IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha -thrombin

Robert S. Mathias1, Katsuhiko Mikoshiba2, Takayuki Michikawa2, Atsushi Miyawaki2, and Harlan E. Ives3,4

Departments of 1 Pediatrics and 3 Medicine and 4 Cardiovascular Research Institute, University of California, San Francisco, California 94143; and 2 Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The effect of inositol 1,4,5-trisphosphate (IP3) receptor blockade on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), endothelin-1 (ET-1), or alpha -thrombin receptor-mediated intracellular Ca2+ (Ca2+i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells and myoblasts. Blockade of the IP3 receptor was achieved by microinjection of heparin or monoclonal antibody (MAb) 18A10 into the IP3 type 1 receptor. Heparin completely inhibited Ca2+i release after flash photolysis with caged IP3 and after exposure to PDGF and FGF. In contrast, heparin failed to block Ca2+i release after alpha -thrombin and ET-1. After application of ligand, IP3 levels were five- to sevenfold higher for alpha -thrombin than for ET-1 or PDGF. IP3 levels after PDGF and ET-1 were comparable. Similar to heparin, MAb 18A10 blocked Ca2+i release after PDGF but failed to block Ca2+i release after ET-1 or alpha -thrombin. These data suggest that the mechanisms of Ca2+i release by tyrosine kinase and certain 7-transmembrane receptors may differ. Although both receptor types use the IP3-signaling system, the ET-1 and alpha -thrombin receptors may have a second, alternative mechanism for activating Ca2+i release.

platelet-derived growth factor; endothelin-1; intracellular microinjection of heparin and monoclonal antibody 18A10; Chinese hamster ovary cells; inositol 1,4,5-trisphosphate

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

CALCIUM SIGNALING in mesenchymal cells is typically a biphasic process: in the first phase, Ca2+ is released into the cytoplasm from intracellular organelles; in the second phase, Ca2+ enters the cytoplasm from outside the cell. Inositol 1,4,5-trisphosphate (IP3), formed by hydrolysis of phosphatidylinositol 4,5-bisphosphate, is believed to be the major mediator of Ca2+ release from intracellular stores (3). IP3 is released into the cytoplasm and binds to specific receptors located on intracellular Ca2+ (Ca2+i)-storing organelles, which activate channel opening and release Ca2+ into the cytoplasm (13). IP3 is produced after activation of tyrosine kinase and 7-transmembrane receptors. Although 7-transmembrane and tyrosine kinase receptors produce IP3 by different isoforms of phospholipase C (51, 54), it has generally been assumed that the IP3 formed by both signaling pathways acts similarly.

It has recently been found that there are at least three isoforms of the IP3 receptor (IP3R) (37). Some of these receptors may be found within a single cell (40, 45, 58) and may localize to different structures within the same cell (16, 45). This raises the possibility of functional heterogeneity among IP3R isoforms. It is possible that the proximity of the IP3R to different signal transduction systems could be an important determinant of the various cellular responses associated with IP3 generation.

In this study we examined the effect of microinjected heparin and an IP3R1 monoclonal antibody (MAb 18A10) on release of Ca2+i stores after activation of tyrosine kinase or 7-transmembrane receptors. We find that Ca2+i release by tyrosine kinase receptors [platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF)] is blocked by heparin and MAb (18A10) to the IP3R1, suggesting that the pathway involves heparin-sensitive IP3R1. In contrast, Ca2+i release by certain 7-transmembrane [endothelin-1 (ET-1) and alpha -thrombin] receptors is not blocked by heparin or MAb 18A10. These findings indicate that there may be alternative mechanisms for Ca2+i release in addition to the traditional IP3-IP3R pathway and that 7-transmembrane receptors may activate one or more such alternative systems for Ca2+i release.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials. Unless otherwise specified, all chemicals were purchased from Sigma Chemical, including ET-1 (E-9262), low-molecular-weight heparin (H-5271), MAb to IgG2alpha (M-9144), and a polyclonal antibody to IgG (I-5381). Fura 2 and Ca2+ green 1 were purchased from Molecular Probes (Eugene, OR). BSA was fatty acid-poor fraction V from Miles (Kankakee, IL). Highly purified human alpha -thrombin was generously supplied by Dr. John Fenton (State Health Department, Albany, NY). PDGF-BB was purchased from Boehringer Mannheim (Indianapolis, IN). Recombinant human basic FGF (bFGF) was generously supplied by Chiron (Emeryville, CA). Caged IP3 was purchased from Calbiochem (San Diego, CA). IP3R type-specific MAbs (18A10, KM1083, and KM1082) were generated as previously described (17).

Preparation of cell lines. CHO-PDGF cells were stable transfectants of Chinese hamster ovary (CHO) cells containing the cloned PDGF-BB receptor (12). CHO-PDGF/FGF cells were stable transfectants containing the chimeric receptor composed of the PDGF receptor extracellular domain ligated to the FGF receptor transmembrane-cytoplasmic domain (11). M-FGF were stable transfectants in rat L6 myoblasts containing the cloned FGF receptor (44). CHET-B cells were stable transfectants of CHO-K1 cells containing the cloned ET-1 receptor (32).

Cell culture. CHO-PDGF and CHO-PDGF/FGF cells were grown in Ham's F-12 medium; M-FGF and CHET-B cells were grown in DMEM in a humidified atmosphere of 5% CO2-95% air at 37°C. All media contained 10% (vol/vol) fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 U/ml). Medium for stable transfectants was supplemented with 400 µg/ml G418. Culture medium was changed every 2-3 days until cells were confluent. CHO-PDGF and CHO-PDGF/FGF cells were made quiescent by replacement of 10% of the serum with BSA (0.5 mg/ml) and transferrin (5 µg/ml).

Measurement of Ca2+i. For single cell measurement of Ca2+i, cells were plated at 500 cells/ml on a microscope cover glass (Fisher, 25-mm circle) and grown in serum-containing medium until 70-80% confluency was achieved. For CHO-PDGF and CHO-PDGF/FGF cells, serum was removed 24 h before Ca2+i measurements to eliminate any residual PDGF activity in the serum. The other cell lines were studied in serum. Cells were mounted on a temperature-controlled chamber at 31°C in assay medium (140 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 25 mM glucose, 25 mM HEPES-NaOH, pH 7.2, and 0.5 mg/ml BSA with or without 2 mM CaCl2) (24). Single cells were microinjected with fura 2-pentapotassium salt (5 mM) in the absence or presence of heparin or MAb 18A10. The cells were allowed to equilibrate for 20 min before the Ca2+i measurements were performed. Fura 2 fluorescence was measured using a Nikon epifluorescence inverted microscope fitted with a rotating holder for excitation filters (340 and 380 nm), as previously described (25). Signals were digitized using a Labmaster interface board (Scientific Solutions, Solon, OH) and recorded in an IBM-style computer with use of the UMANS software package (Chester Regen, Bio-Rad). To calibrate the fluorescence signals, the ratio of fluorescence at 340 to fluorescence at 380 nm was compared with ratios obtained at maximal Ca2+i (achieved by the addition of 10 µM 4BR-A-23187) and 0 Ca2+i (achieved by the addition of 20 mM EGTA). Ca2+i was calculated as previously described (22).

For Ca2+i measurements with caged IP3, single cells were prepared as described above and microinjected with Ca2+ green 1-hexapotassium salt (0.1 mM) with caged IP3 in the absence or presence of heparin. The fluorescence signal for the photorelease experiments was measured with excitation light passed through a narrow-band interference filter centered at 490 nm to excite Ca2+ green 1 fluorescence. Increases in fluorescence emission collected at 515 nm correspond to increases in the free Ca2+i concentration (5).

Photolysis of caged compounds. Photolysis of microinjected caged IP3 was achieved using a modification of the method of Bird et al. (5). Light from a continuously burning xenon arc flash lamp was passed through a broad 120-nm bandwidth filter centered at 350 nm and directed to the specimen for 0.5-2.0 s. The timing and duration of the ultraviolet flashes were controlled by the computer. Immediately after these computer-controlled flashes, Ca2+i measurements were started.

Measurement of IP3 formation. CHO-PDGF and CHET-B cells were plated on six-well culture dishes and grown in serum-containing medium until 70-80% confluency was achieved. For CHO-PDGF cells, serum was removed 24 h before IP3 measurements. Cells were washed twice with PBS containing 0.2% BSA. Cells were then incubated in 1 ml of PBS containing 0.2% BSA and 10 mM LiCl2 for 30 min at 37°C in a shaking bath. At the time of the experiment, the medium in the well was removed and replaced with 0.8 ml of fresh medium in the absence or presence of the known agonist. The experiment was stopped with the addition of 0.2 ml of perchloric acid (20% vol/vol). IP3 levels were determined using the bovine adrenal protein binding assay kit (Amersham).

Microinjection. Cells were microinjected using glass capillary needles (20) held in a Narishige micromanipulator. Pipettes were made from borosilicate glass tubes (0.9 mm) by use of a Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA). By measurement of the diameter of droplets microinjected into oil, the microinjectate volume was determined to be 6 × 10-14 liter. Various concentrations of heparin or the MAb to the IP3R in buffer (27 mM K2HPO4, 8 mM NaH2PO4, 26 mM KH2PO4, pH 7.3) were microinjected with 5 mM fura 2-pentapotassium or 0.1 mM Ca2+ green 1-hexapotassium salt. With the assumption of an intracellular volume of 3 × 10-12 liter (35), intracellular concentrations were estimated to be 2% of the injectate concentrations.

Preparation of total cell lysates. Total cell lysates of CHO-PDGF and CHET-B cells were prepared as described by Monkawa et al. (40). Cells were homogenized in a solution containing 250 mM sucrose, 5 mM Tris · HCl (pH 8.0), 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 0.01 mM pepstatin A, 0.01 mM leupeptin, and 0.01 mM E-64 in a chilled glass-Teflon Potter homogenizer with 10 strokes at 1,000 rpm. The homogenates were centrifuged at 3,000 g for 5 min at 4°C. The supernatants were centrifuged at 105,000 g for 6 min at 2°C. The pellets were resuspended in 50 mM Tris · HCl (pH 8.0), 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 0.01 mM pepstatin A, 0.01 mM leupeptin, and 0.01 mM E-64 and used as total cell lysates. Protein concentrations were measured with a Bio-Rad (Richmond, CA) protein assay with BSA as the standard.

Western blot analysis. Twenty micrograms of total cell lysates were resolved by 5% SDS-PAGE and electroblotted onto a Hybond enhanced chemiluminescence membrane (Amersham). The blot was processed through sequential incubations with blocking solution (5% skim milk and 0.1% Tween 20 in PBS) and IP3R type-specific antibodies [IP3R1- (4C11, 10A6, and 18A10), IP3R2- (KM1083), or IP3R3-specific antibodies (KM1082)]. Secondary antibodies were anti-rat Ig (for IP3R1-specific antibodies), anti-mouse Ig (for KM1082 and KM1083), horseradish peroxidase-linked F(ab')2 fragment (Amersham) of anti-rat Ig (for 18A10), or anti-mouse Ig (for KM1082 and KM1083). Immunodetection signals were visualized by the enhanced chemiluminescence Western blotting system (Amersham). The intensities of the bands from immunoblotting were measured on a Macintosh computer with use of NIH Image (version 1.61) software.

Statistics. IP3 measurements are expressed as means ± SE and compared by Student's unpaired t-test and Fisher's ANOVA. P < 0.05 was considered significant. The Ca2+ data are means ± SD.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Microinjected heparin inhibits release of Ca2+i stores after photolysis of caged IP3. Ca2+ green (0.1 mM) and caged IP3 (0.375 mM) were microinjected into CHO-PDGF cells in the absence or presence of heparin (5-20 mg/ml injectate concentrations) to determine whether microinjected heparin was capable of blocking IP3-mediated Ca2+i release. The microinjected volume was 6 × 10-14 liter, ~2% of cell volume (see EXPERIMENTAL PROCEDURES). Photorelease of IP3 was achieved with flashes of ultraviolet light, as described in EXPERIMENTAL PROCEDURES. Cells in nominally Ca2+-free medium were exposed to multiple flashes (~10) of 0.5- to 1-s duration (Fig. 1A; n = 8). Each flash caused a single Ca2+ spike. Heparin, at injectate concentration of 10 mg/ml, blocked Ca2+i release in nominally Ca2+-free medium after flash photolysis of caged IP3 (Fig. 1B; n = 5).


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Fig. 1.   Heparin blocks intracellular Ca2+ (Ca2+i) release after photorelease of caged inositol 1,4,5-trisphosphate (IP3) in single Chinese hamster ovary (CHO) cells. Before Ca2+ measurements, single CHO-platelet-derived growth factor (PDGF) cells were microinjected with 0.1 mM Ca2+ green and 0.375 mM caged IP3 (A) or with 0.1 mM Ca2+ green, 0.375 mM caged IP3, and 10 mg/ml heparin (B). After 20-30 min, Ca2+i transients (dimensionless fluorescence units) were measured in single CHO-PDGF cells. In A, each spike represents a single Ca2+i transient in response to single flashes of ultraviolet light (each arrow represents a single flash of 0.5- to 1-s duration) in nominally Ca2+-free medium. In B, after microinjection of heparin, Ca2+i failed to increase, despite multiple single flashes of ultraviolet light (0.5- to 4-s duration). Medium Ca2+i concentration was varied between "0" mM (solid line) and 2 mM Ca2+ (filled bar). Traces are representative of at least 5-8 experiments under each condition.

Heparin blocks release of Ca2+i stores by activation of tyrosine kinase receptors. To illustrate the two phases of Ca2+ mobilization, a single CHO-PDGF cell microinjected with fura 2-pentapotassium salt was exposed to 25 ng/ml PDGF-BB in nominally Ca2+-free medium (Fig. 2A). PDGF caused a delayed and transient increase in Ca2+i to 765 ± 210 nM. On addition of 2 mM Ca2+ to the medium, there was a second, sustained increase in Ca2+i to 1,445 ± 296 nM (n = 16). As observed previously in vascular smooth muscle cells (25), microinjected low-molecular-weight heparin (5 mg/ml in the injectate) completely blocked Ca2+ release from intracellular stores after exposure to PDGF (Fig. 2B). Although there is variability in the appearance of the sustained Ca2+ increase after the addition of 2 mM Ca2+ to the medium, on average the peak Ca2+ concentration (1,298 ± 200 nM) was unaffected by heparin (n = 12).


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Fig. 2.   Inhibition of Ca2+ transients by microinjected heparin in single cells expressing tyrosine kinase receptors. Before Ca2+ measurements, single cells were microinjected with 5 mM fura 2 alone (A and C) or 5 mM fura 2 and 5 mg/ml low-molecular-weight heparin (B and D). Twenty to 30 min later, Ca2+i transients were measured in single cells after addition of PDGF (25 ng/ml) to single CHO-PDGF cells (A and B) or basic fibroblast growth factor (bFGF, 5 ng/ml) to single M-FGF cells (C and D). Medium Ca2+ concentration was varied between "0" mM (solid line) and 2 mM (filled bar). Traces are representative of 5-12 experiments for each agonist.

Similar observations were made with bFGF in single M-FGF cells. bFGF (5 ng/ml) caused a delayed and transient increase in Ca2+i to 282 ± 56 nM in nominally Ca2+-free medium and a second, sustained increase in Ca2+i to 657 ± 104 nM on addition of 2 mM Ca2+ to the medium (Fig. 2C; n = 5). As for PDGF, microinjected low-molecular-weight heparin (5 mg/ml injectate concentration) blocked the first phase (Ca2+i mobilization) but not the second phase of Ca2+ mobilization (influx) in an M-FGF cell after exposure to bFGF (Fig. 2D; n = 5). Finally, we examined Ca2+i transients after activation of a chimeric PDGF/FGF receptor. As observed with PDGF and FGF, the chimeric receptor (see EXPERIMENTAL PROCEDURES) elicited two phases of Ca2+ mobilization after exposure to PDGF, but only the first phase was blocked by microinjected heparin (data not shown). Taken together, the data suggest that tyrosine kinase receptor-mediated release of Ca2+i stores occurs via heparin-sensitive IP3R but that Ca2+ entry from the medium is regulated by a different mechanism.

Heparin does not block release of Ca2+i stores after activation by ET-1 and alpha -thrombin. On addition of ET-1 (1 nM) to CHET-B cells in nominally Ca2+-free medium, there was a transient increase in Ca2+i to 833 ± 168 nM that developed more rapidly than in the tyrosine kinase receptors (Fig. 3A). The lowest concentration of ET-1 that generated reproducible Ca2+ transients was 1 nM (approximately twice the receptor dissociation constant). Subsequent addition of 2 mM Ca2+ to the medium resulted in a second, prolonged increase in Ca2+i to 1,883 ± 437 nM (n = 8). In contrast to what was observed with tyrosine kinase receptors, microinjected heparin (5 mg/ml injectate concentration) failed to block ET-1-induced release of Ca2+ from intracellular stores (980 ± 105 nM; Fig. 3B). The sustained, second phase of Ca2+ entry was also unaffected by heparin (1,724 ± 263 nM, n = 10).


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Fig. 3.   Microinjected heparin fails to inhibit Ca2+i transients in single cells expressing 7-transmembrane receptors. Before Ca2+ measurements, single cells were microinjected with 5 mM fura 2 alone (A and D) or 5 mM fura 2 and 5 mg/ml low-molecular-weight heparin (B, C, and E). Twenty to 30 min later, Ca2+i transients were measured after addition of 1 nM endothelin-1 (ET-1) to single CHET-B cells (A-C) or 1 U/ml alpha -thrombin (alpha -thr) to single CHO-PDGF cells (D and E). Medium Ca2+ concentration was varied between "0" mM (solid line) and 2 mM (filled bar). In C, extracellular Ca2+ was lowered further by addition of 1 mM EGTA. Traces are representative of 7-13 experiments for each agonist.

In view of the unexpected failure of heparin to block Ca2+ release with ET-1 in "nominally Ca2+-free" medium, additional controls were performed with the same medium containing 1 mM EGTA to ensure that the first Ca2+ transient was indeed due to Ca2+i release. Extracellular Ca2+ concentration, measured with fura 2-pentapotassium, was 1.5 µM in nominally Ca2+-free solutions and 116 nM after addition of 1 mM EGTA. Despite the dramatically lower extracellular Ca2+ after addition of EGTA to the medium, heparin again failed to block Ca2+i release (Fig. 3C).

Further studies were performed using alpha -thrombin, a ligand to a 7-transmembrane receptor that is native to the CHO cell. alpha -Thrombin (1 U/ml) caused a transient increase in Ca2+i to 933 ± 231 nM in nominally Ca2+-free medium and a second, transient increase in Ca2+i to 971 ± 302 nM on addition of 2 mM Ca2+ to the medium (Fig. 3D; n = 13). As for ET-1, microinjected heparin (5 mg/ml injectate concentration) failed to block alpha -thrombin-induced release of Ca2+ from intracellular stores (889 ± 167 nM) or Ca2+ entry (850 ± 152 nM, n = 7; Fig. 3E).

To demonstrate that the observed differences in Ca2+ transients between 7-transmembrane and tyrosine kinase receptors were not due to cell variability, we examined Ca2+i mobilization by PDGF and alpha -thrombin in a single CHO-PDGF cell microinjected with fura 2 in the absence of heparin (Fig. 4A). PDGF (25 ng/ml) and alpha -thrombin (1 U/ml) caused Ca2+i release and Ca2+ entry (n = 10). In single cells microinjected with heparin (5 mg/ml injectate concentration), there was a complete block of Ca2+ release from intracellular stores after the addition of PDGF (25 ng/ml; Fig. 4B; n = 8). However, Ca2+i release after alpha -thrombin (1 U/ml) was intact. Ca2+ entry from the medium was intact for both ligands. We then examined the effect of microinjected heparin on Ca2+i stores after photorelease of caged IP3 and activation of PDGF and alpha -thrombin receptors in single CHO-PDGF cells. In Fig. 4C, heparin, at injectate concentrations of 10 mg/ml, blocked Ca2+i release in nominally Ca2+-free medium after flash photolysis of caged IP3 (0.375 nM; 4 single flashes of 0.5- to 4-s duration) or PDGF (25 ng/ml) but failed to block Ca2+i release after alpha -thrombin receptor (1 U/ml; n = 5).


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Fig. 4.   Effect of microinjected heparin on Ca2+i transients in single CHO cells expressing PDGF receptor. Before Ca2+ measurements, single CHO-PDGF cells were microinjected with 5 mM fura 2 (A), fura 2 and 5 mg/ml heparin (B), or 0.1 mM Ca2+ green, 0.375 mM caged IP3, and 10 mg/ml heparin (C). Twenty to 30 min later, Ca2+i transients were measured in single cells after sequential additions of PDGF (25 ng/ml) and alpha -thrombin (1 U/ml) in nominally Ca2+-free medium followed by addition of 2 mM Ca2+ (A and B) or after multiple single flashes of ultraviolet light (0.5- to 4-s duration; C). Medium Ca2+ concentration was varied between "0" mM (solid line) and 2 mM (filled bar). Traces are representative of 5-10 experiments under each condition.

IP3 formation after activation of tyrosine kinase and 7-transmembrane receptors. Because heparin is a competitive inhibitor of the IP3R, the results in Figs. 2-4 might be explained by increased production of IP3 in response to activation of 7-transmembrane receptors. We therefore determined the level of IP3 formation after the addition of PDGF, ET-1, or alpha -thrombin (Table 1). Indeed, with alpha -thrombin, IP3 levels were elevated 7.5-fold from baseline at 15 s, higher than the values observed for PDGF (1.5-fold at 60 s). However, with ET-1 the increase in IP3 level was comparable to that produced by PDGF, as demonstrated by ANOVA of the data in Table 1.

                              
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Table 1.   Time course for agonist-mediated IP3 formation

Assessment of competitive inhibition of Ca2+ release by microinjected heparin. To further assess the competitive inhibition of Ca2+ release by heparin, we varied the microinjected heparin concentration before stimulation at fixed agonist concentration (Table 2) or held heparin constant and reduced the alpha -thrombin concentration to the minimum necessary to elicit Ca2+ transients (see below and Fig. 5).

                              
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Table 2.   Dose response of heparin blockade of agonist-induced release of intracellular Ca2+ stores


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Fig. 5.   Microinjected heparin fails to inhibit Ca2+ transients even at reduced alpha -thrombin concentration. Before Ca2+ measurements, single CHO-PDGF cells were microinjected with 5 mM fura 2 (A) or fura 2 and 5 mg/ml heparin (B). Twenty to 30 min later, Ca2+i transients were measured in single CHO-PDGF cells after addition of alpha -thrombin (0.1 U/ml) in nominally Ca2+-free medium followed by addition of 2 mM Ca2+. Medium Ca2+ concentration was varied between "0" mM (solid line) and 2 mM (filled bar). Traces are representative of 14-17 experiments under each condition.

As the microinjected heparin concentration was increased from 0.01 to 1.0 mg/ml (injectate concentration), the percentage of cells that produced a Ca2+ response to PDGF (25 ng/ml) declined in a dose-dependent manner. Half-maximal inhibition of Ca2+ release was 0.1-0.3 mg/ml heparin; nearly complete blockade was observed at heparin concentrations >1.0 mg/ml. In contrast, microinjected heparin failed to block alpha -thrombin-induced release of Ca2+i stores at concentrations as high as 80 mg/ml, ~400-fold higher than those producing detectable inhibition of PDGF-induced Ca2+ release. Because the level of IP3 formation by ET-1 was more comparable to that produced by PDGF (Table 1), we examined the dose-response relationship for inhibition of Ca2+ transients by microinjected heparin with ET-1 as the agonist. As for alpha -thrombin, microinjected heparin failed to block ET-1-induced release of Ca2+i stores at concentrations as high as 100 mg/ml, ~500-fold higher than those producing detectable inhibition of PDGF-induced Ca2+ release. The percentage of cells responding to ET-1 in the presence of microinjected heparin (5-100 mg/ml) was not different from the percentage of cells responding after microinjection with fura 2 alone (~90%).

To further assess the inhibition of Ca2+ release by heparin, alpha -thrombin concentration was reduced at a fixed concentration of microinjected heparin. The lowest concentration of alpha -thrombin that elicited reproducible Ca2+ transients in this system (data not shown) was 0.1 U/ml (0.7 nM). At this concentration, alpha -thrombin caused a transient increase in Ca2+i to 839 ± 197 nM (n = 14) in nominally Ca2+-free medium (Fig. 5A). As for PDGF (Fig. 1A), and unlike higher concentrations of alpha -thrombin, this increase was significantly delayed after agonist exposure. After heparin injection (5 mg/ml), 0.1 U/ml alpha -thrombin elicited Ca2+ release transients (772 ± 202 nM, n = 17) that were not different from the transients in control cells microinjected with fura 2 alone (Fig. 5B). The percentage of cells responding to alpha -thrombin (0.1 U/ml) in the presence of microinjected heparin (23 of 29 cells, ~80%) was also not different from the percentage of cells responding after microinjection with fura 2 alone (16 of 19 cells). Similar results were obtained in six cells microinjected with 80 mg/ml heparin (data not shown). Taken together, these data (Tables 1 and 2, Figs. 3 and 5) argue that the failure of heparin to block Ca2+ transients with alpha -thrombin or ET-1 is not due to the competitive nature of heparin's inhibition of the IP3R.

IP3R1 MAb distinguishes between tyrosine kinase and 7-transmembrane receptor-induced release of Ca2+i stores. Because the specificity of heparin for the IP3R may not be complete (23), we examined the effects on Ca2+ metabolism of a microinjected MAb to the mouse IP3R1 (18A10). Although this MAb is specific for IP3R1, this IP3 isoform is apparently always present as one of the subunits that coassemble to form heterotetramers of the IP3R in CHO-K1 cells and hepatocytes (40). In hamster eggs, MAb 18A10 was shown to be a potent inhibitor of Ca2+i release after injection of IP3 (39). In CHO-PDGF and CHO-PDGF/FGF cells, microinjected MAb 18A10 (0.75 mg/ml) completely blocked PDGF-induced release of Ca2+i stores (Fig. 6, A and B; n = 5-10). MAb concentrations as low as 0.125 mg/ml yielded similar inhibition of Ca2+ release (data not shown). Microinjection of nonspecific antibodies (IgG2alpha or a polyclonal antibody to IgG) had no effect on PDGF-mediated release of Ca2+i stores (data not shown).


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Fig. 6.   Effect of microinjected monoclonal antibody (MAb) 18A10 to IP3 type 1 receptor on Ca2+i transients. Before Ca2+i measurements, single cells were microinjected with 5 mM fura 2 and MAb 18A10 after addition of PDGF (25 ng/ml) to single CHO-PDGF cells (A), PDGF (25 ng/ml) to single CHO-PDGF/FGF cells (B), ET-1 (1 nM) to single CHET-B cells (C), or PDGF (25 ng/ml) and alpha -thrombin (1 U/ml) to single CHO-PDGF cells (D). Cells were microinjected with MAb 18A10 at concentrations of 0.75 mg/ml (A, B, and D) or 2.2 mg/ml (C). Medium Ca2+ concentration was varied between "0" mM (solid line) and 2 mM (filled bar). Traces are representative of at least 5-10 experiments under each condition.

In marked contrast, microinjected MAb 18A10 (0.75-2.2 mg/ml injectate concentration) in CHET-B cells failed to block ET-1-induced release of Ca2+i stores. ET-1 raised Ca2+i to 1,573 ± 509 nM in nominally Ca2+-free medium and to 2,036 ± 303 nM after the addition of 2 mM Ca2+ (Fig. 6C; n = 8). Likewise, microinjected MAb 18A10 at similar concentrations failed to block alpha -thrombin-induced release of Ca2+i stores, raising Ca2+i to 414 ± 55 nM in nominally Ca2+-free medium and to 920 ± 145 after the addition of 2 mM Ca2+ (data not shown). To demonstrate that the distinction between tyrosine kinase and 7-transmembrane receptors could be observed in a single cell, a CHO-PDGF cell was sequentially exposed to PDGF and alpha -thrombin after microinjection of MAb 18A10 (0.75 mg/ml injectate concentration). PDGF-induced release of Ca2+i stores was completely blocked, but alpha -thrombin-induced Ca2+ release was not (Fig. 6D). alpha -Thrombin raised Ca2+i to 489 ± 167 nM in nominally Ca2+-free medium and to 967 ± 241 nM on readdition of Ca2+ to the medium (n = 5). Ca2+ entry from the medium was unaffected by MAb 18A10 for either ligand. Thus MAb 18A10, like heparin, distinguishes the Ca2+i responses after activation of tyrosine kinase and 7-transmembrane receptors.

IP3R isoform profile is similar in CHO cells expressing PDGF or ET-1 receptor. To determine whether the failure of microinjected heparin or MAb 18A10 to block Ca2+i release after activation of the ET-1 or alpha -thrombin receptor was due to different IP3R isoform profiles in cells expressing the PDGF (CHO-PDGF) or ET-1 receptor (CHET-B), total cell lysates from CHO-PDGF and CHET-B cells were analyzed by immunoblot with use of MAbs to IP3R1, IP3R2, and IP3R3 (Fig. 7). Densitometry of the relevant bands revealed that the isoform expression was nearly identical in the two cell lines. With the band density for CHO-PDGF cells set arbitrarily at 1.0, the density for CHET-B cells was 0.74, 0.93, and 1.18 for IP3R1, IP3R2, and IP3R3, respectively.


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Fig. 7.   Expression of IP3R subunits (IP3R1, IP3R2, and IP3R3) in CHO-PDGF and CHET-B cells. Twenty micrograms of total cell lysates from CHO-P (CHO-PDGF, lane A) and CHET-B (lane B) cells were analyzed by Western blot with use of MAb 18A10, 4C11, and 10A6 (anti-IP3R1), KM1083 (anti-IP3R2), and KM1082 (anti-IP3R3). Molecular size markers are shown on left (×103). Results are representative of 2 similar experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The role of IP3 as a major second messenger in the release of Ca2+ from intracellular stores was first shown in permeabilized cells (49) and later in intact cells (6, 25, 26). IP3, formed by hydrolysis of phosphatidylinositol 4,5-bisphosphate, is released into the cytoplasm, where it binds to a receptor on Ca2+i-storing organelles (3). After binding, Ca2+ is released from the stores into the cytoplasm (13). Although 7-transmembrane and tyrosine kinase receptors produce IP3 by different mechanisms and with slightly different time courses (41), it has generally been assumed that the IP3 formed by both signaling pathways acts similarly.

Surprisingly, microinjected heparin failed to block release of Ca2+i stores in single CHO cells after activation of the native alpha -thrombin receptor or a transfected ET-1 receptor. These findings with 7-transmembrane receptors contradict earlier studies in which heparin has been shown to block 7-transmembrane receptor-mediated release of Ca2+i stores. Three distinct experimental methods have been used in this earlier work: permeabilized cells after the addition of heparin to the medium (23, 27, 42), cells microinjected with low-molecular-weight heparin (4, 8, 18, 26), and cells internally perfused with heparin during patch-clamp experiments (6, 52). In permeabilized cells, it is possible that alternative signaling molecules to IP3 that are involved in Ca2+ release were not produced or leaked from the cells during the experiment. Loss of such an alternative molecule could then render the Ca2+ release process dependent on IP3.

Using direct microinjection techniques, several investigators found that microinjected heparin blocked Ca2+ release by cholecystokinin (18), bradykinin (26), isoproterenol (8), and tert-butyl hydroperoxide (4). It is notable that, in many of these reports (4, 8, 26), heparin concentrations >100 mg/ml were used for microinjection. This concentration is three orders of magnitude higher than that needed to block Ca2+ release by PDGF in the present study. With the assumption that the injectate volume was 2% of cell volume (see EXPERIMENTAL PROCEDURES), the heparin concentration we observed for half-maximal inhibition of PDGF-induced Ca2+ release (0.2 mg/ml) corresponds to an intracellular concentration of 4 µg/ml, very close to the heparin inhibition constant of 5-15 µg/ml when Ca2+ release was measured in response to IP3 in permeabilized cells (23, 42). This raises the possibility that inhibition of Ca2+ release at high concentrations of heparin may be due to a mechanism other than interference with the IP3 receptor, such as binding of Ca2+ (27). Artifactual blockade of Ca2+ release is a concern in microinjection studies, where cellular damage is possible. We noted that ~10% of cells failed to respond to ET-1 at any dose of heparin (Table 2), possibly because of damage from the microinjection. By performing a dose-response relationship and comparing the findings with the results using PDGF, we were able to show that the apparent inhibition of Ca2+ release in 10% of the cells by heparin was indeed an artifact. We also found that injectate concentrations >100 mg/ml caused intracellular vacuolization and poor Ca2+ responsiveness in general. Finally, it is possible that the mechanisms by which 7-transmembrane receptors release Ca2+i stores vary from system to system. In some systems this process may depend entirely on IP3, whereas in the system we studied there may be additional mechanisms.

One obvious explanation for the failure of heparin (a competitive inhibitor) to block Ca2+i transients in response to 7-transmembrane receptors could be the larger quantities of IP3 that are produced after activation of 7-transmembrane receptors. For alpha -thrombin, the peak level of IP3 formation was nearly sevenfold greater than that after PDGF. On the other hand, the peak IP3 level after ET-1 was similar to that after PDGF. These results are not substantially different from the findings of others who studied IP3 formation after ET-1 and PDGF. ET-1 was shown to increase IP3 by approximately two- to threefold in vascular smooth muscle cells, endothelial cells, and osteoblastic cells, whereas PDGF has been found to increase IP3 formation by approximately twofold in vascular smooth muscle cells (28) and fibroblasts (9). On the other hand, as we found, other investigators observed that alpha -thrombin increased IP3 levels by four- to sixfold (7, 24, 56). Because we were unable to block Ca2+i release after ET-1 with heparin concentrations that were nearly three orders of magnitude higher than those that did block Ca2+i release with PDGF, it seems unlikely that the failure of heparin to block Ca2+i release after activation of ET-1 receptors is simply due to the competitive interaction between IP3 and heparin at the IP3 receptor. Although for alpha -thrombin it also seems unlikely that the high levels of IP3 produced after activation of this receptor can explain our results, we cannot rule out the possibility that the greater IP3 formation after alpha -thrombin was in fact sufficient to overcome the heparin blockade of the IP3 receptor.

A second potential explanation for our findings is that microinjected macromolecules may have differential access to IP3R associated with the two receptor classes. In nasal epithelial cells, apical and basolateral purinergic receptors appear to access distinct Ca2+i stores (43). Using CHO cells, Chun et al. (10) showed that ET-1 receptors are localized to caveolae, which have been proposed to contain isolated signal transduction systems (53). Although some doubt has been cast on this signaling role for caveolae (48), Fujimoto et al. found a Ca2+ pump (15) and an IP3R-like protein (16) in caveolae. Thus it appears likely that some cell types may have isolated pools of Ca2+-signaling molecules that theoretically could be inaccessible to microinjected reagents.

A final possible explanation for our results is that 7-transmembrane receptors could regulate release of Ca2+i stores via an IP3-independent mechanism. Babich et al. (1) demonstrated that parathyroid hormone (PTH) and alpha -thrombin mobilized Ca2+ from intracellular stores but that only alpha -thrombin caused detectable IP3 formation. Seuwen and Boddeke (47) also found that PTH raised Ca2+i but failed to generate IP3 in HEK cells expressing the PTH receptor. Although the production of IP3 in these studies may have been below the detection limit for the assay used, it is notable that internal perfusion of heparin failed to block PTH-mediated release of Ca2+i stores in this system (47). Other receptors, including neuropeptide (36), alpha 2A-adrenergic (33), cholecystokinin analog (46), prostacyclin (55), purinergic (14), glucagon-like peptide (21), and isoproterenol (57), have been found under certain circumstances to cause mobilization of Ca2+i stores in the absence of measurable IP3 formation. Taken together, these data raise the possibility of an IP3-independent mechanism for the mobilization of Ca2+i stores under certain conditions.

A novel second messenger that might be involved in Ca2+i release by 7-transmembrane receptors is cyclic ADP-ribose (cADPR), an NAD+ metabolite. cADPR introduced by direct addition to homogenates or permeabilized cells or by microinjection into intact cells has recently been found to cause Ca2+i release in neurosecretory cells (29), sea urchin eggs (30), and epithelial cells (2). Furthermore, addition of 8-amino-cADPR, a known antagonist of cADPR, to sea urchin egg homogenates blocks cADPR-induced Ca2+i release (31). The role of cADPR in Ca2+i mobilization after activation of 7-transmembrane receptors has not been determined.

The results of this study show that release of Ca2+i stores by PDGF and FGF, the receptors of which belong to the tyrosine kinase family, is blocked by heparin and an MAb to IP3R1. Strikingly, these maneuvers do not block Ca2+ release after exposure to ET-1 or alpha -thrombin, the receptors of which belong to the 7-transmembrane family. For PDGF and ET-1, this difference is not due to different quantities of IP3 released after activation of the receptors or to differences in IP3R isoform profile. The simplest conclusion that can be drawn from these data is that, in the systems we examined, certain 7-transmembrane receptors are capable of activating Ca2+i by a signaling system that differs spatially or chemically from the IP3-signaling system as utilized by tyrosine kinase receptors. Although it seems certain that IP3 plays an important role in Ca2+ release by all the receptors we examined, it may be that an alternative signaling system comes into play under certain physiological conditions. Future work is required to identify the spatial or chemical nature of this alternative Ca2+-signaling system.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant HL-41210 (H. E. Ives) and Pediatric Clinical Research Center Grant M01-RR-01271 (R. S. Mathias), a grant-in-aid from the American Heart Association (H. E. Ives), and the University of California, San Francisco Research Evaluation and Allocation Committee Blair Fund (R. S. Mathias.).

    FOOTNOTES

Address for reprint requests: R. S. Mathias, Dept. of Pediatrics, Children's Renal Center, UCSF Medical Center, 533 Parnassus Ave., San Francisco, CA 94143-0748.

Received 3 July 1997; accepted in final form 24 January 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

1.   Babich, M., H. Choi, R. Johnson, K. King, G. Alford, and R. Nissenson. Thrombin and parathyroid hormone mobilize intracellular calcium in rat osteosarcoma cells by distinct pathways. Endocrinology 129: 1463-1470, 1991[Abstract].

2.   Beers, K. W., E. N. Chini, H. C. Lee, and T. P. Dousa. Cyclic ADP-ribose (cADPr), a metabolite of NAD+, elicits release of intracellular Ca2+ in opossum kidney (OK) cells (Abstract). J. Am. Soc. Nephrol. 4: 482A, 1993.

3.   Berridge, M. J. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993[Medline].

4.   Bird, G. S. J., G. Burgess, and J. Putney, Jr. Sulfhydryl reagents and cAMP-dependent kinase increase sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes. J. Biol. Chem. 268: 17917-17923, 1993[Abstract/Free Full Text].

5.   Bird, G. S. J., J. F. Obie, and J. W. Putney, Jr. Sustained Ca2+ signaling in mouse lacrimal acinar cells due to photolysis of "caged" glycerophosphoryl-myo-inositol 4,5-bisphosphate. J. Biol. Chem. 267: 17722-17725, 1992[Abstract/Free Full Text].

6.   Blatter, L. A., and W. G. Wier. Agonist-induced Ca2+i waves and Ca2+-induced Ca2+ release in mammalian vascular smooth muscle cells. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H576-H586, 1992[Abstract/Free Full Text].

7.   Brass, L., and M. Woolkalis. Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties. Biochem. J. 281: 73-80, 1992[Medline].

8.   Burgess, G., G. S. J. Bird, J. Obie, and J. Putney, Jr. The mechanism for synergism between phospholipase C- and adenyl cyclase-linked hormones in liver. J. Biol. Chem. 266: 4772-4781, 1991[Abstract/Free Full Text].

9.   Catteneo, M. G., L. Magrini, S. B. Sparber, and L. M. Vicentini. Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts. Cell Calcium 13: 603-614, 1992[Medline].

10.   Chun, M., H. Lin, Y. Henis, and H. Lodish. Endothelin-induced endocytosis of cell surface ETA receptors. Endothelin remains intact and bound to the ETA receptor. J. Biol. Chem. 270: 10855-10860, 1995[Abstract/Free Full Text].

11.   Clyman, R. I., K. G. Peters, Y. Q. Chen, J. Escobedo, L. T. Williams, H. E. Ives, and E. Wilson. Phospholipase Cg activation, phosphatidylinositol hydrolysis, and calcium mobilization are not required for FGF receptor-mediated chemotaxis. Cell Adhes. Commun. 1: 333-342, 1994[Medline].

12.   Fantl, W. J., J. A. Escobedo, G. A. Martin, C. W. Turck, M. DelRosario, F. McCormick, and L. T. Williams. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell 69: 413-423, 1992[Medline].

13.   Ferris, C. D., and S. H. Snyder. Inositol 1,4,5-trisphosphate-activated calcium channels. Annu. Rev. Physiol. 54: 469-488, 1992[Medline].

14.   Frelin, C., J. Breittmayer, and P. Vigne. ADP induces inositol phosphate-independent intracellular Ca2+ mobilization in brain capillary endothelial cells. J. Biol. Chem. 268: 8787-8792, 1993[Abstract/Free Full Text].

15.   Fujimoto, T. Calcium pump of the plasma membrane is localized in caveolae. J. Cell Biol. 120: 1147-1157, 1993[Abstract].

16.   Fujimoto, T., S. Nakade, A. Miyawaki, K. Mikoshiba, and K. Ogawa. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J. Cell Biol. 119: 1507-1513, 1992[Abstract].

17.   Furuichi, T., S. Yoshikawa, A. Miyawaki, K. Wada, N. Maeda, and K. Mikoshiba. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature 342: 32-38, 1989[Medline].

18.   Gaisano, H., D. Wong, L. Sheu, and J. Foskett. Calcium release by cholecystokinin analogue OPE is IP3 dependent in single rat pancreatic acinar cells. Am. J. Physiol. 267 (Cell Physiol. 36): C220-C228, 1994[Abstract/Free Full Text].

19.   Ghosh, T. K., P. S. Eis, J. M. Mullaney, C. L. Ebert, and D. L. Gill. Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin. J. Biol. Chem. 263: 11075-11079, 1988[Abstract/Free Full Text].

20.   Graessmann, A., M. Graessmann, and C. Mueller. Microinjection of early SV40 DNA fragments and T antigen. Methods Enzymol. 65: 816-825, 1980[Medline].

21.   Gromada, J., P. Rorsman, S. Dissing, and B. Wulff. Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK cells induces cAMP-dependent activation of calcium-induced calcium release. FEBS Lett. 373: 182-186, 1995[Medline].

22.   Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450, 1985[Abstract].

23.   Guillemette, G., S. Lamontagne, G. Boulay, and B. Mouillac. Differential effects of heparin on inositol 1,4,5-trisphosphate binding, metabolism, and calcium release activity in the bovine adrenal cortex. Mol. Pharmacol. 35: 339-344, 1989[Abstract].

24.   Huang, C.-L., M. G. Cogan, E. J. Cragoe, Jr., and H. E. Ives. Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. J. Biol. Chem. 262: 14134-14140, 1987[Abstract/Free Full Text].

25.   Huang, C.-L., T. Takenawa, and H. E. Ives. Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors. J. Biol. Chem. 266: 4045-4048, 1991[Abstract/Free Full Text].

26.   Hughes, A. R., G. S. J. Bird, J. F. Obie, O. Thastrup, and J. W. Putney. Role of inositol (1,4,5) trisphosphate in epidermal growth factor-induced Ca2+ signaling in A431 cells. Mol. Pharmacol. 40: 254-262, 1991[Abstract].

27.   Kobayashi, S., T. Kitazawa, A. V. Somlyo, and A. P. Somlyo. Cytosolic heparin inhibits muscarinic and alpha -adrenergic Ca2+ release in smooth muscle. J. Biol. Chem. 264: 17997-18004, 1989[Abstract/Free Full Text].

28.   Kondo, T., F. Konishi, H. Inui, and T. Inagami. Differing signal transductions elicited by three isoforms of platelet-derived growth factor in vascular smooth muscle cells. J. Biol. Chem. 268: 4458-4464, 1993[Abstract/Free Full Text].

29.   Koshiyama, H., H. C. Lee, and A. H. Tashjian. Novel mechanism of intracellular calcium release in pituitary cells. J. Biol. Chem. 266: 16985-16988, 1991[Abstract/Free Full Text].

30.   Lee, H. C. Potentiation of calcium- and caffeine-induced calcium release by cyclic ADP-ribose. J. Biol. Chem. 268: 293-299, 1993[Abstract/Free Full Text].

31.   Lee, H. C., R. Aarhus, and T. F. Walseth. Calcium mobilization by dual receptors during fertilization of sea urchin eggs. Science 261: 352-355, 1993[Medline].

32.   Lin, H. Y., E. H. Kaji, G. K. Winkel, H. E. Ives, and H. F. Lodish. Cloning and functional expression of a vascular smooth muscle endothelin-1 receptor. Proc. Natl. Acad. Sci. USA 88: 3185-3189, 1991[Abstract].

33.   Macrez-Lepetre, N., J.-L. Morel, and J. Mironneau. Effects of phospholipase C inhibitors on Ca2+ channel stimulation and Ca2+ release from intracellular stores evoked by alpha 1A- and alpha 2A-adrenoceptors in rat portal vein myocytes. Biochem. Biophys. Res. Commun. 218: 30-34, 1996[Medline].

34.   Marsden, P. A., N. R. Danthuluri, B. M. Brenner, B. J. Ballermann, and T. A. Brock. Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization. Biochem. Biophys. Res. Commun. 158: 86-93, 1989[Medline].

35.   Merrill, A. H., Jr., E. Wang, and R. E. Mullins. Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway. Biochemistry 27: 340-345, 1988[Medline].

36.   Michel, M., F. Feth, and W. Rascher. NPY-stimulated Ca2+ mobilization in SK-N-MC cells is enhanced after isoproterenol treatment. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E383-E388, 1992[Abstract/Free Full Text].

37.   Mikoshiba, K., T. Furuichi, and A. Miyawaki. Structure and function of IP3 receptors. Semin. Cell Biol. 5: 273-281, 1994[Medline].

38.   Mitsuhashi, T., R. C. Morris, Jr., and H. E. Ives. Endothelin-induced increases in vascular smooth muscle Ca2+ do not depend on dihydropyridine-sensitive Ca2+ channels. J. Clin. Invest. 84: 635-639, 1989[Medline].

39.   Miyazaki, S., M. Yuzaki, K. Nakada, H. P. Shirakawa, S. Nakanishi, S. Nakade, and K. Mikoshiba. Block of Ca2+ wave and Ca2+ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs. Science 257: 251-255, 1992[Medline].

40.   Monkawa, T., A. Miyawaki, T. Sugiyama, H. Yoneshima, M. Yamamoto-Hino, T. Furuichi, T. Saruta, M. Hasegawa, and K. Mikoshiba. Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits. J. Biol. Chem. 270: 14700-14704, 1995[Abstract/Free Full Text].

41.   Nanberg, E., and E. Rozengurt. Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin. EMBO J. 7: 2741-2747, 1988[Abstract].

42.   Nilsson, T., J. Zwiller, A. L. Boynton, and P.-O. Berggren. Heparin inhibits IP3-induced Ca2+ release in permeabilized B-cells. FEBS Lett. 229: 211-214, 1988[Medline].

43.   Paradiso, A. M., S. J. Mason, E. R. Lazarowski, and R. C. Boucher. Membrane-restricted regulation of Ca2+ release and influx in polarized epithelia. Nature 377: 643-646, 1995[Medline].

44.   Peters, K. G., J. Marie, E. Wilson, H. E. Ives, J. Escobedo, M. DelRosario, D. Mirda, and L. T. Williams. An FGF receptor point mutation that abolishes PI turnover and Ca2+ mobilization but not mitogenesis. Nature 358: 678-681, 1992[Medline].

45.   Ross, C. A., S. K. Danoff, M. J. Schell, S. H. Snyder, and A. Ullrich. Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues. Proc. Natl. Acad. Sci. USA 89: 4265-4269, 1992[Abstract].

46.   Saluja, A. K., R. K. Dawra, M. M. Lerch, and M. L. Steer. CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini. J. Biol. Chem. 267: 11202-11207, 1992[Abstract/Free Full Text].

47.   Seuwen, K., and G. W. M. Boddeke. Heparin-insensitive calcium release from intracellular stores triggered by the recombinant human parathyroid hormone receptor. Br. J. Pharmacol. 114: 1613-1620, 1995[Abstract].

48.   Stan, R.-V., W. G. Roberts, D. Predescu, K. Ihida, L. Saucan, L. Ghitescu, and G. E. Palade. Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae). Mol. Biol. Cell 8: 595-605, 1997[Abstract].

49.   Streb, H., R. F. Irvine, M. J. Berridge, and I. Schulz. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 306: 67-69, 1983[Medline].

50.   Tatrai, A., P. Lakatos, S. Thompson, and P. H. Stern. Effects of endothelin-1 on signal transduction in UMR-106 osteoblastic cells. J. Bone Miner. Metab. 7: 1201-1209, 1992.

51.   Taylor, S. J., H. Z. Chae, S. G. Rhee, and J. H. Exton. Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. Nature 350: 516-518, 1991[Medline].

52.   Thorn, P., and O. Peterson. Calcium oscillations in pancreatic acinar cells, evoked by the cholecystokinin analogue JMV-180, depend on functional inositol 1,4,5-trisphosphate receptors. J. Biol. Chem. 268: 23219-23221, 1993[Abstract/Free Full Text].

53.   Travis, J. Cell biologists explore "tiny caves." Science 262: 1208-1209, 1993[Medline].

54.   Ullrich, A., and J. Schlessinger. Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203-212, 1990[Medline].

55.   Vassaux, G., D. Gaillard, G. Ailhaud, and R. Negrel. Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP and Ca2+-elevating agent. J. Biol. Chem. 267: 11092-11097, 1992[Abstract/Free Full Text].

56.   Vittet, D., M.-N. Mathieu, J.-M. Launay, and C. Chevillard. Thrombin inhibits proliferation of the human megakaryoblastic MEG-01 cell line: a possible involvement of a cyclic AMP-dependent mechanism. J. Cell. Physiol. 150: 65-75, 1992[Medline].

57.   Wayman, G. A., T. R. Hinds, and D. R. Storm. Hormone stimulation of type III adenylyl cyclase induces Ca2+ oscillations in HEK-293 cells. J. Biol. Chem. 270: 24108-24115, 1995[Abstract/Free Full Text].

58.   Wojcikiewicz, R. Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J. Biol. Chem. 270: 11678-11683, 1995[Abstract/Free Full Text].


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