstrated increases in Ins, GroPIns and Ins(1,3,4,5,6)P5 levels during the differentiation of HL60 cells towards neutrophils in response to DMSO and of GroPIns during the monocytoid differentiation of normal primitive myeloid blast cells in response to PMA. These observations suggest that deacylation of phosphatidylinositol by a phospholipase A/lysophospholipase pathway, forming GroPIns and probably also regulatory arachidonate metabolites, has some role in haemopoietic cell differentiation. The reasons why Ins(1,3,4,5,6)P5 and Ins accumulate during haemopoietic differentiation remain unknown.

Potentiation of myeloid differentiation by anti-inflammatory agents, by steroids and by retinoic acid involves a single intracellular target, probably an enzyme of the aldoketoreductase family.

HL60 cells are human promyeloid cells that can be induced to differentiate by physiological stimuli (e.g. all-trans retinoic acid (ATRA), 1 alpha,25-dihydroxyvitamin D3 (D3), granulocyte colony-stimulating factor (G-CSF)) and by non-physiological agents such as dimethysulphoxide (DMSO) and protein kinase C-activating phorbol esters. The sensitivity of HL60 cells to physiological differentiating agents, but not to DMSO, is enhanced when cells are exposed to 'anti-inflammatory agents' (e.g. indomethacin) or are 'primed' (pretreated) with a small amount of ATRA: alone, neither treatment induces differentiation. We earlier suggested that indomethacin might act by inhibiting the endogenous formation of a differentiation-suppressing prostanoid (Bunce, C.M., et al. (1994) Leukemia 8, 595-604). Studies of the formation of prostanoids by HL60 cells and of the effects of prostanoids on these cells failed to identify any prostanoid that could be implicated in sensitization by indomethacin. 3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) is another target of such 'anti-inflammatory agents'. Steroid inhibitors of 3 alpha-HSD sensitized HL60 cells to inducers of differentiation in a manner similar to indomethacin. 3 alpha-HSD is a member of the aldoketoreductase enzyme family, which comprises many enzymes of similar size and primary sequence. A protein that was recognised by an antiserum to 3 alpha-HSD was found in HL60 cells, but the cells showed no detectable 3 alpha-HSD activity. The 3 alpha-HSD-like protein was strikingly down-regulated by 'priming' doses of ATRA. When treatment with a differentiation-sensitizing 'anti-inflammatory agent' or steroid was combined with ATRA "priming', the effects of the different treatments were not additive: the resulting increase in sensitivity equalled that achievable by either treatment alone. We conclude that interference with a single intracellular regulatory mechanism underlies the increases in sensitivity of cells to differentiating agents that are caused by anti-inflammatory agents, by certain steroids and by 'priming' with ATRA. Decreased activity of a yet-to-be-identified member of the aldoketoreductase family of dehydrogenases is likely to be a central feature of a previously unrecognised mechanism that controls the responsiveness of cells to environmental stimuli such as retinoids and D3.

Indomethacin potentiates the induction of HL60 differentiation to neutrophils, by retinoic acid and granulocyte colony-stimulating factor, and to monocytes, by vitamin D3.

We have confirmed previous observations that HL60 cells treated with a combination of 10 nM retinoic acid (RA), and 30 ng/ml granulocyte colony-stimulating factor (G-CSF) differentiate efficiently towards neutrophils, as characterized by their growth arrest and acquisition of phagocytic ability. Such low concentrations of RA alone provoked only a small proportion of HL60 cells to differentiate, and G-CSF alone provoked no differentiation. In the presence of 30 microM indomethacin (an inhibitor of the enzyme cyclooxygenase that catalyses the first step of prostanoid synthesis), the onset of differentiation provoked by RA plus G-CSF was more rapid, but the final proportion of mature cells was unchanged. Indomethacin also potentiated the growth arrest and differentiation of cells in response to 10 nM RA alone. Although the potentiating effect of indomethacin on RA-induced differentiation occurred at several indomethacin and RA concentrations, it was only apparent when the RA concentration used was alone sufficient to induce a small proportion of cells to differentiate. Indomethacin shifted the G-CSF dose-response curve of cells treated with 10 nM RA to lower G-CSF concentrations. 1 alpha,25-dihydroxy vitamin D3 (VitD3) induces HL60 cells to differentiate to monocytes and indomethacin also potentiated the differentiation of HL60 cells in response to low doses of VitD3 5,8,11-eicosatriynoic acid, an inhibitor of 5-lipoxygenase and 12-lipoxygenase, neither potentiated neutrophil differentiation of HL60 cells, nor prevented indomethacin potentiation of the differentiation of RA-primed cells. Treatment of cells with dexamethasone, a steroid whose effects include inhibition of arachidonate mobilization by phospholipase A2, potentiated RA-primed neutrophil differentiation in a manner similar to indomethacin. These observations suggest that an arachidonate metabolite formed downstream of cyclooxygenase suppresses differentiation of HL60 cells both to neutrophils and monocytes, probably by inhibiting some event essential to commitment to differentiation.

Rat testicular myoid cells express vasopressin receptors: receptor structure, signal transduction, and developmental regulation.

Detection of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT) in the testis of several species has led to the proposal that these peptides may have a physiological role in the regulation of testicular function. Therefore, we investigated whether the contractile myoid cells of rat seminiferous tubules express functional receptors for AVP or OT and, thus, constitute a target for these hormones. This study used primary cultures of purified peritubular myoid cells derived from rats both before and after puberty. By several criteria, myoid cells prepared from adult rats expressed vasopressin receptors (VPRs). We detected specific and saturable [3H]AVP binding to a single population of sites with a Kd of 7.5 nM and a binding capacity of 145 fmol/mg protein. AVP stimulated the accumulation of inositol phosphates in a dose-dependent manner with an EC50 of 1.7 nM. Cloning and sequencing of the myoid cell VPR confirmed it to be the V1a subtype of VPR. VPR expression by myoid cells is under developmental control, as the receptors are present in the adult rat, but absent before puberty. In contrast, OT receptors were not expressed at any stage of development. Peritubular myoid cells are also responsive to endothelin-1 (ET-1), which potently stimulated phosphoinositidase-C. However, unlike AVP, the ET-1 responses were observed both before and after sexual maturity, suggesting different roles for AVP and ET-1 in the control of myoid cell function. Our data establish that the myoid cells of the adult rat seminiferous tubule are a target for AVP. This indicates an additional role for AVP in the regulation of testicular function and male fertility in the adult rat.

Stimulation of tyrosine phosphorylation without inositol lipid hydrolysis in human B lymphocytes on engaging CD72.

Occupancy of CD72 on resting tonsillar B cells by monoclonal antibodies (mAb) promotes entry into the G1 phase of the cell cycle with an accompanying increase in MHC Class II expression and provides a co-stimulus to immobilized anti-mu for driving DNA synthesis. We now report that engagement of CD72 by mAb stimulates tyrosine phosphorylation in B cells with a peak of activity seen at 5-10 min. Two major substrates of 29 and 57 kDa showed a basal level of phosphorylation which increased with time, while a 40 kDa protein and several other minor components were phosphorylated de novo on the addition of mAb to CD72. Inositol lipid hydrolysis was found to be unperturbed, although a shallow rise in the basal level of intracellular free Ca2+ was provoked on engaging CD72. Receptor cross-linking was not a requirement for signaling human B cells through CD72: simple occupancy by univalent antibody was sufficient both to trigger the rise in basal [Ca2+]i and to promote DNA synthesis.