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ABSTRACT: The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common beta (betac) receptor is shared by the three cytokines and functions together with cytokine-specific alpha subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human betac (hbetac) and unidentified cysteine residues in the N-terminal domains of the alpha receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hbetac is part of the cytokine binding epitope of this receptor and that an IL-3Ralpha isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hbetac and the related mouse receptor, beta(IL-3), could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hbetac led to both a complete loss of high affinity binding with the human IL-3Ralpha SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, beta(IL-3), not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Ralpha SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hbetac and beta(IL-3) in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.
Journal of Biological Chemistry 08/2010; 285(32):24759-68. · 4.77 Impact Factor
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ABSTRACT: The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF),
exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by
the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is
that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and
unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new
data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an
IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated
whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, βIL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of
the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream
signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, βIL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling
with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and
βIL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.
Journal of Biological Chemistry 08/2010; 285(32):24759-24768. · 4.77 Impact Factor
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ABSTRACT: The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific alpha-subunit and common beta-subunit (beta c; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a beta-subunit that specifically binds IL-3 (beta(IL-3); present in mice but not humans). We recently identified two splice variants of the alpha-subunit of the IL-3 receptor (IL-3R alpha) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length ("SP1" isoform) and a novel isoform (denoted "SP2") lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) R alpha isoform can direct mIL-3 binding to two distinct sites on the beta(IL-3) subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of beta(IL-3) recognition and whether the human IL-3R alpha SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human beta c subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu(23), in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the beta(IL-3) and mIL-3R alpha SP2 subunits, whereas an overlapping cluster was required for binding and activation of beta(IL-3) in the presence of mIL-3R alpha SP1. Similarly, our studies of human IL-3 indicate that two different modes of beta c binding are utilized in the presence of the hIL-3R alpha SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.
Journal of Biological Chemistry 07/2010; 285(29):22370-81. · 4.77 Impact Factor
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ABSTRACT: The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its
effects on target cells via receptors comprising an IL-3-specific α-subunit and common β-subunit (βc; shared with IL-5 and
granulocyte-macrophage colony-stimulating factor) or a β-subunit that specifically binds IL-3 (βIL-3; present in mice but not humans). We recently identified two splice variants of the α-subunit of the IL-3 receptor (IL-3Rα)
that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length (“SP1” isoform) and a
novel isoform (denoted “SP2”) lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3
(mIL-3) Rα isoform can direct mIL-3 binding to two distinct sites on the βIL-3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of βIL-3 recognition and whether the human IL-3Rα SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites
on the human βc subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix
residue, Glu23, in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the βIL-3 and mIL-3Rα SP2 subunits, whereas an overlapping cluster was required for binding and activation of βIL-3 in the presence of mIL-3Rα SP1. Similarly, our studies of human IL-3 indicate that two different modes of βc binding are
utilized in the presence of the hIL-3Rα SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative
orientations of receptor subunits are modulated to achieve distinct signaling outcomes.
Journal of Biological Chemistry 07/2010; 285(29):22370-22381. · 4.77 Impact Factor
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ABSTRACT: GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.
Biochemical Journal 03/2010; 426(3):307-17. · 4.90 Impact Factor
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ABSTRACT: Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor alpha (IL-3Ralpha), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with beta(IL-3) mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBPalpha and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.
Journal of Biological Chemistry 01/2009; 284(9):5763-73. · 4.77 Impact Factor
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ABSTRACT: Hepatocytes are highly differentiated and spatially polarised cells which conduct a wide range of functions, including intermediary metabolism, protein synthesis and secretion, and the synthesis, transport and secretion of bile acids. Changes in the concentrations of Ca(2+) in the cytoplasmic space, endoplasmic reticulum (ER), mitochondria, and other intracellular organelles make an essential contribution to the regulation of these hepatocyte functions. While not yet fully understood, the spatial and temporal parameters of the cytoplasmic Ca(2+) signals and the entry of Ca(2+) through Ca(2+)-permeable channels in the plasma membrane are critical to the regulation by Ca(2+) of hepatocyte function. Ca(2+) entry across the hepatocyte plasma membrane has been studied in hepatocytes in situ, in isolated hepatocytes and in liver cell lines. The types of Ca(2+)-permeable channels identified are store-operated, ligand-gated, receptor-activated and stretch-activated channels, and these may vary depending on the animal species studied. Rat liver cell store-operated Ca(2+) channels (SOCs) have a high selectivity for Ca(2+) and characteristics similar to those of the Ca(2+) release activated Ca(2+) channels in lymphocytes and mast cells. Liver cell SOCs are activated by a decrease in Ca(2+) in a sub-region of the ER enriched in type1 IP(3) receptors. Activation requires stromal interaction molecule type 1 (STIM1), and G(i2alpha,) F-actin and PLCgamma1 as facilitatory proteins. P(2x) purinergic channels are the only ligand-gated Ca(2+)-permeable channels in the liver cell membrane identified so far. Several types of receptor-activated Ca(2+) channels have been identified, and some partially characterised. It is likely that TRP (transient receptor potential) polypeptides, which can form Ca(2+)- and Na(+)-permeable channels, comprise many hepatocyte receptor-activated Ca(2+)-permeable channels. A number of TRP proteins have been detected in hepatocytes and in liver cell lines. Further experiments are required to characterise the receptor-activated Ca(2+) permeable channels more fully, and to determine the molecular nature, mechanisms of activation, and precise physiological functions of each of the different hepatocyte plasma membrane Ca(2+) permeable channels.
Biochimica et Biophysica Acta 06/2008; 1783(5):651-72. · 4.66 Impact Factor
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ABSTRACT: Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).
Cytokine 06/2008; 42(2):234-42. · 3.02 Impact Factor
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ABSTRACT: The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2++Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Cao2+) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Cao2+-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Cao2+-induced [Ca2+]cyt spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Cao2+-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2++Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.
Cell Calcium 05/2004; 35(4):317-31. · 3.77 Impact Factor
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ABSTRACT: The TRPC1 (transient receptor potential canonical 1) protein, which is thought to encode a non-selective cation channel activated by store depletion and/or an intracellular messenger, is expressed in a number of non-excitable cells. However, the physiological functions of TRPC1 are not well understood. The aim of these studies was to investigate the function of TRPC1 in liver cells using small interfering RNA (siRNA) to ablate the TRPC1 protein. Treatment of H4-IIE liver cells with siRNA targeted against TRPC1 caused an approx. 50% decrease in expression of the human TRPC1 protein in cells transfected with cDNA encoding human TRPC1, and a 50% decrease in expression of the endogenous TRPC1 protein (assessed by Western blot and immunofluorescence). The decrease in endogenous TRPC1 protein in cells transfected with TRPC1 siRNA was associated with a greater increase in cell volume (compared with the increase observed in control cells) immediately after cells were placed in a hypotonic medium, and an enhanced regulatory cell volume decrease after exposure to hypotonic medium. Treatment with siRNA targeted against TRPC1 also led to a 25% inhibition of thapsigargin-stimulated Ca(2+) inflow, a 40% inhibition of ATP and maitotoxin-stimulated Ca(2+) inflow, and a 50% inhibition of maitotoxin-stimulated Mn(2+) inflow. The idea that, in liver cells, TRPC1 encodes a non-selective cation channel involved directly or indirectly in the regulation of cell volume is consistent with the results obtained.
Biochemical Journal 08/2003; 373(Pt 2):327-36. · 4.90 Impact Factor
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ABSTRACT: Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80 kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92 kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the alpha- and beta-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92 kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120 kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92 kDa or 120 kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated beta-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.
Biochemical Journal 07/2002; 364(Pt 3):641-8. · 4.90 Impact Factor
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ABSTRACT: Hepatocytes are highly differentiated and spatially polarised cells which conduct a wide range of functions, including intermediary metabolism, protein synthesis and secretion, and the synthesis, transport and secretion of bile acids. Changes in the concentrations of Ca2+ in the cytoplasmic space, endoplasmic reticulum (ER), mitochondria, and other intracellular organelles make an essential contribution to the regulation of these hepatocyte functions. While not yet fully understood, the spatial and temporal parameters of the cytoplasmic Ca2+ signals and the entry of Ca2+ through Ca2+-permeable channels in the plasma membrane are critical to the regulation by Ca2+ of hepatocyte function. Ca2+ entry across the hepatocyte plasma membrane has been studied in hepatocytes in situ, in isolated hepatocytes and in liver cell lines. The types of Ca2+-permeable channels identified are store-operated, ligand-gated, receptor-activated and stretch-activated channels, and these may vary depending on the animal species studied. Rat liver cell store-operated Ca2+ channels (SOCs) have a high selectivity for Ca2+ and characteristics similar to those of the Ca2+ release activated Ca2+ channels in lymphocytes and mast cells. Liver cell SOCs are activated by a decrease in Ca2+ in a sub-region of the ER enriched in type1 IP3 receptors. Activation requires stromal interaction molecule type 1 (STIM1), and Gi2α, F-actin and PLCγ1 as facilitatory proteins. P2x purinergic channels are the only ligand-gated Ca2+-permeable channels in the liver cell membrane identified so far. Several types of receptor-activated Ca2+ channels have been identified, and some partially characterised. It is likely that TRP (transient receptor potential) polypeptides, which can form Ca2+- and Na+-permeable channels, comprise many hepatocyte receptor-activated Ca2+-permeable channels. A number of TRP proteins have been detected in hepatocytes and in liver cell lines. Further experiments are required to characterise the receptor-activated Ca2+ permeable channels more fully, and to determine the molecular nature, mechanisms of activation, and precise physiological functions of each of the different hepatocyte plasma membrane Ca2+ permeable channels.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research.
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ABSTRACT: The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca2+, Mn2+ and Na+, have a high selectivity for Na+ compared with Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 1 μM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca2+ concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase–polymerase chain reaction in poly(A)+ RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca2+ inflow and a modest enhancement of thapsigargin-initiated Ca2+ inflow (measured using fura-2) and no enhancement of the highly Ca2+-selective store-operated Ca2+ current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na+ and Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 3 μM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na+ compared with Ca2+; (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca2+-selective store-operated Ca2+ channels.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research.
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ABSTRACT: The treatment of H4-IIE cells (an immortalised liver cell line derived from the Reuber rat hepatoma) with thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or pretreatment with EGTA, stimulated Ca2+ inflow (assayed using intracellular fluo-3 and a Ca2+ add-back protocol). No stimulation of Mn2+ inflow by thapsigargin was detected. Thapsigargin-stimulated Ca2+ inflow was inhibited by Gd3+ (maximal inhibition at 2 μM Gd3+), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca2+ channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca2+ inflow and release of Ca2+ from intracellular stores. No inhibition of either Ca2+ inflow or Ca2+ release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca2+ inflow by calmidazolium was only observed when the inhibitor was added before thapsigargin. Pretreatment of H4-IIE cells with pertussis toxin, or treatment with brefeldin A, did not inhibit thapsigargin-stimulated Ca2+ inflow. Compared with freshly isolated rat hepatocytes, H4-IIE cells exhibited a more diffuse actin cytoskeleton, and a more granular arrangement of the endoplasmic reticulum (ER). In contrast to freshly isolated hepatocytes, the arrangement of the ER in H4-IIE cells was not affected by pertussis toxin treatment. Western blot analysis of lysates of freshly isolated rat hepatocytes revealed two forms of Gi2α with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of Gi2α and substantially less total Gi2α than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca2+ channels which do not require calmodulin for activation and exhibit properties similar to those in freshly isolated rat hepatocytes, including susceptibility to inhibition by relatively high concentrations of voltage-operated Ca2+ channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require Gi2α for activation. Possible explanations for differences in the requirement for Gi2α in the activation of Ca2+ inflow are briefly discussed.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research.
