beta-Sitosterol activates Fas signaling in human breast cancer cells.

beta-Sitosterol is the most abundant phytosterol. Phytosterols are enriched in legumes, oil seeds and unrefined plant oils as found in foods such as peanut butter, pistachios and sunflower seeds. beta-Sitosterol inhibits the growth of several specific types of tumor cells in vitro and decreases the size and the extent of tumor metastases in vivo. The effects of beta-sitosterol on the extrinsic apoptotic programmed cell death pathway in human breast MCF-7 and MDA-MB-231 adenocarcinoma cells were examined, along with the extent of its incorporation into cellular membranes and its effects on cell growth, expression of Fas receptor pathway proteins, and caspase-8 activity. The results show that beta-sitosterol exposure promotes its enrichment in transformed cell membranes and significantly inhibits tumor cell growth. Concurrently, Fas levels and caspase-8 activity are significantly increased. These actions are specific, as expression of other proteins of the Fas receptor pathway, including Fas ligand, FADD, p-FADD and caspase-8, remain unchanged. These findings support the hypothesis that beta-sitosterol is an effective apoptosis-promoting agent and that incorporation of more phytosterols in the diet may serve a preventive measure for breast cancer.

Single column discrepancy and dynamic max-mini optimizations for quickly finding the most parsimonious evolutionary trees.

MOTIVATION: In the maximum parsimony (MP) method, the tree requiring the minimum number of changes (discrepancy) to explain the given set of DNA or amino acid sequences is chosen to represent their evolutionary relationships. To find the MP tree, the branch-and-bound algorithm is normally used. For a partial phylogenetic-tree (one that has a subset of the organisms) the traditional algorithm assigns a cost equal to the discrepancy of the partial phylogenetic-tree. We propose a single column discrepancy heuristic which increases this cost by predicting a minimum additional discrepancy needed to attach the sequences yet to be added to the partial phylogenetic-tree. A dynamic Max-mini order of sequence addition is also proposed to quickly terminate branch-and-bound search paths that are guaranteed to lead to suboptimal solutions. RESULTS: We studied the running time of 47 problems generated from 17 data sets. The use of single column discrepancy heuristic speeded up the computation to 2.4-fold for static and 18.2-fold for dynamic search order. The improvement appeared to increase exponentially with the number of sequences. The proposed strategies are also likely to be useful in speeding up the MP tree search using heuristic searches that are based on branch-and-bound-like algorithms. CONTACT: s.kumar@asu.edu

IL-1 beta increases type 1 inositol trisphosphate receptor expression and IL-6 secretory capacity in osteoblastic cell cultures.

Acute IL-6 secretion from osteosarcoma cells induced by the PI-linked hormones PTH(1-34) and endothelin-1 is potentiated by IL-1 beta. The present findings indicate that this potentiation is accompanied by increased signal transduction capacity. Specifically, IL-1 beta (30 pM) increased the B(max) of InsP(3) receptor binding (2. 7-fold) and immunoblot showed a 2.4-fold increase specifically in the type 1 InsP(3) receptor protein. Northern analyses of IL-1 beta-treated G-292 cells showed an 1.8-fold increase in type 1 InsP3 receptor mRNA and, in IL-1 beta-treated murine MC3T3-E1 osteoblastic cells, an 8.4-fold enhancement of the type 1 InsP(3) receptor gene transcription. Promoter reporter assays confirmed the mRNA measurements and showed the effect of IL-1 beta to be mediated by the major transcriptional regulatory region of the type 1 InsP(3) receptor promoter. The findings support the hypothesis that chronic regulators of osteoblast function, such as IL-1 beta, affect the capacity of cellular signal transduction through changes in InsP(3) receptor levels.

The effect of bone morphogenetic protein-7 on the expression of type I inositol 1,4,5-trisphosphate receptor in G-292 osteosarcoma cells and primary osteoblast cultures.

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus-secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, alpha1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and beta-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206+/-8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretory hormones during osteoblast differentiation.

Molecular characterization of the alpha1 subunit of the L type voltage calcium channel expressed in rat calvarial osteoblasts.

Voltage-activated calcium channels (VACCs) regulate extracellular calcium influx in many cells. VACCs are composed of five subunits. The alpha1 subunit is considered the most important in regulating channel function. Three isoforms of this subunit have been described: skeletal, cardiac, and neuroendocrine. It was the purpose of the present study to determine the molecular identity of the alpha1 subunit of the VACCs in rat calvarial osteoblasts and to study the nature of the regulation of these channels as a function of cellular growth. We also attempted to identify which isoform of the alpha1 subunit of the VACCs mediates the effects of epidermal growth factor (EGF) on osteoblastic cell proliferation. Reverse transcription-polymerase chain reaction was used to detect the isoforms of the VACCs that are expressed in osteoblastic cells. These analyses showed that the proliferative state of the cell and the time in culture influence RNA expression. The only alpha1 subunit detected in osteoblasts corresponds to the cardiac isoform. In additional experiments, the effects of EGF on cytosolic calcium and osteoblast proliferation were determined. For these experiments, the synthesis of the different isoforms of the VACCs was selectively blocked by antisense oligonucleotides prior to EGF stimulation. These studies showed that the cardiac isoform mediates the effects of EGF on cytosolic calcium and cellular proliferation in rat calvarial osteoblasts.

Characterization of inositol 1,4,5-trisphosphate receptor isoform mRNA expression and regulation in rat pancreatic islets, RINm5F cells and betaHC9 cells.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that plays a role in the regulation of insulin secretion. In rat isolated pancreatic islets the expression of types I, II and III InsP3R mRNA was identified by reverse transcriptase-polymerase chain reaction and confirmed by cDNA cloning and sequencing. The islet ratios of types I, II and III InsP3R mRNA to beta-actin mRNA were 0.08 +/- 0.02, 0.08 +/- 0.03 and 0.25 +/- 0.04 respectively. Types I, II and III InsP3R mRNA were also expressed in rat (RINm5F) and mouse (betaHC9) pancreatic beta-cell lines, and rat cerebellum. Type III InsP3R mRNA was quantitatively the most abundant form in rat islets and RINm5F cells. In betaHC9 cells, types II and III InsP3R mRNA were expressed at similar levels, and in much greater abundance than type I mRNA. Type III was the least abundant InsP3R mRNA in cerebellum. Culture of betaHC9 cells for 5 days at 2.8 and 25 mM glucose, or RINm5F cells for 7 days at 5.5 and 20 mM glucose, resulted in significantly enhanced expression of type III, but not types I and II, InsP3R mRNA in the cells at the higher glucose concentrations. During short-term (0.5-2 h) incubations, betaHC9 cell type III InsP3R mRNA levels increased in response to glucose in a time- and concentration-dependent manner. Actinomycin D inhibited the glucose response. Alpha-ketoisocaproic acid also stimulated betaHC9 cell type III InsP3R mRNA expression in a concentration-dependent manner, whereas 2-deoxyglucose and 3-O-methylglucose were without effect. The different levels of expression of mRNA for three InsP3R isoforms in islets and insulinoma cells, and the influence of glucose and alpha-ketoisocaproic acid on the expression of type III mRNA, suggests that nutrient metabolism plays a role in the regulation of this gene and that the function of InsP3R subtypes may be unique with each playing a distinct role in beta-cell signal transduction and insulin secretion.

Cloning and characterization of the type I inositol 1,4,5-trisphosphate receptor gene promoter. Regulation by 17beta-estradiol in osteoblasts.

The inositol 1,4,5-trisphosphate (InsP3) receptor is essential for signal Ca2+ release from intracellular stores and for capacitative Ca2+ entry. We have isolated the promoter and proximal DNA segments of the human type I InsP3 receptor gene. Transcription initiation in human G-292 osteosarcoma and HL-60 promyelocytic leukemia cells was shown to occur predominantly from an adenine residue located 39 base pairs downstream of a consensus TATA box element. Upstream DNA including the TATA box promoted directional transcription of a chloramphenicol acetyltransferase reporter gene when transfected into G-292 cells. A negative regulatory element in the distal promoter and a positive element in the proximal region were identified by deletion mapping and transcription assays. The proximal region enhanced transcription in response to 12-O-tetradecanoylphorbol-13-acetate or serum, but conferred transcriptional repression in response to 1,25-dihydroxyvitamin D3 or 17beta-estradiol. The repressive effect of 17beta-estradiol was mediated by the nuclear estrogen receptor, as estrogen-dependent transcriptional repression was inhibited by the antiestrogen tamoxifen and the estrogen receptor antagonist ICI 182,780. This is the first study of the type I InsP3 receptor gene promoter, and the results suggest a mechanism by which chronic estrogen treatment of osteoblasts affects type I InsP3 receptor gene expression, signal transduction, and secretion.

Inositol trisphosphate receptor gene expression and hormonal regulation in osteoblast-like cell lines and primary osteoblastic cell cultures.

The inositol trisphosphate receptor (IP3R) is an intracellular calcium channel that mediates the cellular actions of a wide variety of hormones, growth factors, and cytokines. In osteoblastic cell cultures, many bone resorbing hormones increase phosphoinositide turnover, inositol trisphosphate production, mobilization of intracellular calcium, and the secretion of osteoclast recruitment and activating factors. In this study, the effects of 17 beta-estradiol, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), phrobol ester, and serum on IP3R mRNA levels were evaluated in osteogenic-osteosarcoma cells and in primary osteoblastic cultures derived from neonatal rat calvaria. Type-specific reverse transcription polymerase chain reaction (RT-PCR) indicated that all cell types evaluated (G-292, U-2 OS, Saos-2, MC3T3-E1, UMR-106, and calvarial osteoblastic cells) express IP3R mRNA type I; G-292, U-2 OS, MC3T3-E1, and calvarial osteoblastic cells also express type II IP3R mRNA; and UMR-106 and the calvarial osteoblastic cells express type III IP3R mRNA. Northern blot and RT-PCR analyses of human G-292 osteosarcoma cells and rat calvarial osteoblastic cells showed that phorbol ester and serum increase IP3R mRNA levels, whereas 17 beta-estradiol and 1,25(OH)2D3 decrease these levels. In G-292 cells, the effect of 17 beta-estradiol was not due to accelerated IP3R mRNA degradation and required continued protein synthesis. The results show that multiple IP3R types are expressed in osteoblasts and osteoblastic osteosarcoma cells and that this expression is regulated by 17 beta-estradiol and other osteoporotic and antiosteoporotic hormones. These findings indicate that hormonal control of IP3R expression may be relevant in the chronic regulation of osteoblast secretory activity.

Isolation and functional characterization of the human gene encoding the myeloid zinc finger protein MZF-1.

The expression of the human myeloid zinc finger gene (MZF-1) by human bone marrow cells is necessary for granulopoiesis. We have analyzed the structure and function of the MZF-1 gene by diagnostic polymerase chain reaction, genomic cloning, and promoter analysis. Comparison of human promyelocytic HL-60 cell cDNA with isolated MZF-1 genomic clones indicated that the human MZF-1 gene is without introns and spans approximately 3 kb. Restriction enzyme mapping and Southern analysis indicated further that the human MZF-1 gene is a single-copy gene. Primer extension studies identified the major transcription start site as a thymidine residue located 1102 bp upstream of the ATG translation start codon. A putative TATA box sequence (TAAAAA) was found at -66 bp and a CCAAT box at -130 bp relative to the transcription initiation site. In HL-60 cells, MZF-1 mRNA levels are increased by granulopoietic inducers including retinoic acid and GM-CSF. DNA upstream of the transcription start site contains tandem-repeated consensus retinoic acid response elements at -666 through -696 bp and paired putative GM-CSF-responsive sequences centered at -50 and -100 bp. CAT reporter gene constructs containing these DNA regions promoted transcription and conferred transcriptional responsiveness to retinoic acid and GM-CSF when transfected into HL-60 cells. Additional putative regulatory binding sites included conserved MZF-1 zinc finger binding sequences, the importance of which was suggested by the enhanced expression of the endogenous MZF-1 gene following vector-driven expression of MZF-1 constructs in K562 myeloblastic leukemia cells. These findings provide a clearer basis for understanding the role of MZF-1 gene expression in myeloid cell growth and differentiation.

1,25-dihydroxyvitamin D3 enhances the transcription and expression of the inositol trisphosphate receptor gene in HL-60 cells.

Human leukemic HL-60 cells were treated with 1,25-dihydroxyvitamin D3 (VitD3) to induce monocytic cell differentiation. Concomitant with differentiation there was increased inositol-1,4,5-trisphosphate (InsP3) receptor expression, as assessed by both [3H]InsP3 binding site density and maximal InsP3-mediated Ca2+ mobilization from intracellular stores. Within 8 hr after VitD3 treatment the steady state level of a 10-kilobase InsP3 receptor mRNA was specifically elevated, and it continued to rise for 1-2 days. Nuclear run-off assays indicated a higher transcription rate of the InsP3 receptor gene in response to VitD3. The increased rate of transcription was sufficient to account for the increased steady state level of InsP3 receptor mRNA in VitD3-treated cells. VitD3 had no effect on the decay of InsP3 receptor mRNA in transcriptionally arrested cells; however, InsP3 receptor mRNA decay was dependent upon continued protein synthesis. Moreover, in cycloheximide-treated cells VitD3 was still able to induce an increase in the steady state level of InsP3 receptor mRNA, indicating that protein synthesis was not required for the enhanced transcriptional response. The results suggest that VitD3 directly enhances the transcription of the InsP3 receptor gene in HL-60 cells.

Transcriptional regulation and increased functional expression of the inositol trisphosphate receptor in retinoic acid-treated HL-60 cells.

The inositol trisphosphate (InsP3) receptor is an essential regulator of intracellular calcium in many cells including chemoattractant- and cytokine-stimulated neutrophils and differentiated promyelocytic leukemic (HL-60) cells. We examined the expression and function of the InsP3 receptor and the transcriptional regulation of the InsP3 receptor gene in HL-60 cells and in HL-60 cells treated for 1-5 days with 1 microM retinoic acid. Radioligand binding studies using membranes from control and retinoic acid-treated HL-60 cells showed that the Bmax of InsP3 receptor increased progressively from 0.24 to 0.69 pmol/mg protein during 5 days retinoic acid treatment with no change in KD (19 nM). During this period, maximal InsP3-stimulated Ca2+ mobilization increased 2-3-fold. InsP3 receptor mRNA was present at low levels in HL-60 cells but was increased significantly after treatment with retinoic acid, reaching maximal levels of approximately 4-fold greater than untreated cells after 4 days treatment with retinoic acid. Nuclear run-on assays indicated that the elevated steady state level of InsP3 receptor mRNA in retinoic acid-treated HL-60 cells was primarily the result of enhanced transcription of the InsP3 receptor gene. Furthermore, the transcriptional enhancing effect of retinoic acid was seen in the presence of cycloheximide, suggesting that the InsP3 receptor gene is directly regulated by retinoic acid. The studies also demonstrate that the InsP3 receptor mRNA is rapidly degraded in HL-60 cells by a mechanism that also requires protein synthesis.

IL-3 and GM-CSF induce the expression of the inositol trisphosphate receptor in K562 myeloblast cells.

When treated with IL-3 plus GM-CSF, K562 myeloblast cells acquired the ability to mobilize nonmitochondrial stores of intracellular Ca2+ in response to added Ins (1, 4, 5) P3. Untreated K562 cells are capable of sequestering intracellular Ca2+ but released none of this Ca2+ in response to Ins (1, 4, 5) P3. Untreated K562 cells were shown to have no detectable specific [3H] Ins (1, 4, 5) P3 binding sites and no InsP3 receptor mRNA as assayed by Northern blot and PCR. However, following IL-3 and GM-CSF treatment, both a single class of low nM KD Ins (1, 4, 5) P3 binding site and a 10 kb InsP3 receptor mRNA were detectable. The results suggest that IL-3 and GM-CSF regulate the expression of the Ins (1, 4, 5) P3 receptor gene.

Increased expression of the inositol 1,4,5-trisphosphate receptor in human leukaemic (HL-60) cells differentiated with retinoic acid or dimethyl sulphoxide.

The Ins(1,4,5)P3 receptor was examined in human promyelocytic leukaemic cells (HL-60) and in HL-60 cells differentiated towards granulocytes with either retinoic acid (RA) or dimethyl sulphoxide (Me2SO). HL-60 cell membranes enriched in marker enzyme activities of the endoplasmic reticulum and the plasma membrane possess a high-affinity binding site for [3H]Ins(1,4,5)P3 (KD = 22 nM). Electrotransfer studies indicate that Ins(1,4,[32P]5)P3 binds specifically to a 260 kDa protein of HL-60 cell membranes. This Ins(1,4,5)P3-binding protein selectively binds Ca(2+)-mobilizing inositol phosphates and other inositol phosphates which also bind to the purified InsP3 receptor, suggesting that the Ins(1,4,5)P3-binding protein of HL-60 cell membranes is the InsP3 receptor. When HL-60 cells are incubated with 1 microM-RA or with 1.25% Me2SO the cells differentiate within 5-7 days into cells resembling neutrophils in both structure and function. Treated cells cease to proliferate, acquire the ability to reduce Nitro Blue Tetrazolium dye, and undergo morphological changes typical of differentiated granulocytes. Concomitant with HL-60 cell differentiation, the maximal [3H]Ins(1,4,5)P3 binding in membranes increases 3-4-fold, with no change in KD. The results suggest that there is an absolute increase in the level of the InsP3 receptor during HL-60 cell differentiation and that the expression of this signal-transducing protein may be specifically regulated by differentiation factors.

Specific binding sites for [3H]inositol(1,3,4,5)tetrakisphosphate on membranes of HL-60 cells.

Membranes of HL-60 cells were shown to possess saturable binding sites for [3H]inositol(1,3,4,5)tetrakisphosphate, with nanomolar affinity (KD = 90 nM) and a density of 250 fmol/mg protein. The specificity of the binding sites for Ins(1,3,4,5)P4 was assessed by competition studies utilising a variety of inositol polyphosphates; results indicated that both the presence and the correct grouping of the phosphates were important for high affinity recognition. The apparent affinity of the binding sites for Ins(1,3,4,5)P4 was over 200-fold greater than for Ins(1,4,5)P3. The possibility is discussed that this binding site represents the receptor which mediates the action of Ins(1,3,4,5)P4 as a putative intracellular second messenger.

Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells.

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils.

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.

Guanine nucleotide regulation of phospholipase C activity in permeabilized rabbit neutrophils. Inhibition by pertussis toxin and sensitization to submicromolar calcium concentrations.

Rabbit neutrophils labelled with [3H]inositol and permeabilized with saponin produced [3H]inositol trisphosphate (InsP3) when incubated with stable analogues of GTP or millimolar concentrations of Ca2+. [3H]InsP3 production elicited by guanosine 5'-[gamma-thio]triphosphate was enhanced by the chemoattractant formylmethionyl-leucyl-phenylalanine and inhibited by pertussis-toxin pretreatment. A pertussis-toxin-sensitive stimulation of [3H]InsP3 concentration was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with guanosine 5'-[beta-thio]diphosphate or GTP. Millimolar Ca2+ alone was sufficient to stimulate [3H]InsP3 production; however, in the presence of guanosine 5'-[gamma-thio]triphosphate, the Ca2+ dose-response curve was shifted to submicromolar concentrations. These findings directly confirm the role of a pertussis-toxin-sensitive guanine nucleotide regulatory protein (G protein) in chemoattractant-stimulated phospholipase C activity in rabbit neutrophils. Moreover, the ability of guanine nucleotides to sensitize phospholipase C to physiologically relevant Ca2+ concentrations suggests that the role of the activated G protein may be to enhance the apparent affinity of phospholipase C for Ca2+ and thus to activate the enzyme without an increase in the Ca2+ concentration.

A saturable receptor for 32P-inositol-1,4,5-triphosphate in hepatocytes and neutrophils.

Several receptors for neurotransmitters, hormones and growth factors cause accelerated phosphodiesteratic breakdown of polyphosphoinositides when activated. One of the soluble products of this reaction, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) is thought to act as a second messenger signalling the release of Ca2+ from intracellular stores. In support of this hypothesis, several studies have shown that Ins(1,4,5)P3 releases sequestered Ca2+ from permeable cells and microsomes. On the basis of certain structural requirements for Ca2+-releasing activity by inositol phosphates, it has been postulated that Ins(1,4,5)P3 acts by binding to a specific intracellular receptor, probably on a component of the endoplasmic reticulum. Here we report that 32P-Ins(1,4,5)P3 binds to a specific saturable site in permeabilized guinea pig hepatocytes and rabbit neutrophils, and that the properties of this binding site suggest that it is the physiological receptor for Ins(1,4,5)P3.

The differential effects of nedocromil sodium and sodium cromoglycate on the secretory response of rabbit peritoneal neutrophils.

Activation of neutrophils, through the formation of inositol triphosphate (IP3) and diacylglycerol, results in lysosomal enzyme secretion, a release process which involves calcium and protein kinase C. Rabbit peritoneal neutrophils were used in this study to compare the effects of nedocromil sodium cromoglycate on enzyme release and IP3 accumulation induced by the activators fMLP (a chemotactic peptide which generates diacylglycerol and produces a rise in cytosolic Ca2+) and phorbol dibutyrate (PDBu), which activates only protein kinase C. Both drugs produced a small but significant (p less than 0.05) inhibition of fMLP-induced lysozyme secretion. With PDBu-induced enzyme secretion, however, nedocromil sodium caused a 25% decrease in the secretory response whilst sodium cromoglycate had no effect, suggesting that the two compounds have different mechanisms of action or that nedocromil sodium has an additional mode of action not shown by sodium cromoglycate. In addition, nedocromil sodium did not block fMLP-induced IP3 accumulation. These results indicate that nedocromil sodium inhibits the secretory response of neutrophils via an effect on protein kinase C rather than on calcium homeostasis.