beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).     

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8.     

Effects of rare earth compounds on growth and apoptosis of leukemic cell lines

To explore a new agent for inhibiting leukemic cells, we investigated the effects of rare earth compounds (lanthanum chloride and cerium chloride) on the growth and apoptosis of HL-60 and NB4 cells. The growth of HL-60 and NB4 cells was tested by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) colorimetric assay. The apoptosis was measured by light microscopy, flow cytometry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) method. The effect of LaCl(3) on normal bone marrow hematopoietic progenitor cells was evaluated by colony-forming unit-granulocyte-macrophage (CFU-GM) assay. Under our experimental conditions, MTT assay showed that 48-h treatment with 1, 2, and 3 mM LaCl(3) or 48- and 72-h treatments with 1 mM LaCl(3) could significantly inhibit the growth of HL-60 cells. Treatment with 2 and 4 mM CeCl(3) for 72 h could significantly inhibit the growth of NB4 cells. Apoptosis could be detected on treatment with 2 mM LaCl(3) for 24 h in HL-60 cells by light microscopic morphology examination, flow cytometric analysis, and TUNEL method. Apoptosis could be also detected on treatment with 2 mM CeCl(3) for 72 h in NB4 cells. Treatment with 1 mM LaCl(3) could arrest the transitions from G0/G1 to S phase. The granulocyte-macrophage colony formation of normal bone marrow cells was not significantly inhibited at lower concentrations of LaCl(3) (0.5 to 2 mM). Our results indicate that at certain concentrations, the rare earth compounds may inhibit the growth of leukemic cells, induce them to apoptosis, and have no significant inhibitory effects on normal bone marrow hematopoietic progenitor cells (CFU-GM). The mechanism needs to be further investigated.     

Thymosin beta4 and thymosin beta4-derived peptides induce mast cell exocytosis

The peptide thymosin beta4 (Tbeta4) promotes angiogenesis and wound healing. Mast cells are involved in these processes as well and therefore we investigated the effect of Tbeta4 on mast cells. Exposure to 0.2-2000nM Tbeta4 induced mediator release (up to 23%) in murine peritoneal and human HMC-1 mast cells in a concentration-dependent manner. While the peptide AcSDKP, matching the 4 N-terminal amino acid residues of Tbeta4, mediated low but detectable mediator release, peptides corresponding to the Tbeta4 amino acid sequences 16-38 and 17-23 stimulated mast cells mediator release on a level equal to or higher than that observed with native Tbeta4. These observations and certain characteristics of Tbeta4-mediated mast cell activation suggest that the actin-binding motif LKKTET present in Tbeta4 (amino acid 17-22) might be implicated in this process. Thus, Tbeta4 activates mediator release in mast cells by a process that possibly involves an actin-binding motif and this could be important for understanding the mechanisms of Tbeta4-mediated effects in vivo.     

CaMKII-dependent reactivation of SR Ca(2+) uptake and contractile recovery during intracellular acidosis

In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca(2+) response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 +/- 0.3% of diastolic length to 0.2 +/- 0.1% (means +/- SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 +/- 0.3% at 10 min (P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) transient (CaT) amplitude increased, and the twitch [Ca(2+)](i) decline prolonged significantly (P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca(2+)](i) decline were observed. The increase in diastolic [Ca(2+)](i) was less extensive than the increase in the cells that did not recover (n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 microM) and thapsigargin (1 microM) or a selective inhibitor of Ca(2+)-calmodulin kinase II, 2-[N- (2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (1 microM) completely abolished the reacceleration of twitch [Ca(2+)](i) decline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca(2+)-calmodulin kinase II-dependent reactivation of SR Ca(2+) uptake could increase SR Ca(2+) content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca(2+) response, but may also cause Ca(2+) overload after returning to physiological pH(i).     

Real-time measurement of cytosolic free calcium concentration in HL-60 cells during static magnetic field exposure and activation by ATP

Calcium ions are involved in a number of important signal transduction pathways in cells. Cytosolic calcium concentration ([Ca(2+)](c)) can be affected by the activation of Ca(2+) channels through the action of ligands such as ATP. The response of [Ca(2+)](c) to ligands may be affected by external factors like magnetic fields. The purpose of this study was to determine if exposure to a static magnetic field (SMF) for 800 s altered the [Ca(2+)](c) response to ATP in undifferentiated HL-60 cells. We sham exposed or field exposed fura-2 loaded HL-60 cells to a SMF of 1, 10, and 100 mT. Cells were activated with ATP 300 s into the exposure. The level of [Ca(2+)](c) was followed before, during, and after field or sham exposure with a ratiometric fluorescence spectroscopy system. It was found that high concentrations of ATP resulted in greater [Ca(2+)](c) responses, but faster recovery to near basal levels. The application of 1, 10, or 100 mT SMF did not affect the [Ca(2+)](c) response to ATP. Future work could examine the effect of a longer SMF exposure on the [Ca(2+)](c) response to ATP. Longer exposures might provide sufficient time for morphological changes in the plasma membrane to occur.     

Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering

Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.     

Inhibition of H2 histamine receptor-mediated cation channel opening by protein kinase C in human promyelocytic cells

Histamine, through H(2) receptors, triggers a prominent rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in addition to an elevation of cAMP level in HL-60 promyelocytes. Here we show that the histamine-induced [Ca(2+)](i) rise was due to influx of Ca(2+) from the extracellular space, probably through nonselective cation channels, as incubation of the cells with SKF 96365 abolished the histamine-induced [Ca(2+)](i) rise, Na(+) influx, and membrane depolarization. The Ca(2+) influx was specifically inhibited by pretreatment of the cells with PMA or extracellular ATP with 50% inhibitory concentrations of 0.12 +/- 0.03 nM and 185 +/- 17 microM, respectively. Western blot analysis of protein kinase C (PKC) isoforms revealed that PMA (< or =1 nM) and ATP (300 microM) caused selective translocation of PKC-delta to the particulate/membrane fraction. Costimulation of the cells with histamine and SKF 96365 partially reduced histamine-induced granulocytic differentiation, which was evaluated by looking at the extent of fMet-Leu-Phe-induced [Ca(2+)](i) rise and superoxide generation. In conclusion, nonselective cation channels are opened by stimulation of the H(2) receptor, and the channels are at least in part involved in the induction of histamine-mediated differentiation processes. Both effects of histamine were selectively inhibited probably by the delta isoform of PKC in HL-60 cells.     

Diverse effects of sphingosine on calcium mobilization and influx in differentiated HL-60 cells

Sphingosine induces a biphasic increase in cytosolic-free Ca(2+)([Ca(2+)](i)) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (beta-aminoethyl ether) N, N, N', N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca(2+)influx does not appear due to Ca(2+)influx through store-operated Ca(2+)(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca(2+)influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca(2+)](i)elicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca(2+)from intracellular Ca(2+)stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca(2+)influx through an undefined Ca(2+)channel and cause a blockade of SOC channels.     

白藜芦醇降低大鼠心室肌细胞内游离钙浓度

实验旨在研究白藜芦醇(resveratrol)对大鼠心室肌细胞内钙浓度(intracellular calcium concentratoin,[Ca2+]i)的影响.应用激光共聚焦显微镜技术记录心室肌细胞内的钙荧光强度.结果表明在正常台氏液和无钙台氏液中,白藜芦醇(15～60μmol/L)呈浓度依赖性地降低[Ca2+]i.蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate,1.0 mmol/L)和L型Ca2+通道激动剂Bay K8644(10 μmol/L)可部分抑制正常台氏液中白藜芦醇的效应.但NO合酶阻断剂L-NAME(1.0 mmol/L)对白藜芦醇的作用无影响.白藜芦醇也能明显抑制无钙台氏液中由低浓度ryanodine(1.0 nmol/L)引起的[Ca2+]i增加.当细胞外液钙浓度由1 mmol/L增加到10 mmol/L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,白藜芦醇(60 μmol/L)可降低钙波的传播速度和持续时间,最终阻断钙波.结果提示,白藜芦醇能够降低心室肌细胞内游离钙浓度,此作用可能与其抑制电压依赖性Ca2+通道、酩氨酸激酶和肌浆网内钙释放有关.     

KB-R7943 inhibits store-operated Ca(2+) entry in cultured neurons and astrocytes

We have studied cyclopiazonic acid (CPA)-sensitive store-operated Ca(2+) entry (SOCE) in cultured neurons and astrocytes and examined the effect of 2-[2-[4-(4-nitrobenzyloxy)phenyl]]isothiourea (KB-R7943), which is often used as a selective inhibitor of the Na(+)-Ca(2+) exchanger (NCX), on the SOCE. CPA increased transiently intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a sustained increase in [Ca(2+)](i) in neurons and astrocytes. The sustained increase in [Ca(2+)](i) depended on the presence of extracellular Ca(2+) and inhibited by SOCE inhibitors, but not by a Ca(2+) channel inhibitor. CPA also caused quenching of fura-2 fluorescence when the cells were incubated in Mn(2+)-containing medium. KB-R7943 at 10 microM inhibited significantly CPA-induced sustained increase in [Ca(2+)](i) in neurons and astrocytes. KB-R7943 also inhibited CPA-induced quenching of fura-2 fluorescence in the presence of extracellular Mn(2+). These results indicate that cultured neurons and astrocytes possess SOCE and that KB-R7943 inhibits not only NCX but also SOCE.     

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.     

Direct cytoplasmic CA(2+) responses to gastrin-releasing peptide in single beta cells

The neuropeptide gastrin releasing peptide (GRP) stimulates insulin secretion and induces oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) in clonal insulinoma cells. It is not known whether GRP affects [Ca(2+)](cyt) in normal beta cells. We investigated, in single, normal, mouse islet beta cells, the effects of GRP on [Ca(2+)](cyt), by dual wavelength spectrophotofluorometry. Beta cells were identified by their typical response to glucose or tolbutamide. At 15 mM glucose, GRP (100 nM) evoked an immediate [Ca(2+)](cyt) transient to 423 +/- 48 nM compared to 126 +/- 18 nM before GRP (P < 0.001). After the initial transient, [Ca(2+)](cyt) exhibited either a sustained elevation or oscillations. At 3.3 mM glucose, in cells with a non-oscillating [Ca(2+)](cyt), GRP stimulated a prompt increase in [Ca(2+)](cyt) (from 60 +/- 6 to 285 +/- 30 nM, P = 0.024) followed by either a sustained increase in [Ca(2+)](cyt) or [Ca(2+)](cyt) oscillations. We conclude that GRP promptly elevates [Ca(2+)](cyt) by a direct action in normal mouse pancreatic beta cells.     

Effect of timosaponin A-III,from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.     

C2-Ceramide increases cytoplasmic calcium concentrations in human parathyroid cells

Effects of extracellular calcium ([Ca(2+)](ext)) on parathyroid cells are mainly due to the activation of a plasma membrane calcium receptor (CaR) coupled with release of intracellular calcium. In addition, high [Ca(2+)](ext) activates the sphingomyelin pathway in bovine parathyroid cells, generating ceramides and sphingosine. This study explored the direct effects of synthetic ceramides on [Ca(2+)](i) in human parathyroid cells. Cells from five parathyroid adenomas removed from patients with primary hyperparathyroidism were dispersed and maintained in primary culture. Intracellular calcium concentration ([Ca(2+)](i)) [Ca(2+)](i) was monitored using standard quantitative fluorescence microscopy in Fura-2/AM-loaded cells. Laser scanning microscopy was used to monitor the intracellular distribution of a fluorescent ceramide analogue (BODIPY-C5). After addition of 10 microM C2-ceramide (N-acetyl-d-erythro-sphingosine), [Ca(2+)](i) increased rapidly (30-60 s) to a peak three times above basal levels in 70% of cells (37/55 cells in four experiments). This effect appeared to be due to release of Ca(2+) from intracellular stores rather than Ca(2+) entry from the extracellular medium. C2-responsive cells had a smaller [Ca(2+)](i) response to subsequent stimulation with the CaR agonist-neomycin (1 mM). These responses were specific to C2 since C6-ceramide (N-hexanoyl-d-erythro-sphingosine) did not affect basal [Ca(2+)](i) nor the responses to an increase in [Ca(2+)](ext) and to neomycin. C5-BODIPY generated intense perinuclear fluorescence, suggesting targeting of the ceramides to the Golgi apparatus. These data demonstrate that endogenous generation of ceramides has the potential to modulate changes in [Ca(2+)](i) and secretion in response to [Ca(2+)](ext) in human parathyroid cells.     

Nongenomic mechanism of glucocorticoid inhibition of bradykinin-induced calcium influx in PC12 cells: possible involvement of protein kinase C

Many stimulants, including bradykinin (BK), can induce increase in [Ca(2+)](i) in PC12 cells. Bradykinin induces an increase in [Ca(2+)](i) via intracellular Ca(2+) release and extracellular Ca(2+) influx through the transduction of G protein, but not through voltage-sensitive calcium channels. In this experiment, We analyzed how corticosterone (Cort) influences BK-induced intracellular Ca(2+) release and extracellular Ca(2+) influx, and further studied the mechanism of glucocorticoid's action. To dissociate the intracellular Ca(2+) release and extracellular Ca(2+) influx induced by BK, the Ca(2+)-free/Ca(2+)- reintroduction protocol was used. The results were as follows: (1) The Ca(2+) influx induced by BK could be rapidly inhibited by Cort, but intracellular Ca(2+) release could not be affected significantly. (2) The inhibitory effect of Cort-BSA (BSA -conjugated Cort) on Ca(2+) influx induced by BK was the same as the effect of free Cort. (3) Protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) could mimic and PKC inhibitor G?6976 could reverse the inhibitory effect of Cort. (4) There was no inhibitory effect of Cort on Ca(2+) influx induced by BK when pretreated with pertussis toxin. The results suggested, for the first time, that Cort might act via a putative membrane receptor and inhibit the Ca(2+) influx induced by BK through the pertussis toxin -sensitive G protein-PKC pathway.     

Follicle-stimulating hormone mediated calcium signaling by the alternatively spliced growth factor type I receptor

Ovarian granulosa cell and testicular Sertoli cell functions are regulated by the tropic action of the pituitary follicle-stimulating hormone (FSH), which may exert pleiotropic effects using a variety of signaling pathways. The effects of FSH on the mobilization of Ca(2+) into granulosa and Sertoli cells have been widely studied, but whether all the effects of the hormone are mediated by the single G-protein-coupled (G(s)) receptor with the seven-transmembrane structure (R1) has remained an enigma. With the object of resolving this mystery, we have compared the hormonal responses of HEK 293 cells transfected with three different cloned FSH receptor cDNAs of testis/ovary, designated R1 (G(s)), R2 (similar to R1 but having a shorter carboxyl terminus), and R3, a novel FSH receptor exhibiting a growth factor type I receptor motif. The latter two that use the same DNA segment for alternative splicing of the single large 80- to 100-kilobase gene create different structural motifs and carboxyl termini. Of the three receptors, only the FSH-R3 type induced a significant rise in intracellular free calcium concentration ([Ca(2+)](i)), as measured by single cell fluorescence digital imaging with the Ca(2+) sensitive dye fura-2AM. FSH induced a rapid [Ca(2+)](i) response that was concentration dependent. The response was hormone-specific, as neither its individual alpha/beta subunits nor the related glycoprotein hormone LH were effective. To determine whether the [Ca(2+)](i) response was due to Ca(2+) influx or to intracellular Ca(2+) mobilization, cells were exposed to Ca(2+)-free buffer and to the Ca(2+)-channel blocker diltiazem (10(-5) M). FSH-Induced [Ca(2+)](i) responses were inhibited in Ca(2+)-free buffer and abrogated in the presence of diltiazem. These novel data demonstrate that FSH can increase [Ca(2+)](i) through L-type voltage-dependent Ca(2+) channels via the growth factor type 1 receptor. Our findings support the concept that different receptor motifs act to integrate intracellular signaling events.     

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.