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
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ABSTRACT |
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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 -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
-thrombin and
ET-1. After application of ligand, IP3 levels were five- to sevenfold
higher for
-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
-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
-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
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INTRODUCTION |
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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 -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.
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EXPERIMENTAL PROCEDURES |
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Materials.
Unless otherwise specified, all chemicals were purchased from Sigma
Chemical, including ET-1 (E-9262), low-molecular-weight heparin
(H-5271), MAb to IgG2 (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
-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 × 1014 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.
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RESULTS |
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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 × 1014 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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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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Heparin does not block release of
Ca2+i stores
after activation by ET-1 and -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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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 -thrombin (Table 1).
Indeed, with
-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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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 -thrombin concentration to the minimum necessary to elicit Ca2+
transients (see below and Fig. 5).
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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 (IgG2 or a
polyclonal antibody to IgG) had no effect on PDGF-mediated release of
Ca2+i stores (data not shown).
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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 -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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DISCUSSION |
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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 -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 -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
-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
-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
-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 -thrombin mobilized Ca2+ from intracellular stores but
that only
-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),
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 -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.
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ACKNOWLEDGEMENTS |
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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.).
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FOOTNOTES |
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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.
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