The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells.

The pharmacological inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway have been proposed in the treatment of leukemia based on their antiproliferative effects. However, several studies demonstrated the activation of PI3K in the nuclei of all-trans-retinoic acid (ATRA) - differentiated HL-60 cells, raising the possibility that PI3K/Akt-inhibitors may block antitumor properties of retinoids. The aim of the present study was to investigate the possible activation of nuclear Akt in ATRA-treated cells and to test the effects of Akt-inhibitors on ATRA-mediated differentiation. The Akt-activity was found to be increased in the nuclei and lysates of ATRA-differentiated HL-60 and NB4 cells. The down-modulation of the expression of Akt protein in HL-60 cells using siRNA reduces the CD11b expression in ATRA-treated cells. The treatment of both cell lines with the commercially available Akt inhibitors inhibited the growth of both control and ATRA-treated cells. Akt-inhibitors had no inhibitory effects on ATRA-mediated growth arrest and the expression of CD11b in HL-60 cells, but increased the percentage of control cells expressing CD11b. In contrast, the presence of Akt inhibitors reduced the expression of CD11b in ATRA-treated NB4 cells.

Extracellular Ca2+ influences 1,2-diacylglycerol and inositol monophosphate formation in renal cortical slices stimulated with plasma obtained from uninephrectomized rats.

Formation of 1,2-diacylglycerol (DAG) and inositol phosphates was investigated in renal cortical slices after addition of plasma obtained 30 min after either uninephrectomy or sham operation. Plasma from uninephrectomized rats increased DAG concentration about 100% above control value during 5 min of incubation, but when extracellular Ca2+ had been chelated with 4 mM EGTA, the increase of DAG was only about 50% and the duration of the effect was 2 min only. Furthermore, chelation of extracellular Ca2+ did not affect the formation of InsP3 and InsP2 during 20 min of incubation, while the formation of InsP was significantly reduced. Results indicate that extracellular Ca2+ is needed for sustained increase in DAG concentration and increase in InsP radioactivity, suggesting phosphatidylinositol as their probable source.

Epidermal growth factor stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides but does not affect phosphoinositide breakdown by phospholipase C in renal cortical slices.

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidic acid and phosphoinositides were examined using renal cortical slices labelled with either sodium [32P]orthophosphate or myo-[3H]inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid and phosphoinositides. This effect is not dependent on external calcium and is inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). When phospholipids were prelabelled, EGF did not decrease the level of 32P in phosphatidic acid and phosphoinositides, and EGF did not affect the formation of inositol phosphates or the concentration of cAMP and cGMP in renal tissue. The results show that EGF stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides, but does not affect breakdown of phosphoinositides by phospholipase C in renal cortical slices.

Insulin-like growth factor I stimulates phospholipid synthesis in renal cortical slices without production of inositol phosphate.

The effects of insulin-like growth factor I (IGF-I) on the metabolism of phospholipids in renal cortical slices were examined using either sodium [32P]orthophosphate or myo-[3H]inositol. IGF-I was found to increase the incorporation of phosphate into phospholipids about 2-3-times above control values, leading to an increase in the concentration of total phospholipid phosphorus of 20% above control value after 1 h of incubation. The increased incorporation of phosphate into phospholipids could be prevented by 10 microM cycloheximide, while with 1 microM TPA (12-O-tetradecanoylphorbol 13-acetate) it could not. Insulin was also found to increase the incorporation of phosphate into phospholipids, but only if its concentration was at least 100-times higher than that of IGF-I. When phospholipids were prelabelled, IGF-I neither decreased the level of 32P in phospholipids nor stimulated the formation of inositol phosphates. The results show that IGF-I stimulates phospholipid synthesis without production of inositol phosphates in renal cortical slices.

Stimulation of ATP-driven Ca2+ pump in the basal-lateral plasma membranes of kidney cortex during compensatory renal growth.

During compensatory renal growth 45Ca2+ transport in basal-lateral plasma membrane vesicles isolated from the rat renal cortex have been investigated. Stimulation of Ca2(+)-ATPase activity was observed, without an effect of compensatory renal growth on Na+/Ca2+ exchanger activity and on passive Ca2+ permeability of the vesicles. Twelve hours following unilateral nephrectomy about 40% increase of Ca2(+)-ATPase activity above control value was observed and this effect was present until the end of the experimental period (7 days). When kinetic parameters for Ca2(+)-ATPase were studied in native membranes, an increase of Vmax was observed, whereas the Km for Ca2+ was similar in control vesicles and vesicles isolated from the remnant kidney. Depletion of endogenous calmodulin resulted in a decrease of Vmax and an increase of Km (Ca2+), while its addition reversed these parameters and increased the Hill coefficient from about 1 to about 2. Once again, only a significant increase of Vmax in vesicles isolated from the remnant kidney above the control value was observed. Finally, increase of Ca2(+)-ATPase activity during compensatory renal growth could be abolished by actinomycin D, indicating that its stimulation is due to protein synthesis.

Incorporation of 32P into phospholipids in vivo during compensatory renal growth.

Incorporation of 32P into phospholipids, RNA and DNA was studied in adult male C57BL/GoZgb mice. Left nephrectomy was performed under diethyl ether anesthesia, and the remaining right kidney was excised 10 min to 28 days later. Sham-operated animals were used as controls. 2 h before killing, animals were injected intraperitoneally with 37 kBq (1 microCi) 32P (as sodium orthophosphate) per g of body weight. In the right kidney, incorporation of 32P into total phospholipids, and five phospholipid fractions (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diphosphatidylglycerol and sphingomyelin) was increased by 25-35% between 20 and 72 h after uninephrectomy. The incorporation of 32P into RNA showed a similar pattern. However, incorporation of 32P into phosphatidylinositol and phosphatidic acid was already increased 20 min after uninephrectomy, reached a peak about 110-120% above control values 4 h after uninephrectomy, and then slowly returned to the control value at about 84-96 h. These results indicate that an early alteration in phospholipid metabolism, particularly of inositol lipids, may play a role in initiation of compensatory renal growth.

The nuclear phosphoinositide cycle--does it play a role in nuclear Ca2+ homoeostasis?

The probable answer to this question is no. Much of the current evidence summarised elsewhere in this issue points to nuclear Ca2+ changes changing in response to cytosolic Ca2+, with little evidence for an independently controlled nuclear Ca2+ homeostasis. There are InsP3 receptors in the nuclear membrane, and it is possible that during nuclear membrane assembly the InsP3 acting on these (Sullivan and Wilson, this issue) is formed by an inositide cycle located on the assembling nuclear skeleton. But our current experimental data suggest that when the nucleus is intact, InsP3 generated by this cycle would have to exit through the nuclear pores to act on any known InsP3 receptors. Thus the nuclear inositide cycle appears more likely to serve to generate diacylglycerol to activate protein kinase C, and/or to generate inositol phosphates such as InsP2, which may have distinct intranuclear functions.

Acetylcholine and cholecystokinin receptors functionally couple by different G-proteins to phospholipase C in pancreatic acinar cells.

We have studied the involvement of GTP-binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin-octapeptide (CCK-OP) and GTP gamma S but not carbachol (CCh)-induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP-analogue, 8-bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist-induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP-ribosylated a 40 kDa protein, which was inhibited by CCK-OP but not by CCh. We conclude from these data that both CCK- and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP-binding proteins.

The effect of big endothelin-1 in the proximal tubule of the rat kidney.

1. An obligatory step in the biosynthesis of endothelin-1 (ET-1) is the conversion of its inactive precursor, big ET-1, into the mature form by the action of specific, phosphoramidon-sensitive, endothelin converting enzyme(s) (ECE). Disparate effects of big ET-1 and ET-1 on renal tubule function suggest that big ET-1 might directly influence renal tubule function. Therefore, the role of the enzymatic conversion of big ET-1 into ET-1 in eliciting the functional response (generation of 1,2-diacylglycerol) to big ET-1 was studied in the rat proximal tubules. 2. In renal cortical slices incubated with big ET-1, pretreatment with phosphoramidon (an ECE inhibitor) reduced tissue immunoreactive ET-1 to a level similar to that of cortical tissue not exposed to big ET-1. This confirms the presence and effectiveness of ECE inhibition by phosphoramidon. 3. In freshly isolated proximal tubule cells, big ET-1 stimulated the generation of 1,2-diacylglycerol (DAG) in a time- and dose-dependent manner. Neither phosphoramidon nor chymostatin, a chymase inhibitor, influenced the generation of DAG evoked by big ET-1. 4. Big ET-1-dependent synthesis of DAG was found in the brush-border membrane. It was unaffected by BQ123, an ETA receptor antagonist, but was blocked by bosentan, an ETA.B-nonselective endothelin receptor antagonist. 5. These results suggest that the proximal tubule is a site for the direct effect of big ET-1 in the rat kidney. The effect of big ET-1 is confined to the brush-border membrane of the proximal tubule, which may be the site of big ET-1 sensitive receptors.

Neutrophil signal transduction in Met-enkephalin modulated superoxide anion release.

The present study explored the involvement of signal transduction system(s) in Met-enkephalin (MENK) modulated superoxide anion (O2-) release from human neutrophils. This opioid pentapeptide stimulated the O2- release in all samples if present at 10(-8) M concentration while in lower concentrations the stimulatory concentration was donor-dependent. The most abundant product of MENK degradation, Tyr-Gly-Gly (TGG), suppressed O2- release over a wide range of concentrations (10(-12)-10(-8) M). MENK induced O2- release was associated with a dose-dependent increase of diacylglycerol (DAG) concentration and protein-kinase C (PKC) translocation to the neutrophil membranes, with an increase of cytosolic Ca++, and could be abolished by H7, a PKC inhibitor. On the contrary, the suppressive effect of TGG was not associated with alteration of DAG concentration in neutrophil membranes. Superoxide anion release induced by low concentrations of MENK (10(12)-10(-10) M), could be blocked by NDGA, an inhibitor of the lipoxygenase pathway. We concluded that MENK-induced O2- release results mainly due to DAG/PKC pathway activation, although other secondary messengers might be involved.

Plasma from uninephrectomized rats stimulates production of inositol trisphosphates and inositol tetrakisphosphate in renal cortical slices.

Metabolism of inositol phosphates in renal cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats stimulated production of InsP3 (inositol trisphosphate) when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response of InsP3 production was obtained. This effect could be prevented by 0.1 microM-TPA (12-O-tetradecanoylphorbol 13-acetate). When analysis of inositol phosphates was performed by h.p.l.c., plasma from uninephrectomized rats stimulated a rapid increase in Ins(1,4,5)P3 radioactivity, whereas the increase in inositol 1,3,4,5-tetrakisphosphate and Ins(1,3,4)P3 radioactivity was slower. Plasma from uninephrectomized rats decreased cyclic AMP concentration in renal cortical slices. Similar effect was obtained when slices were incubated with TPA (0.05 microM). Plasma from uninephrectomized rats increased cyclic GMP concentration in renal cortical slices, but this effect was abolished when extracellular Ca2+ had been chelated with 4 mM-EGTA. Results indicate that plasma from uninephrectomized rats stimulates phospholipase C, increases cyclic GMP concentration and decreases cyclic AMP concentration in renal cortical slices. Increases in cyclic GMP depend on extracellular Ca2+, whereas the decrease in cyclic AMP concentration is mediated by protein kinase C.

Incorporation of 32P into renal phospholipids of mice during postnatal growth.

During the first 40 days of life the rate of incorporation of 32P into total phospholipids and into phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, diphosphatidylglycerol, phosphatidic acid and phosphatidylinositol of mouse kidney was by some 25-35% higher than in older animals. Results suggest a different involvement of cellular membranes during of normal and compensatory renal growth.

Plasma from uninephrectomized rats stimulates phospholipid methylation and arachidonic acid release in renal-cortical slices.

Phospholipid methylation and arachidonic acid release in renal-cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats ('uni-plasma') stimulated phospholipid methylation when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response in phospholipid methylation was obtained. Addition of 50 nM-12-O-tetradecanoylphorbol 13-acetate for 10 min to intact slices also stimulated phospholipid methylation, whereas incubation of slices before addition of 'uni-plasma' with 100 microM-1-(5-isoquinolinylsulphonyl)-2-methylpiperazine prevented it, suggesting that protein kinase C stimulates phospholipid methylation in renal-cortical slices. Plasma from uninephrectomized rats also stimulates [3H]arachidonic acid release from phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) via activation of phospholipase A2. Two mechanisms of phospholipase A2 activation are proposed: first, in which it is activated by protein kinase C and releases 3H radioactivity from PtdCho, and second, in which phospholipase A2 is stimulated by Ca2+ ions and releases 3H radioactivity from PtdEtn.

Plasma from uninephrectomized rats increases 32P incorporation into phospholipids of renal cortical slices.

Incorporation of 32P into phospholipids of renal cortical slices was studied in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats increased the labeling of phospholipids when obtained within the first hour after uninephrectomy. With different amounts of added plasma a graded response in labeling of phosphatidic acid and phosphatidylinositol was obtained. Phosphatidylcholine labeling also showed a graded response but only if the incubation was prolonged. The results suggest that an early alteration in phospholipid metabolism, particularly of inositol lipids, may play a role in initiation of compensatory renal growth by humoral factor(s).

Arachidonic acid mediates interferon-gamma-induced sphingomyelin hydrolysis and monocytic marker expression in HL-60 cell line.

The biochemical signaling mechanisms involved in transducing the effects of interferon-gamma (IFN-gamma) on human leukemia-derived HL-60 cell differentiation are not completely understood. Recent studies established the existence of a sphingomyelin (SM) cycle that operates in response to the action of IFN-gamma on HL-60 cells, but the mechanisms by which IFN-gamma induces the SM hydrolysis remain unexplored. In this study, biochemical events mediating IFN-gamma effects on SM turnover and their specificity and role in HL-60 differentiation were investigated. The activation of the SM cycle by IFN-gamma occurred rapidly, with a decrease of approximately 20% in the SM level observed after 60 minutes with a concomitant increase in ceramide level. Treatment of HL-60 cells with IFN-gamma did not influence the 1,2-diacylglycerol concentration, intracellular Ca2+ concentration, or phospholipase D activity. IFN-gamma stimulated a rapid release of arachidonic acid (AA) from HL-60 cells; the effect was abolished by the pretreatment of cells with pertussis toxin, suggesting a role for a pertussis-toxin-sensitive G protein in IFN-gamma-mediated activation of phospholipase A2 (PLA2). At 4 to 120 hours after the stimulation of the cells with IFN-gamma, a significant increase in the particulate and soluble PLA2 activity was observed, corresponding to an increase in the level of immunoreactive cPLA2 in both cytosol and membrane fractions. The treatment of cells with tyrosine kinase inhibitor herbimycin A completely abolished the effect of IFN-gamma on PLA2 activity in membrane and cytosolic fractions, but had no effect on IFN-gamma-mediated early AA release suggesting dual mechanism of PLA2 activation. Melittin, potent activator of PLA2, and AA mimicked the effect of IFN-gamma on SM hydrolysis. Pretreatment of HL-60 cells with the PLA2 inhibitor, bromophenacyl bromide (BPB), or pertussis toxin abolished the effect of IFN-gamma on SM hydrolysis; exogenous addition of AA overcame the effects of BPB and pertussis toxin. Long-term exposure (5 days) of HL-60 cells to IFN-gamma caused an increase in nitroblue tetrazolium (NBT)-reducing and nonspecific esterase (NSE) activity and induced expression of Fc gamma RI (CD64) without significant effects on cell number, adherence, or phagocytic activity. The treatment of cells with AA or melittin induced NBT, NSE, and CD64 expression to the level similar to that observed with IFN-gamma, and no further increase was observed with the combination of IFN-gamma and AA or IFN-gamma and melittin. Treatment of HL-60 cells with indomethacin, an inhibitor of cyclo-oxygenase, and nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, had no effects on IFN-gamma-mediated induction of CD64 expression. These studies indicate a key role for the phospholipase A2/AA pathway, as an early biochemical signal elicited by the occupation of IFN-gamma-receptor, in mediating IFN-gamma induction of the SM cycle and phenotypic changes associated with differentiation of HL-60 along monocytic lineage.

Different roles of protein kinase C alpha and delta isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells.

The signalling mechanisms responsible for the hydrolysis of sphingomyelin mediated by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] and interferon gamma (IFN-gamma) in HL-60 cells were investigated. IFN-gamma was found to increase selectively the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The treatment of HL-60 cells with the combination of 1,25(OH)(2)D(3) and IFN-gamma had an additive effect on sphingomyelin hydrolysis, ceramide release and the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The pretreatment of HL-60 cells with staurosporine, chelerythrine chloride and bisindolylmaleimide abolished the activity of sphingomyelinase in response to 1,25(OH)(2)D(3) and IFN-gamma. Calphostin C, which acts on the regulatory site of protein kinase C (PKC), and Gö 6976, a selective inhibitor of Ca(2+)-dependent PKC isoforms, inhibited the effect of 1,25(OH)(2)D(3) but had no effect on the IFN-gamma-mediated increase in activity of sphingomyelinase. Isoform-specific antibodies were used to deplete different PKC isoforms from cytosol before the treatment of the cytosolic fraction with 1,25(OH)(2)D(3), arachidonic acid (AA) and PMA. The depletion of PKC isoforms beta(1), beta(2), epsilon, eta, mu, zeta and lambda had no effect on the activation of sphingomyelinase induced by 1,25(OH)(2)D(3) or by AA. The depletion of PKC alpha from the cytosol completely abolished the effect of 1,25(OH)(2)D(3) on sphingomyelinase activity but had no effect on the AA-induced activity of sphingomyelinase. PMA had no effect on the activity of sphingomyelinase in either untreated or alpha-depleted cytosol but significantly increased the activity of sphingomyelinase when added to cytosol depleted of PKC delta. Moreover, PMA inhibited the effect of 1,25(OH)(2)D(3) on sphingomyelinase activation but the inhibitory effect was abolished by prior depletion of PKC delta from the cytosol. These studies demonstrate that 1,25(OH)(2)D(3)-induced activation of sphingomyelinase is mediated by PKC alpha. Furthermore, PKC delta had an inhibitory effect on sphingomyelinase, suggesting that the difference between the 1,25(OH)(2)D(3)- and PMA-mediated effects on sphingomyelin turnover depends on the specific regulation of the PKC alpha and PKC delta isoforms.

Endothelin mediates phospholipase C stimulation in the proximal tubule during initiation of compensatory renal growth in adult rats.

AIM: Mechanisms that initiate compensatory renal growth following unilateral nephrectomy are incompletely understood. An early event following unilateral nephrectomy is the activation of phospholipase C in the apical membrane of the proximal tubule, mediated by an unknown agonist. We tested the hypothesis that endothelin is responsible for the stimulation of phospholipase C in rat proximal tubule following unilateral nephrectomy. METHODS: Compensatory renal growth was induced in adult male rats by unilateral nephrectomy. 1,2-Diacylglycerol, a product of phospholipase C activation, was measured in renal cortical slices and isolated proximal tubules, respectively, 20 min following unilateral nephrectomy, or after incubation of the slices or proximal tubules with plasma from unilaterally nephrectomized or sham-operated rats. RESULTS: Twenty min following unilateral nephrectomy, an increase in 1,2-diacylglycerol concentration occurred in the renal cortex. Bosentan, a nonselective endothelin receptor antagonist, as well as an anti-endothelin-1 antibody administered intravenously, completely inhibited this 1,2-diacylglycerol accumulation in renal cortex. Incubation of renal cortical slices with plasma from unilaterally nephrectomized or bilaterally nephrectomized rats, stimulated 1,2-diacylglycerol production in isolated proximal tubule apical membranes. Again, bosentan prevented the increase evoked by incubation with plasma from unilaterally nephrectomized rats. Finally, concentration of endothelin-1 increased in renal cortex in response to unilateral nephrectomy. CONCLUSION: These results make evident the role of endothelin in stimulation of phospholipase C in proximal tubule following unilateral nephrectomy, suggesting participation of the endothelin system during the initiation of the compensatory renal growth.

The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor (for IGF-I) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C to the nucleus.

When Swiss 3T3 cells are treated with Insulin-like Growth Factor I, a rapid decrease in the mass of polyphosphoinositol lipids (phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate) occurs within the nuclei, with a concomitant increase in nuclear diacylglycerol and translocation of protein kinase C to the nuclear region. This is in contrast to the effects of the regulatory peptide, bombesin, which causes similar inositol lipid changes in the plasma membrane, has no effect on nuclear inositide levels and causes a translocation of protein kinase C to post-nuclear membranes. These results suggest the existence of a discrete nuclear polyphosphoinositide signalling system entirely distinct from the well-known plasma membrane-located system, which is under regulatory control by cell surface-located receptors.