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Endocrinology Vol. 142, No. 5 1968-1974
Copyright © 2001 by The Endocrine Society

ARTICLES

Receptor-Operated Osteoclast Calcium Sensing1

Brian D. Bennett, Ulises Alvarez and Keith A. Hruska

Renal Division (B.D.B., U.A., K.A.H.), Departments of Medicine and Cell Biology, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Brian Bennett, Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, 216 South Kingshighway, Mailstop #90–32-648, St. Louis, Missouri 63110. E-mail: bbennett{at}im.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclasts "sense" elevated extracellular calcium, which leads to cytoskeletal changes that may be linked to phospholipase C (PLC) activation and the associated rise in intracellular calcium ([Ca2+]i). Since PLC is linked to transient receptor potential channels (trp), we hypothesized that receptor activated calcium influx due to this channel type would be activated by osteoclasts sensing [Ca2+]e. We found that high [Ca2+]e induced similar intracellular Ca2+ rises in chicken osteoclasts with or without intracellular Ca2+ store depletion by either TPEN or thapsigargin, thus defining store-insensitive Ca2+ influx. This store-insensitive calcium sensing component was blocked by the PLC antagonist U73122. Also, the calcium channel inhibitor SKF 96365, a blocker of store-independent trp-like channels, was effective in inhibiting calcium sensing in the presence of thapsigargin. Thus, a store-independent component of calcium sensing was associated with ion channels linked to PLC. Since receptor activated transient receptor potential (trp) family cation channels open in a PLC-dependent and store-independent manner, we suggest that receptor operated channels are activated in osteoclasts stimulated by high extracellular Ca2+.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCLASTS function to support calcium homeostasis and to remodel bone. During bone resorption, osteoclasts generate very high extracellular Ca2+ concentrations ([Ca2+]e) in the resorption space (1). High [Ca2+]e induces diverse responses in osteoclasts (OCs), including a rise in intracellular Ca2+ ([Ca2+]i), IL-6 secretion (2) and dissipation of sealing zone cytoskeletal assemblies required for resorption (3). Secretion and regulation of the cytoskeleton are two of the many intracellular functions affected by intracellular Ca2+. Such diverse function requires keeping different Ca2+ signaling pools separate via tight spatial and temporal regulation (4). Therefore, finding discrete intracellular Ca2+ pools that are increased by elevating [Ca2+]e is of great interest, especially in [Ca2+]e-producing cells like osteoclasts.

Phospholipase C may mediate [Ca2+]e-induced effects in OCs, because [Ca2+]e increases production of inositol 1,4,5-triphosphate (IP3) in GCT23 osteoclast-like cells (5) and chicken OCs (Miyauchi, A., and K. A. Hruska, unpublished data). IP3 activates receptor linked PLC mediated Ca2+ signaling by binding IP3 receptors, which release Ca2+ from internal stores. Evidence exists for [Ca2+]e-induced release of Ca2+ from intracellular stores in OCs, beginning with studies showing marked reduction of [Ca2+]e-induced [Ca2+]i rises by the release inhibitor TMB-8 (6). Release via IP3 receptors is often accompanied by stimulation of other intracellular channels such as the ryanodine receptor, which may explain why the ryanodine receptor (RYR) inhibitor dantrolene reduces [Ca2+]e-induced [Ca2+]i rises in chicken OCs (3). PLC mediated release of intracellular stores initiates refilling of stores by store-operated influx through transient receptor potential (trp) channels (7, 8, 9). Receptor driven release through either the IP3 receptor or the RYR initiates store-operated influx through trp channels putatively via direct coupling of these intracellular release channels to the plasma membrane influx channel (9, 10). However, trp channels also mediate receptor operated influx that is not dependent on thapsigargin sensitive stores (11, 12, 13). Trp3 can operate in a store-sensitive or store-insensitive mode (14) and trp6 is regulated by DAG or DAG metabolites after emptying internal stores (12). Also, receptor operated trp4 and trp5 channels are activated by agonist in a store insensitive manner (13). Preliminary results in OCs derived from the RAW 264.7 rodent macrophage cell line showed expression of several trp homologs including trp3, trp4 and trp6 (15), leading us to ask if osteoclast calcium sensing requires filled stores. The data presented here demonstrate that Ca2+ entry into chicken osteoclasts stimulated by high [Ca2+]e did not require release of store contents, occurred while stores remained empty, and maintained PLC dependency, all hallmarks of store-independent influx supported by trp channels. Thus, PLC activated intracellular Ca2+ store independent channels are a mechanism of extracellular Ca2+ sensing by osteoclasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and solutions
Perfusion buffer was a modified HEPES buffered Kreb Ringer solution (KRB-HEPES) containing, 130 mM NaCl, 5.0 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 17 mM HEPES, pH 7.2, 300 mosmol/kg. Calcium additions were isotonic with NaCl. Calcium free buffer was KRB-HEPES without added calcium plus 4 mM EGTA (Sigma, St. Louis, MO). N,N,N',N'-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN) was purchased from Calbiochem (San Diego, CA). TPEN and SKF-96363 were dissolved in KRB-HEPES buffer. U73122 and thapsigargin solutions were 2000x in DMSO. 0.05% DMSO had no effect on [Ca2+]e-induced [Ca2+]i changes.

Preparation of avian osteoclasts
Avian osteoclasts were prepared as described previously in accordance with protocols approved by the Animal Studies Committee of the Washington University School of Medicine (16). Briefly, osteoclast precursors were isolated from bone marrow of egg-laying hens maintained on Ca2+-deficient diets. Partially purified preparations of mononuclear cells were recovered from the interface of Ficoll/Hypaque gradients. Nonadherent cells were separated from the adherent population after 18–24 h in culture. The nonadherent cells were sedimented, resuspended in fresh medium, and plated on 25-mm glass coverslips in six-well plates at 5 x 105/well. After culture in the presence of cytosine arabinoside (5 µg/ml), multinucleated osteoclast giant cells (>100 µm diameter) formed between 3 and 6 days in culture. Ninety-five percent of the cells in the preparation were tartrate-resistant acid phosphatase-positive cells, of which 90% were multinucleated giant cell osteoclasts.

[Ca2+]i imaging and analysis
Cells plated on circular coverslips were rinsed and incubated with 2 mM Fura2/AM and 0.01% Pluronic-127 (Molecular Probes, Inc., Eugene, OR) in KRB-HEPES, 130 mM NaCl, 5.0 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 17 mM HEPES at 25 C for 1 h. After one change in KRB-HEPES, the coverslip was transferred to a PDMI-2 thermostated perifusion chamber (Medical Systems Corp., Greenvale, NY) mounted on a Carl Zeiss Axiovert 35 microscope (Carl Zeiss Inc., Thornwood, NY). Imaging was performed using an IM-4000 system (Georgia Instruments, Roswell, GA). Fura-2 ratio images were acquired using excitation wavelengths of 340 and 380 nm. [Ca2+]i in individual cells was monitored during 37 C perifusion at 1 ml/min in either a 0.5 ml (for TPEN experiments) or 1 ml chamber. FURA-2 was calibrated by the standard two-point method (17). Individual Ca2+ dynamics from osteoclasts were taken across three separate osteoclast preparations used on the same day after initiation of differentiation. When monocytes were compared with osteoclasts they were paired within each of three separate preparations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To relate internal Ca2+ stores to [Ca2+]e-induced [Ca2+]i elevation in chicken osteoclasts, we depleted stores with thapsigargin before increasing [Ca2+]e. Thapsigargin (THG) blocks the sarco-endoplasmic reticulum calcium ATPase pump (SERCA) located in the membrane of the endoplasmic reticulum (ER) and other intracellular vesicular store compartments. Inhibiting Ca2+ re-uptake of Ca2+ into the ER with THG breaks the cycle of Ca2+ uptake and re-release through ER imbedded Ca2+ release channels (18). Chicken osteoclasts were perifused with 1 mM [Ca2+]e buffer before addition of THG (Fig. 1Go). In the giant cell OC shown, THG induced the expected transient rise in [Ca2+]i due to passive Ca2+ extrusion from the ER. Next [Ca2+]e was taken from 1 mM (Ca1) to 20 mM (Ca20), a change that induces IP3 production in OCs (5) and activates heterologously expressed calcium sensing receptors (19). Ca20 evoked a significant increase in [Ca2+]i in this osteoclast even after Ca2+ uptake into the ER was inhibited. Similar Ca20 evoked responses were seen in 30 osteoclasts across multiple preparations. However, the THG-induced Ca2+ transient occurred in only 5 of these 30 osteoclasts, suggesting that THG would not normally cause passive depletion of Ca2+ stores in our OCs. Ionomycin was not able to induce Ca2+ release from stores when THG was added first (Fig. 1BGo). However, without THG pretreatment ionomycin caused a significant [Ca2+]i rise (Fig. 1CGo). Thus, ionomycin reveals the THG block on SERCA in our OCs. The effect of THG on ionomycin associated stores was not seen unless the calcium-free incubation period was short, suggesting again that the OC stores were prone to depletion. The data suggest that [Ca2+]i rises linked to [Ca2+]e contain a component that does not require filled thapsigargin-sensitive Ca2+ stores and concur with previous observations, which also suggest that influx is an important component of osteoclast calcium sensing (3, 5, 20). Further, since Ca2+ store-operated influx (SOI) is intimately linked to intracellular stores and release from them, it appears that the osteoclast calcium sensing seen above bypasses SOI regulation.



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  		Figure 1. Osteoclast calcium sensing after store depletion. Cytosolic free calcium was measured by FURA-2 imaging in avian osteoclast giant cells. Thapsigargin (1 µM) was added to induce release of Ca2+ stores, whereas osteoclasts were bathed in 1 mM extracellular calcium. After release was complete, 20 mM extracellular calcium was added (A; n = 8). Ionomycin (2 µM) was added to giant cell OCs after switching to calcium free buffer (Ca0) containing thapsigargin (B; 1 µM, n = 4). Ionomycin did not induce release of intracellular stores with thapsigargin pretreatment, but was effective with Ca0 alone (C; n = 4).

 
Because THG activates store-operated channels (9), a possible path for a direct mode of influx exists that could generate calcium sensing independent of PLC signaling. For example, SOI is amplified by [Ca2+]e in T lymphocytes by direct interaction with PM calcium channels (21). Therefore, we determined the [Ca2+]i changes in cells with active SOI, but inactive Ca2+ signaling associated with IP3 to see if SOI could support calcium sensing levels seen in giant cell OCs. We found the needed cells adjacent to giant cells. Mononuclear cells, most likely unfused osteoclast precursors, were scattered around giant cells, making immediate comparisons possible. Mononuclear cells did not respond to Ca20, whereas osteoclasts did (Fig. 2Go, left). Also, repeated Ca20 volleys gave the same result seen for OCs in Fig. 2Go. The results demonstrate that mononuclear cells do not sense calcium, and thus, PLC catalyzed IP3 production in response to [Ca2+]e is likely limited. To activate SOI in these monocytes in a PLC independent manner we depleted stores with TPEN. TPEN activates SOI in RBL cells by selectively chelating high Ca2+ stored in internal calcium stores (22). In Fig. 2Go, cells were challenged with Ca20 again after treatment with 1 mM TPEN in calcium free buffer (Fig. 2Go, right). After TPEN treatment mononuclear cells now responded to Ca20. In contrast, TPEN treatment did not affect the Ca20-induced [Ca2+]i rise in giant cells. For giant cells, this again demonstrates the store-independence of osteoclast calcium sensing as shown by the THG result. However, monocytes did respond to TPEN with an increase in influx when Ca20 was readded, providing an internal control for TPEN-induced SOI. That TPEN evoked similar SOI in both monocytes and giant cells was demonstrated by readding Ca1 rather than Ca20. After TPEN treatment, Ca1 readdition led to the same increase in [Ca2+]i in OCs and monocytes (Fig. 3AGo). Also, the increased influx was TPEN specific since Ca0 alone did not elicit the large increase in [Ca2+]i upon Ca1 readdition (Fig. 3BGo).



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  		Figure 2. TPEN activation of SOI promotes calcium sensing in mononuclear cells but not osteoclasts. Ca20 addition to mononuclear cells (filled circles) had no effect on [Ca2+]i until 1 mM TPEN was added first (n = 8/8). In osteoclasts (open circles), TPEN had no effect on calcium sensing (n = 6/6).

 


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  		Figure 3. TPEN activates similar SOI levels in monocytes and osteoclasts. Osteoclasts (open circles) and monocytes (closed circles) were exposed to 1 mM TPEN in calcium free buffer then Ca1 was readded (A; n = 6 each). Ca1 raised [Ca2+]i by similar amounts. Also, OCs were treated with Ca0 alone (B; filled squares, n = 4) or Ca0 plus 1 mM TPEN (B, line trace, n = 4). Though Ca0 elicited some influx, TPEN evoked a much larger increase in [Ca2+]i upon Ca1 readdition.

 
Because mononuclear cells did not truly sense [Ca2+]e, but support SOI due to TPEN induction, the effect of [Ca2+]e on [Ca2+]i linked solely to SOI could be determined. Figure 4AGo demonstrates that, after TPEN treatment, changing the [Ca2+]e from Ca0 to Ca1 raised [Ca2+]i by 130 nM in mononuclear cells, whereas a 160 nM increase was seen after Ca20. Ca1 raised [Ca2+]i by the same amount in osteoclasts as in mononuclear cells. However, Ca20 increased [Ca2+]i by 400 nM in osteoclasts after TPEN treatment. Quantitative analysis (Fig. 4BGo) shows that the [Ca2+]i rise due to SOI alone, as represented by monocytes, was rather shallow. However, osteoclasts gave a much steeper response. The data demonstrate that a calcium sensing effector must be activated to increase influx; calcium activation of SOI alone is not enough.



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  		Figure 4. Calcium sensing dose response during CCE activation. [Ca2+]i responses to Ca1 and Ca20 were determined independently, each after prior exposure to 1 mM TPEN (A) in giant cell osteoclasts (filled circles) and mononuclear cells (filled squares). Peak Ca2+ elevations were then quantitated (B). Only osteoclasts sensing Ca2+ afford increased [Ca2+]i significantly greater than that seen in monocytes (P < 0.01) n = 10 mononuclear and 9 giant cells.

 
Because store-independent influx is often receptor activated and linked to PLC (11, 23) we determined the effect of PLC inhibition by U73122 on osteoclast calcium sensing. Ca1 increased [Ca2+]i by similar amounts (75 nM) in control osteoclasts (Fig. 5AGo), osteoclasts exposed to THG (Fig. 5BGo), and osteoclasts treated with U73122 in addition to THG (Fig. 5CGo). Thus the increase in [Ca2+]i caused by basal extracellular calcium (Ca1) was unaffected by store depletion or PLC inhibition. Also, the [Ca2+]i increase due to Ca20 was unaffected by THG (Fig. 5BGo), thus confirming store-insensitive calcium sensing. However, the store-insensitive influx due to Ca20 was blocked by 10 µM U73122 (Fig. 5CGo). In Fig. 6Go, quantitative analysis shows that THG alone did not significantly reduce calcium sensing due to Ca20, but addition of U73122 at 1 µM or 10 µM produced significant inhibition. Because store-insensitive PLC dependent influx is a functional hallmark of trp channel-mediated receptor operated influx, we tested the effects of SKF-96365, a known blocker of receptor operated trp3 channel activity (24). Figure 7Go demonstrates that store depleted osteoclasts do not respond to Ca20 in the presence of 50 µM SKF-96365.



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  		Figure 5. Thapsigargin-insensitive PLC dependent calcium sensing. [Ca2+]i rises in OCs were measured as extracellular calcium was taken from Ca0 to Ca1 and then Ca20 (A; n = 4). This change in dose was repeated for OCs pretreated with 1 µM thapsigargin (B; n = 4) and thapsigargin plus 10 µM U-73122 (C; n = 9). Only U73122, not thapsigargin, decreased the rise in [Ca2+]i caused by Ca20. The Ca1-mediated change was not affected.

 


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  		Figure 6. Quantitation of THG and U73122 effects on calcium sensing. Calcium sensing is expressed as a response ratio ([Ca2+]i rise induced by Ca20/[Ca2+]i increase by Ca1). THG (1 µM) did not significantly change the [Ca2+]i response due to Ca20. However, when U73122 was added at 1 µM or 10 µM, the response was significantly reduced compared with THG alone. (*, P < 0.02 for THG vs. THG + 1 µM U73122; **, P < 0.01 for THG vs. THG + 10 µM U73122).

 


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  		Figure 7. SKF-96365 inhibition of release independent influx. SKF-96365 (50 µM) was added along with 1 µM THG to osteoclasts. [Ca2+]e was raised from Ca0 to Ca1 and then Ca20 (n = 8).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data demonstrate that osteoclasts recognize elevations ("sense") in extracellular calcium and increase cytosolic Ca2+ by a PLC dependent intracellular Ca2+ store independent influx of Ca2+ across the plasma membrane. The probable mechanism of the Ca2+ influx is a conductance through a transient receptor potential channel (trp). We used two separate methods to block store-operated Ca2+ influx pathways that operate through the cycle of store uptake of Ca2+, release, and influx activation.

Store depletion by TPEN is known to initiate SOI in RBL-1 cells (22). Thus, TPEN "preactivates" store-operated channels, which leads to enhanced influx of Ca2+. Increasing [Ca2+]e to high levels associated with calcium sensing should lead to increased flux through "preactivated" SOI channels, while flux through channels activated simultaneously by store independent means would add to the influx through preactivated SOI. The change in [Ca2+]i due to influx of 1 mM [Ca2+]e after TPEN addition was similar for giant cell OCs and mononuclear cells. Therefore, TPEN appeared to activate similar levels of SOI in each cell type. However, the effects of 20 mM [Ca2+]e were quite different for giant cells compared with mononuclear monocytes. With TPEN-activated SOI present, 20 mM [Ca2+]e produced about the same [Ca2+]i rise as 1 mM [Ca2+]e in monocytes. In contrast, 20 mM [Ca2+]e caused a much greater response than 1 mM [Ca2+]e in giant cell osteoclasts. The monocyte data suggests that influx follows increased [Ca2+]e monotonically when SOI is preactivated, thus changing [Ca2+]i by increased mass action. The much higher level of giant cell response to 20 mM [Ca2+]e under SOI activating conditions are not easily explained by mass action. Though SOI contributes to some influx of 20 mM [Ca2+]e under SOI preactivation conditions, it cannot account for the large [Ca2+]i changes seen in giant cells. Most likely, 20 mM [Ca2+]e produces some form of signaling. We suggest PLC linkage to cation channels enhances the influx over and above that possible by SOI alone because high [Ca2+]e activates PLC in OCs (5) and PLC is linked to such influx activation (25). That monocytes give a muted response to Ca20 during SOI activation conditions compared with giant cells may reflect the lack of intracellular signal generation in monocytes, which is consistent with the monocyte insensitivity to Ca20 seen without TPEN pretreatment.

We also related SERCA store pumps to OC calcium sensing by blocking SERCA with thapsigargin. In Ca1, SERCA inhibition with THG increased [Ca2+]i in only 20% of the giant cell OC population, indicating that SERCA stores are prone to depletion in giant cells. However, ionomycin released Ca2+ from intracellular stores when OCs were bathed in zero calcium buffer, and this ionomycin-sensitive pool was not released when THG was added first. Thus, SERCA associated Ca2+ stores in ER and Golgi lumen were affected by THG. The difficulty detecting passive Ca2+ efflux from THG sensitive stores may arise due to slow efflux or close proximity of SERCA in the ER with PM CaATPase efflux pumps or rapid basal release that may leave SERCA stores relatively empty. An alternative, in which OCs express THG resistant SERCA pumps seems unlikely, because all known SERCA isoforms are THG sensitive, and ionomycin-linked pools were emptied by THG. The latter concept originally led us to a rationale where basal store depletion would engender chicken giant cell OCs with "constitutively" active store-operated influx through which elevated [Ca2+]e could flow and promote calcium sensing. This idea was influenced by studies showing that cells cloned during prolonged thapsigargin treatment have constitutively open Ca2+ entry channels coupled to empty or partially emptied thapsigargin-sensitive Ca2+ stores (26). However, we showed here that store-operated influx "constitutively" activated by TPEN cannot account for the bulk of OC calcium sensing. Rather, Ca20 produces similarly large increases in [Ca2+]i with or without interference from THG or TPEN. Combined, the data from THG/TPEN store perturbation methods reveal a calcium sensing component that does not require the normal operation of filling and maintenance of high calcium in intracellular stores.

Because PLC is activated by [Ca2+]e in OCs, a variety of PLC linked influx pathways may be involved in calcium sensing. For example, Falsolato et al. (27) have shown that receptors operating through G{alpha}q and PLC concurrently mediate both store and store-independent influx pathways. These original functional data have received considerable molecular description by the activities attributed to the trp family of ion channels. For example, several trp homologous support store and store-insensitive receptor-mediated influx. Often the same trp homolog is store sensitive in one heterologous expression system, but store-insensitive in another (28). The rules of trp regulation are currently unfolding. Because OCs derived from mouse RAW 264.7 macrophages express several trp homologs that support store-insensitive influx, we took clues from receptor-operated trp3. trp3 supports store-independent, PLC-dependent, and SKF-96363-sensitive influx (24). We suggest that PLC activation by our OCs sensing high calcium increases [Ca2+]i by both store and store-independent influx. The modes of store perturbation used here preactivate SOI so that concurrent store-independent influx adds to SOI much like that seen for store-independent trp3 mediated influx (24).

The PLC dependency we see in chicken OC calcium sensing suggest the possible involvement of a plasma membrane delimited calcium sensing receptor, such as the parathyroid (pCaR). However, OCs do not respond to the CaR agonist ion, Gd3+, suggesting that the pCaR is not functional in OCs. This is consistent with the lack of pCaR expression in osteoclast like GCT23cells (5). But, other reports show expression of the pCaR in osteoclasts and osteoclast precursors (29, 30). Added complexity arises from data showing that osteoblasts from CaR knock-out mice respond to high [Ca2+]e and Gd3+ (31). If involved in calcium sensing, such a receptor would likely elicit signaling through Gaq and PLCß (32). Here, we provide more evidence that PLC is involved in OC calcium sensing and look to modes of [Ca2+]i regulation known to be mediated by the GPCR-PLC couple.

Calcium signals mediated by receptor activation are often modified by RYR channels. [Ca2+]e-induced RYR channel activation in OCs was originally indicated by sensitivity to dantrolene (14). Kiselyov et al. 2000 (10) have demonstrated functional and physical coupling of trp3 to ryanodine receptors, suggesting that the PM channel-ER-RYR structure of muscle cells is also present in nonexcitatble cells. Also, synthetic catalysis of cyclic ADP-ribose, the RYR agonist, by CD38 appears to be activated downstream of GPCRs because the first phase of the [Ca2+]i rise due to acetylcholine-induced muscarinic receptor activation is muted by CD38 knockout (33). Therefore, a complex relationship between trp, RYR, and G protein-coupled receptors can be envisioned. However, in OCs special consideration of the RYR is required, because the RYR-type2 isoform (RYR2) is expressed in OCs (34). Though the bulk of RYR2 localizes throughout the OC in other nonexcitable cells, the peripheral orientation seen in OCs may impart a topology that puts a low affinity Ca2+ binding site in the extracellular space where it could sense high [Ca2+]e. Such a PM topography has was also suggested for the IP3-receptor intracellular release channel in an early proposal for IP3R-mediated SOI. Ion conductances for neither IP3R or RYR for ions flowing from the extracellular space to the cytosol have been demonstrated, indicating that functional pools are relegated to intracellular membrane delimited stores. Further support for an RYR-mediated calcium sensing component comes from the increase in [Ca2+]i caused by extracellular application of the potent RYR agonist cADP-ribose (35) and by OC expression of CD38 (36), the ubiquitious enzyme that catalyzes synthesis of cADP-ribose (37). cADP-ribose is cell impermeant and usually requires cell permeabilization to effectively activate the cytoplasmic binding domain of RYRs. However, cADP-ribose has also been shown to be transported to the cytosol where it can diffuse to its binding domain on the RYRs (38). Possibly uptake of cADP-ribose by OCs could explain its effects on OC calcium signaling. Overall, OC calcium sensing appears to be mediated by effectors normally associated with receptor activation, but specific molecular approaches are lacking to date.

In summary, the published literature before this report indicated that OC Ca sensing is related to a Ca sensor/receptor induced release of intracellular stores and Ca2+ entry through undefined pathways (3, 5, 15). The data reported here are the first to characterize a Ca2+ sensor activated influx as Ca2+-store independent, activities supported by various trp receptor-activated channels. We propose that the calcium sensing receptor of the OC activates PLCß and releases Ca2+ stores through the action of IP3 and concurrently stimulates Ca2+ entry even when stores are empty, through the action of Ca2+ store independent trp channels.


   Acknowledgments
 
We thank Dr. Thomas Steinberg (Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, MO) for the use of the calcium imaging system and helpful advice; and Kathy Jones and Helen Odle for administrative assistance.


   Footnotes
 
1 This work was supported by NIH Grant DK-49728–05. Back

Received May 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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