Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells.

alpha-Thrombin, a G-protein-coupled receptor agonist, is mitogenic for neonatal vascular smooth muscle (VSM) cells, but it also causes secretion of the tyrosine kinase-coupled receptor agonist platelet-derived growth factor (PDGF). In order to determine the role of growth factors with tyrosine kinase-coupled receptors in thrombin's mitogenic signal transduction cascade, the synergistic effect of basic fibroblast growth factor (bFGF) in this system was examined. While bFGF itself is a growth factor for VSM cells, it causes a 1.7-fold synergistic effect when added together with thrombin. Herbimycin A, a specific tyrosine kinase inhibitor, both decreases thrombin-induced mitogenesis by greater than 90% and abolishes tyrosine phosphorylation of phospholipase C (PLC)-gamma-1. The magnitude and time course of the increase in intracellular free calcium concentration in response to thrombin is comparable in both the presence and absence of herbimycin A. These results provide evidence that herbimycin A specifically inhibits PLC-gamma-1 tyrosine phosphorylation without affecting VSM cell viability or calcium release. Furthermore, tyrosine phosphorylation is a necessary step in thrombin's mitogenic signal transduction cascade, but it is not essential for thrombin-induced release of calcium from intracellular stores. These data suggest that a tyrosine kinase, possibly supplied by the bFGF receptor, plays an essential role in thrombin-induced mitogenesis.     

Noise enhanced hormonal signal transduction through intracellular calcium oscillations.

In a wide range of non-linear dynamical systems, noise may enhance the detection of weak deterministic input signals. Here, we demonstrate this phenomenon for transmembrane signaling in a hormonal model system of intracellular Ca(2+) oscillations. Adding Gaussian noise to a subthreshold extracellular pulsatile stimulus increased the sensitivity in the dose-response relation of the Ca(2+) oscillations compared to the same noise signal added as a constant mean level. These findings may have important physiological consequences for the operation of hormonal and other physiological signal transduction systems close to the threshold level.     

The role of tyrosine phosphorylation in regulation of signal transduction pathways in unicellular eukaryotes

The review summarizes for the first time the current concepts of the role of tyrosine phosphorylation in regulation of signal transduction pathways in unicellular eukaryotes. Evolutionary concepts are developed about the origin of protein tyrosine kinases (PTK)-signaling.     

The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

Antigen- and ionophore-induced signal transduction in rat basophilic leukemia cells involves protein tyrosine phosphorylation

Treatment of rat basophilic leukemia cells (RBL-2H3) with antigen or ionophore leads to an increase in cellular protein tyrosine phosphorylation. Three major proteins of molecular mass of 72, 92, and 110 kDa are targeted by antigen and a 110-kDa species by ionophore, A23187. The antigen- and ionophore-induced tyrosine phosphorylation responses are dose-dependent and correlate with increases in serotonin release from activated cells. The presence of extracellular Ca2+ is required to sustain the antigen- and ionophore-stimulated tyrosine phosphorylation as well as mediator release. A protein tyrosine kinase inhibitor, RG 50864, differentially inhibits the antigen-stimulated tyrosine phosphorylation in the decreasing order of 72, 91, and 110-kDa proteins. The compound inhibition of the 72-kDa protein tyrosine phosphorylation correlates with that of serotonin release. In ionophore-stimulated cells, the inhibition of the 110-kDa protein tyrosine phosphorylation and serotonin release by RG 50864 occurs in parallel. These results suggest that the 72- and 110-kDa phosphoproteins may represent the respective regulators of serotonin release in antigen- and ionophore-activated cells. The 110-kDa tyrosine phosphorylated proteins from antigen- and ionophore-stimulated cells exhibit identical electrophoretic mobility and V8 protease-generated phosphopeptide maps, suggesting that these two proteins may be the same. These results provide new evidence that both the stimulatory actions of antigen and ionophore on mediator release are mediated through enhanced protein tyrosine phosphorylation in RBL-2H3 cells. Significantly, the present study suggests the presence of multiple tyrosine phosphorylation signaling pathways in RBL cells and that their selective utility may be determined by the nature of the stimulus.     

Inhibition of calcium signaling in ultraviolet-irradiated fibroblasts: role of tyrosine phosphorylation and protein kinase C.

Our aim was to study whether ultraviolet radiation produced any alterations in the subsequent signaling response of V79 fibroblasts to mitogenic stimulus. In ultraviolet C (UVC)-irradiated V79 fibroblasts, increase in cytosolic calcium in response to thrombin was nearly abolished in the presence of 3 mM external Ca(2+). UVC-treated V79 cells showed a greatly enhanced permeability to Ca(2+) which was reversed by pretreatment with genistein, a tyrosine kinase inhibitor. Genistein also alleviated the inhibition of thrombin response caused by UVC. In UVC-treated cells, significant activation of protein kinase C (PKC) occurred only on exposure to 3 mM external calcium and PKC inhibitors (H-7 or staurosporine) reversed UVC-induced adverse effects on the thrombin response. Therefore, it is likely that protein tyrosine phosphorylation by UVC may play a role in the subsequent inhibition of thrombin response in V79 cells through increased calcium influx and activation of PKC.     

Signal transduction of human interleukin 3 and granulocyte-macrophage colony-stimulating factor through serine and tyrosine phosphorylation.

To elucidate the rapid events in signal transduction of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL 3), we examined phosphorylation of proteins on both serine and tyrosine residues in a cytokine-stimulated human myeloid cell line. We found increases in tyrosine phosphorylation within 30 s of stimulation with GM-CSF or IL 3, with peak responses occurring within 2 min. IL 3 and GM-CSF also induced serine phosphorylation, though 10 min of stimulation was required for maximum phosphate incorporation. Interestingly, both IL 3 and GM-CSF stimulated phosphate incorporation in identical substrates, a 68 kDa seryl-phosphoprotein (p68) and a 140 kDa tyrosyl-phosphoprotein (p140). Treatment of AML 193 cells with phorbol myristate acetate resulted in serine phosphorylation of p68; however, p140 was not phosphorylated on tyrosine. Depletion of protein kinase C isoenzymes with high concentrations of phorbol myristate acetate resulted in p68 phosphorylation, which was not further increased by IL 3 or GM-CSF. In contrast, cytokine-induced phosphorylation on tyrosine of p140 was observed after protein kinase C depletion. These data demonstrate the co-ordinate yet independent serine and tyrosine phosphorylation in IL 3- and GM-CSF-treated human myeloid cells, and thus suggest a common set of protein kinases stimulated by each separate ligand.     

Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal transduction pathways.

The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.     

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.     

The role of calcium in hypoxia-induced signal transduction and gene expression

Mammalian cells require a constant supply of oxygen in order to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. Sophisticated mechanisms have therefore evolved which allow cells to respond and adapt to hypoxia. Specialized oxygen-sensing cells have the ability to detect changes in oxygen tension and transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in a wide variety of different organisms. An increase in intracellular calcium levels is a primary response of many cell types to hypoxia/ischemia. The response to hypoxia is complex and involves the regulation of multiple signaling pathways and coordinated expression of perhaps hundreds of genes. This review discusses the role of calcium in hypoxia-induced regulation of signal transduction pathways and gene expression. An understanding of the molecular events initiated by changes in intracellular calcium will lead to the development of therapeutic approaches toward the treatment of hypoxic/ischemic diseases and tumors.     

Respiratory burst in human B lymphocytes. Triggering of surface Ig receptors induces modulation of chemiluminescence signal.

B lymphocytes have been shown to proliferate and release oxygen metabolites when surface Ig is cross-linked and when stimulated with phorbol ester. Biochemical evidence has been provided for the presence of a superoxide generating system in B cells, which seems to be identical to the well-characterized NADPH-oxidase of phagocytes. In this report, we show that normal and EBV-transformed B cells produce superoxide anions after stimulation with phorbol ester and when surface Ig was cross-linked, as detected by lucigenin-dependent chemiluminescence. Anti-surface IgG antibodies induced a significant respiratory burst whereas those directed against surface IgM had no effect on B cell oxidative metabolism. Prestimulated B lymphocytes responded to further triggering by the same or another ligand. Pretreatment with Staphlococcus aureus Cowan I strain (SAC) or anti-IgM antibodies resulted in complete unresponsiveness to subsequent SAC or anti-IgG stimulation, but it did not affect PMA- and ionomycin-mediated B cell chemiluminescence. In contrast to preincubation with anti-IgM antibodies, the pretreatment of B cells with SAC induced a transient inhibitory effect on B cell signaling. In fact, SAC-pretreated B lymphocytes could be restimulated with the same ligand when blast cells were isolated. Furthermore, a 24-h incubation of the pretreated B cells in the absence of SAC completely restored the SAC-mediated respiratory burst. These results suggest that two distinct mechanisms may account for SAC- and anti-IgM-induced inhibition: a transient and reversible modulation of surface Ig, induced by SAC, and a long-lasting desensitization of the surface Ig receptors, respectively. These findings may have interesting implications for understanding the transduction of negative signals in B lymphocytes.     

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides

Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since release of EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium response in cultured endothelial cells. Aggregating platelets caused a transient increase in intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adenine nucleotides ADP and ATP, but not other platelet-derived mediators, mimicked the platelet-induced calcium response, and inhibition of adenine nucleotides impaired the response to aggregating platelets. Thus, aggregating platelets release adenine nucleotides and stimulate a rise in intracellular calcium in cultured endothelial cells. This calcium response may represent the intracellular transduction mechanism by which aggregating platelets induce endothelial release of EDRF and subsequent relaxation of coronary arteries.     

Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation.

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.     

Thrombin- and histamine-induced signal transduction in human endothelial cells. Stimulation and agonist-dependent desensitization of protein phosphorylation

Treatment of human endothelial cells with thrombin, histamine, or dioctanoylglycerol (DiC8), a synthetic diacylglycerol, resulted in the rapid and transient phosphorylation of a Mr = 29,000 protein (P29) in a dose-dependent manner. Various tumor promoters also promoted P29 phosphorylation while the adenylate cyclase activator, forskolin, did not. The level of phosphorylation with all three agonists was similar (2.5-4 fold), and analysis of P29 by two-dimensional gel electrophoresis revealed identical patterns in each case. Receptor specificity was demonstrated for the histamine-stimulated changes; pyrilamine (10(-6) M; H1) but not cimetidine (10(-4); H2) blocked the response. The thrombin effect was active site-dependent. Phosphorylation induced by thrombin and histamine occurred within 1 min, peaked between 5 and 10 min, and returned to control levels by 1 h. DiC8-induced phosphorylation occurred more slowly but was also reduced by 1 h while phorbol ester treatment prolonged phosphorylation for at least 4 h. Treatment of these cells with thrombin or histamine for 1 h desensitized P29 to further phosphorylation by the homologous agonist although secondary phosphorylation could occur with heterologous compounds. However, if the primary agonist was removed following the onset of a desensitized state, secondary phosphorylation of P29 could be stimulated by the same compound. These same results were observed with two other phosphoproteins Mr = 18,000 (P18) and 80,000 (P80) which became more highly phosphorylated in response to thrombin treatment and with histamine/thrombin-stimulated prostaglandin I2 production. In contrast, homologous down-regulation of P29 phosphorylation was not observed with DiC8-treated cells, and the decline in phosphorylated P29 was associated with the loss of functional DiC8. The protein kinase inhibitors staurosporine and H-7 blocked P18 and P80 phosphorylation by thrombin but had no effect on P29 phosphorylation by histamine, thrombin, or DiC8 suggesting distinct pathways leading to the phosphorylation of these different proteins. These data suggest that multiple and independent thrombin/histamine-induced events are susceptible to receptor occupancy-dependent homologous down-regulation.     

Inducible nuclear expression of NF-kappa B in primary B cells stimulated through the surface Ig receptor

Constitutive expression of NF-kappa B has been associated with developmental maturity in B cells on the basis of studies using continuously growing cell lines and plasmacytomas; however, little is known about the behavior of NF-kappa B in primary, mature B cells. In the present work, the regulation of NF-kappa B expression was studied by analyzing subcellular fractions of adult murine splenic B cells with the electrophoretic mobility shift assay using a kappa B-containing oligonucleotide. Although nuclear extracts from resting B cells contained kappa B-binding activity, additional kappa B-binding activity was present in cytosolic fractions in a form that became apparent after treatment with detergent. Competition analysis indicated that the DNA binding activity detected by electrophoretic gel mobility shift assay was specific for the kappa B motif, and UV photo-cross-linking showed the molecular size of kappa B-binding protein to be similar to that of the DNA binding subunit of NF-kappa B. Nuclear expression of kappa B-binding activity was markedly induced by treatment of B cells with phorbol ester or with LPS. Most notably, kappa B-binding activity was induced after surface IgR cross-linking, and the mechanism of this induction involved PKC. Further, anti-Ig-induced activity was superinduced in the presence of cycloheximide. These results indicate that nuclear NF-kappa B is rapidly induced as a result of B cell stimulation, and further suggest that NF-kappa B may play a specific role in mature B cells after ligand binding to surface Ig distinct from its postulated developmental role as a stage-specific factor involved in kappa-enhancer function.     

Superoxide anion increases intracellular pH, intracellular free calcium, and arachidonate release in human amnion cells.

We determined the effects of superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, on the intracellular pH (pHi) and intracellular free calcium concentration ([Ca2+]i) and release of arachidonate in human cultured amnion cells. Superoxide anion induced a prompt increase of pHi and subsequent increase of [Ca2+]i. The evoked pHi was inhibited by pretreatment with anion channel blockers but not affected by omission of extracellular Na+ or addition of amiloride. The increase of [Ca2+]i was inhibited significantly by the absence of extracellular calcium or by the addition of a calcium channel blocker, cobalt. NH4Cl, which can generally increase pHi, also increased [Ca2+]i of amnion cells. But the increase of [Ca2+]i induced by the NH4Cl was significantly less than that induced by the amount of superoxide anion causing a similar increase in pHi. These results show that superoxide anion, crossed through anion channel in membrane, increased [Ca2+]i at least partially via increase of pHi and that the calcium mobilization was dependent on both extracellular and intracellular sources. Superoxide anion induced the release of arachidonate in a dose-dependent manner and this induction was inhibited by omission of extracellular calcium. These data suggest that the release of arachidonate was dependent on the increase of [Ca2+]i. We also determined the viability of cells in the presence of superoxide anion by flow cytometry. Superoxide anion at the levels used in these experiments did not change the percentage of viable cells. These findings suggested that superoxide anion may regulate biological functions in amnion cells via pHi, [Ca2+]i mobilization, and the release of arachidonate without damaging the cells.     

The functional expression of calcium-sensing receptors in BRL cells and related signal transduction pathway responsible for intracellular calcium elevation

The calcium-sensing receptors (CaSRs) exist in a variety of tissues and cells. In 2001, Canaff et al. first identified its expression in liver tissue and primary cultured hepatocytes, and demonstrated that GdCl₃ (a specific agonist of CaSR) can cause an increase in intracellular calcium and bile flow. However, authors did not elucidate its mechanisms. Therefore, this study sought to detect CaSR expression in BRL cell line, which is derived from buffalo rat liver, and to reveal the cellular signal transduction pathway by which the CaSR activation results in increased intracellular calcium by BRL cells. In this study, the expression and distribution of CaSR were detected by RT-PCR, Western blotting, and immunofluorescence, and the intracellular calcium concentration [Ca²⁺]_i was measured using LCSM. The results showed that CaSR mRNA and protein were expressed in BRL cells and mainly distributed in cell membrane and cytoplasm. Increased extracellular calcium or GdCl₃ could increase intracellular calcium concentration and CaSR expression. Moreover, this increase of [Ca²⁺]_i could be inhibited or even abolished by U73122 (a specific inhibitor of PLC), 2-APB (an inhibitor of IP₃ receptor), and thapsigargin (an inhibitor of endoplasmic reticulum calcium pump). In conclusion, CaSR is functionally expressed in BRL cells, and activation of CaSR involves in increased intracellular calcium through Gq–PLC–IP₃ pathway.