1 Department of Oral Cell Biology, ACTA-Vrije Universiteit, 1081 BT Amsterdam, The Netherlands; and 2 Department of Molecular Cell Biology, Leiden University Medical Centre, 2301 CD Leiden, The Netherlands
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ABSTRACT |
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To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E2 (PGE2). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF; 0.7 ± 0.03 Pa at 5 Hz), and PGE2 release was measured. Blockers of Ca2+ entry into the cell or Ca2+ release from internal stores markedly inhibited the PFF-induced PGE2 release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca2+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE2 release. These results are consistent with the hypothesis that PFF raises intracellular Ca2+ by an enhanced entry through mechanosensitive ion channels in combination with Ca2+- and inositol trisphosphate (the product of phospholipase C)-induced Ca2+ release from intracellular stores. Ca2+ and protein kinase C then stimulate phospholipase A2 activity, arachidonic acid production, and ultimately PGE2 release.
osteocytes; mechanotransduction; prostaglandin E2
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INTRODUCTION |
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BONE TISSUE responds to mechanical stress with adaptive changes in mass and structure. Increases in bone loading lead to increased bone mass (18), whereas reductions in loading, as in bed rest or weightlessness, are associated with bone loss (28, 55). These studies have established that mechanical forces are essential for the maintenance of the structural integrity of bone tissue. However, little is known about the cellular and molecular mechanisms whereby mechanical stimuli are translated into changes in bone structure and mass. Osteocytes, the predominant cells of adult bone tissue, are generally believed to act as the mechanosensors in this process (see current review in Ref. 8). The question remains how they are mechanically activated. An attractive and plausible hypothesis is that loading of bone tissue causes flow of interstitial fluid in the lacuno-canalicular network that interconnects osteocytes and bone lining cells, and that this flow provides the stress-derived mechanical signal for the osteocytes (56, 59). We have shown earlier that osteocytes, and to a lesser extent osteoblasts, respond to fluid flow with increased production of signaling molecules, such as prostaglandins (3, 26) and nitric oxide (25). The importance of prostaglandins in the mechanoregulation of bone is stressed by the in vivo findings of Forwood et al. (15) and Chow and Chambers (9) showing that mechanical loading-induced bone formation could be inhibited by the administration of cyclooxygenase inhibitors.
Although the mechanism for the initial detection and the conversion of mechanical force into a biochemical signal has yet to be determined, several candidate mechanotransducers have been proposed. These include the integrin-cytoskeleton structure (23), mechanosensitive cation channels within the cell membrane (26), G protein-dependent pathways (5, 47), phospholipase C (PLC) (7), and protein kinase C (PKC) (46). Several of these cellular structures have been shown to be involved in the fluid shear stress-induced prostaglandin (PG) production. We have shown earlier that the fluid flow-induced prostaglandin E2 (PGE2) production by chicken osteocytes was abolished after the actin cytoskeleton had been disrupted with cytochalasin B, suggesting that this flow-induced prostaglandin response was mediated by the cytoskeleton (3). Others have shown that PKC mediates the fluid flow-induced PGE2 response in osteoblasts (46). Fluid flow has also been reported to stimulate the production of inositol trisphosphate (IP3) through the action of PLC (37). Because IP3 is known to induce Ca2+ elevations by mobilizing Ca2+ from intracellular stores (4), intracellular Ca2+ increase may act as a secondary response. This is supported by several studies showing that fluid flow induced transient increases in intracellular Ca2+ in osteoblasts (20, 21). Both intracellular Ca2+ stores and extracellular Ca2+ were found to be required for the increases in intracellular Ca2+ (20).
Studies in endothelial cells have suggested a close relationship between a rise in intracellular Ca2+ and PG production. Prostacyclin release in response to thrombin or Ca2+ ionophores is decreased by agents such as TMB-8, which can inhibit mobilization of intracellular Ca2+ (19). Furthermore, when endothelial cells were subjected to fluid flow in the presence of an intracellular calcium chelator, there was a markedly reduced PG response compared with cells that had not been pretreated with this chelator (6). It is therefore likely that Ca2+ functions as a second messenger mediating the flow-induced PG response.
The elevation of intracellular Ca2+ may result from IP3-induced Ca2+ release from intracellular stores or may result from the entrance of Ca2+ via membrane-associated, mechanosensitive ion channels. These mechanosensitive channels have been found on a variety of cells, including osteoblast-like cells (11), and are prime candidates for mechanotransduction. The involvement of these channels in the PG response of osteocytes to pulsating fluid flow (PFF) is, therefore, an attractive hypothesis.
In this study, we subjected isolated chicken osteocytes to 10 min of fluid flow and then determined PGE2. The experiments were carried out in the presence of various specific blockers acting at various cellular structures that were possibly involved in the induction of PG production. Specifically, we addressed the involvement of phospholipase A2 (PLA2), PLC, and PKC in the fluid flow-induced PGE2 response. We also studied the involvement of the cytoskeleton, mechanosensitive channels, and the possible role of intracellular Ca2+ concentration in the fluid flow-induced PG synthesis.
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MATERIALS AND METHODS |
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Isolation and Culture of Chicken Osteocytes
Fetal chicken calvarial cells were obtained as previously described (35). Briefly, calvariae of 18-day-old chicken fetuses were aseptically dissected, and the periostea were removed. A mixture of osteoblasts and osteocytes was then isolated by sequential collagenase-EDTA digestion (34, 58). The cell fractions were pooled and precultured for 1 day inPFF
PFF was generated by flow apparatus containing a parallel-plate flow chamber, as described earlier (3, 16). The apparatus contained 15 ml of medium that was pumped over the cells in a pulsatile manner by a revolving pump. This resulted in a pulsating (5 Hz) fluid shear stress of 0.7 ± 0.03 Pa and an estimated peak stress rate of 12.2 Pa/s. A glass slide on which 2.5 × 104 OCY were seeded served as the bottom of the parallel-plate flow chamber (16). During an experiment the apparatus was placed in a 37°C incubator. The flow medium, also used in the stationary controls, consisted of HEPES (10 mM, pH 7.4)-buffered Hanks' balanced salt solution, 0.1% bovine serum albumin (BSA; Sigma), 1.4 mM L-glutamine, 0.3 mM L-ascorbic acid, 5.6 mM glucose, and 0.5 µg/ml gentamycin. Cells were incubated for 10 min in the absence (stationary control) or presence of PFF. After termination of the experiment, the circulating and the stationary media were collected for PG extraction, and the cells were harvested for DNA determination.Experimental Design
Inhibitors. The inhibitor studies were performed with four experimental groups: 1) control (+ vehicle) group, 2) control + inhibitor group, 3) PFF (+ vehicle) group, and 4) PFF + inhibitor group. For every inhibitor used in this study, cell viability was assessed by the trypan blue exclusion procedure. Toxicity of all inhibitors was found negligible at the concentration and under the circumstances used. The vehicle was DMSO for all inhibitors [final concn <0.1% (vol/vol)] except for EGTA (Sigma), tetraethylammonium (TEA; Sigma), and gadolinium chloride (GdCl3; Sigma). Stock solutions of EGTA, TEA, and GdCl3 were prepared in water. To study the role of extracellular Ca2+, the response to PFF in flow medium was compared with the response in flow medium from which Ca2+ was left out and to which 2 mM EGTA were added. To determine the role of intracellular Ca2+, cells were either preincubated with 30 µM quin 2-AM (CalBiochem) or 20 µM TMB-8 (Biomol) (24) for 30 min, and the experiment was carried out in quin 2-AM-free flow medium or medium containing TMB-8.
To study the role of ion channels, cells were preincubated with 5 mM TEA or 10 µM GdCl3 (32) for 30 min, and the experiments were carried out in the presence of the inhibitors. To determine the involvement of the PLC-PKC pathway, cells were preincubated with 30 µg/ml D-609 (Biomol), a PLC inhibitor, or 3.4 µM Hypericin (Biomol) (54), a PKC inhibitor, for 60 min, and the experiments were carried out in the presence of the inhibitor. To investigate the role of the cytoskeleton in the PG response of OCY to PFF, cells were preincubated for 60 min with 1 µM cytochalasin B (Sigma), an actin filament-disrupting agent, and the experiments were carried out in the presence of cytochalasin B. To determine the routes for substrate (arachidonic acid) release in the acute PG response of OCY to PFF, cells were either preincubated for 30 min with 30 µM AACOCF3 (Biomol) (52), a cytosolic PLA2 inhibitor, or with 10 µM RHC-80267 (Biomol) (27), a diacylglycerol-lipase inhibitor, and the experiments were carried out in AACOCF3-free medium or medium containing RHC-80267, respectively.Calcium ionophore experiments. OCY were seeded and cultured for 24 h. The next day, cells were washed 2 times with PBS and incubated for 10 min at 37°C in flow medium containing either vehicle or ionomycin (Biomol). After this period, the medium was collected for PG extraction, and cells were harvested for DNA measurements.
PG extraction. PGs were extracted from culture media with octadecylsilyl-silica columns according to Powell (41). Medium samples were acidified to pH 3 with 1 N HCl and passed through a Sep-Pak cartridge (C8 125 Å, Waters, Millipore, MA), which was pretreated with two-column volumes of methanol, followed by a 1-column volume of water. The cartridges were placed on a vacuum container, the column was washed with 3 × 2 ml of water, 1 ml of petroleum ether, and PGs eluted with 2 × 1 ml of chloroform. The chloroform fractions were combined and evaporated under nitrogen, and the residue was dissolved in 200 µl of assay buffer consisting of 0.1 M phosphate buffer, pH 7.5, containing 0.01% thimerosal, 0.9% NaCl, and 0.1% BSA.
Medium PGE2 determination. Extracted samples of 50 µl were assayed for PGE2 by use of a nonradioactive enzyme immunoassay system (Amersham, Buckinghamshire, UK). The detection limit for PGE2 was 16 pg/ml. The absorbance was read at 450 nm with a Dynatech MR7000 (Billinghurst, UK) microplate reader.
DNA content. DNA content of the cell layers was determined according to Rao and Otto (43). Bisbenzimid H 33258 (Hoechst reagent; Riedel-De-Haën, Seelze-Hannover, Germany) was used as a color reagent for these determinations. DNA content was quantitated by measuring the fluorescence at 458 nm with a spectrophotometer and purified calf thymus DNA (Merck) as a standard.
Statistical Analysis
The data from several independent experiments were pooled and are expressed as means ± SE. Significance of differences among means was determined using Student's t-test for paired observations. ![]() |
RESULTS |
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As was previously shown (3), PFF treatment for 10 min results in a significant increase of PGE2 production by cultured chicken OCY. The requirement of extracellular Ca2+ for this response was demonstrated by a 50% reduction of the flow-induced response in the presence of a calcium-free perfusate containing 2 mM EGTA (Fig. 1A). A possible route for the entry of extracellular Ca2+ is through mechanosensitive channels. Gadolinium, a blocker of mechanosensitive Ca2+-permeable channels, inhibited the fluid flow-induced PGE2 response by 54% (Fig. 1B), suggesting that these channels are, at least in part, responsible for the Ca2+ entry into OCY subjected to PFF. Potassium channels have also been implicated in cellular responses to mechanical stimulation (38, 39), including calcium dependent-potassium channels (KCa), which potentiate Ca2+ entry into cells by a Ca2+-activated hyperpolarizing potassium efflux (60). To determine the possible role of KCa, cells were preincubated with TEA, a potassium channel blocker. TEA did not affect the fluid flow-induced PGE2 response (Fig. 1C).
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To determine the role of intracellular Ca2+ stores and intracellular Ca2+ availability in the PGE2 response of OCY to PFF, cells were pretreated with TMB-8, an inhibitor of intracellular calcium release, or quin 2-AM, a chelator of intracellular Ca2+ (Fig. 2). TMB-8 caused a 40% reduction in the PFF-induced PGE2 response (Fig. 2A), whereas treatment with quin 2-AM resulted in a 45% reduction (Fig. 2B). To confirm the calcium requirement for inducing PGE2 production, OCY were exposed to a Ca2+ ionophore (ionomycin) at 0.25 and 2.5 µM. Ionomycin stimulated PGE2 production at both concentrations tested (Fig. 3).
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PLC generates diacylglycerol (DAG) and IP3 (the latter releases Ca2+ from internal stores) and has been implicated in cellular responses to mechanical stimulation (7). Because PLC is also activated by a rise in intracellular Ca2+ (14), we estimated its involvement in PFF-induced PGE2 production by OCY. D-609, a blocker of PLC, reduced the PFF-induced PGE2 response by 45% (Fig. 4A). DAG together with Ca2+ activates PKC (36). Because previous studies have shown the involvement of PKC in cellular responses to mechanical loading, including enhanced PGE2 production (46), we studied the involvement of PKC in the PFF-induced PGE2 production in OCY. Hypericin, a blocker of PKC, reduced the PFF-induced PGE2 response by 40% (Fig. 4B). Combination of GdCl3 and D-609 caused a marked, but not additive, reduction of 60% in the PFF-induced PGE2 production (PGE2 production, means ± SE of 4 experiments: static control, 352 ± 131 pg/µg DNA; PFF treated, 1,893 ± 378 pg/µg DNA; PFF treated with GdCl3 and D-609, 844 ± 251 pg/µg DNA; PFF treated with GdCl3, 1,080 ± 337 pg/µg DNA; PFF treated with D-609, 997 ± 477 pg/µg DNA).
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To determine the role of the actin cytoskeleton in the PFF-induced PGE2 production by OCY, the actin cytoskeleton was disrupted by cytochalasin B (1 µM) pretreatment. Next, the cells were exposed to PFF. Cytochalasin B reduced the PFF-induced PGE2 response in OCY by 40% but did not affect PGE2 production in stationary cultures (Fig. 5).
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Arachidonic acid substrate generation in PFF-induced PGE2 production. The main routes of arachidonic acid (AA) generation are 1) the hydrolysis of phospholipids from the plasma membrane through the action of PLA2 and 2) the sequential action of PLC and DAG-lipase. To determine which of these pathways is involved in the acute PGE2 response to PFF in OCY, cells were either preincubated with AACOCF3, a blocker of cytosolic PLA2, or RHC-80267, a DAG-lipase blocker (Fig. 6). AACOCF3 (30 µM) caused a marked reduction of 80% of the PFF-induced PGE2 response (Fig. 6A), whereas RHC-80267 (10 µM) inhibited this response by 40% (Fig. 6B). The values presented in Fig. 6A are given as PFF values (with or without AACOCF3 preincubation) corrected for their respective paired control (Con) values, because trace amounts of AACOCF3 still present between the cells despite a wash leaked into the media during flow or stationary incubation and resulted in high background values (from cross-reactivity) in the PG assay.
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DISCUSSION |
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In this study we investigated the acute PGE2 response of isolated OCY to PFF. We used OCY derived from calvarial bones, which have a different origin and biomechanical loading pattern from the bones of the axial and appendicular skeleton. Generally speaking, the latter seem to receive higher strains as a result of their load-bearing function than calvarial bones (22). An important part of the dynamic strain in calvarial bone is derived from muscular activity during mastication and incision, and steep functional strain gradients have been measured in the face of baboons and macaques (22). Low-magnitude high-frequency strains seem to have effects on bone adaptation similar to high-magnitude low-frequency strains (48), suggesting that, regardless of the bone's origin or location, stress-sensitive cell populations perceive and respond to parameters of the mechanical environment. Therefore, we assume that our findings in calvarial OCY also reflect mechanotransduction in long bone OCY, which in our experience are much more difficult to isolate and purify.
It has been shown that fluid flow results in acute stimulation of PG production in various cell types, including OCY (3), osteoblasts (3, 26), and endothelial cells (16). A response to mechanical stress may be either direct or indirect, in the sense that cells respond to mechanical stress with the production of an autocrine/paracrine factor that stimulates a secondary response. The increased production of insulin-like growth factor in response to mechanical stimulation is an example of such an indirect effect, as this response was found to be inhibited by indomethacin (30). In the PFF setup used in this study, the time of stimulation is relatively short and the ratio of cells to medium volume is very small. It is therefore most unlikely that the PGE2 response used here as a measure for the response of OCY to PFF is other than a direct response. Here, we addressed the question of how in OCY this external physical stimulus is transduced into the cellular biochemical response. To this aim we used a panel of blockers to study the involvement of various intracellular and membrane-associated enzymes and structures.
Intracellular Ca2+ acts in many agonist-activated processes as a second messenger. Intracellular Ca2+ may also be involved in mechanotransduction (20). We therefore first studied whether Ca2+ is a second messenger mediating the initial PGE2 response of OCY to mechanical loading, and whether it is derived from the extracellular space or intracellular stores. When OCY were subjected to PFF in nominally Ca2+-free media containing a Ca2+ chelator (EGTA), we observed a marked decrease of PFF-induced PGE2 production compared with that of cells that had been perfused with normal (Ca2+-containing) media. These findings suggest that Ca2+ influx is, at least in part, necessary for the flow-induced PGE2 production, and they agree with the results of studies involving endothelial cells in which, under similar circumstances, a 74% decrease in shear-induced prostacyclin production rate was observed (6). In endothelial cells, elevation of intracellular Ca2+ concentration mediates agonist-induced PG synthesis (19). In this study, the addition of ionomycin, a Ca2+ ionophore, to the Ca2+-containing culture medium stimulated PGE2 release in a dose-dependent manner, suggesting that, also in OCY, increases in intracellular Ca2+ mediate PGE2 synthesis.
Intracellular Ca2+ modulates the functions of various enzymes linked to the PG production cascade. These include cPLA2, which releases AA, a substrate for PG synthesis, from the plasma membrane, and PKC, which is coactivated by DAG. Also, constitutive nitric oxide synthase, which has been shown to be linked to the flow-induced PGE2 production in mouse osteoblasts (25), is Ca2+ dependent (33). Therefore, it is likely that one (or more) of these enzymes will not function optimally when Ca2+ influx is impaired, thus leading to an attenuated PG response.
We speculated that the putative Ca2+ influx into OCY in response to fluid flow involved the action of mechanosensitive Ca2+-permeable ion channels. GdCl3, a well known inhibitor of these channels, reduced at low concentrations (10 µM) the PFF-induced PGE2 response in OCY. Mechanosensitive channels have been found on a wide variety of cells. These include vascular endothelial cells (29), chick heart cells (51), and osteoblast-like cells (11). Whether the mechanosensitive channels in OCY are voltage activated or voltage independent is not yet clear. Ypey et al. (61) were unable to demonstrate the presence of voltage-independent stretch-activated (SA) channels in chicken OCY by using patch-clamp techniques. On the other hand, Hung et al. (20) found that blockers against these SA channels inhibited a flow-induced rise in intracellular Ca2+ in osteoblasts, whereas blockers against voltage-sensitive calcium channels did not. In addition, Rawlinson et al. (44) reported recently that GdCl3 abolished loading-related increase in the PGI2 release and OCY glucose-6-phosphate dehydrogenase activity in loaded bones in vitro, whereas nifedipine, a voltage-activated Ca2+ channel inhibitor, had no effect. Several other ion channels, in particular potassium channels, may also be involved in cellular responses to mechanical stimulation. TEA-sensitive potassium channels were shown to mediate flow-induced nitric oxide production in endothelial cells (10). However, we were unable to demonstrate the involvement of these channels, because blocking of potassium channels by TEA did not affect the flow-induced PGE2 production in OCY. This suggests that these channels do not mediate the flow-induced Ca2+ entry into OCY that leads to increased PG release. This observation is supported by studies involving human osteoblast cells, where TEA had no effect on the flow-induced intracellular Ca2+ elevations (31).
Intracellular Ca2+ elevations may result from Ca2+ influxes derived from the extracellular space but also from the release of Ca2+ from intracellular stores. Inhibition of Ca2+ release from intracellular stores by TMB-8 reduced the PGE2 response, suggesting that the Ca2+ availability from these stores is, at least in part, necessary for the flow-enhanced PGE2 synthesis. Studies involving Ca2+ measurements in bone cells that were subjected to flow in the presence of specific blockers of Ca2+ release from intracellular stores support this view (20, 21, 31). The requirement of a rise in intracellular Ca2+ concentration for the PFF-induced PGE2 was demonstrated by subjecting OCY to flow in the presence of quin 2-AM, an intracellular calcium chelator. Quin 2-AM significantly reduced the fluid flow-induced PGE2 response in OCY. These findings agree with other studies in which quin 2-AM markedly reduced the flow-induced prostacyclin production in endothelial cells (6).
Ca2+ release from internal stores may be mediated by Ca2+ influx into the cell and may also be triggered by IP3, a breakdown product of membrane phospholipids released through the action of PLC (4). D-609, a specific blocker of PLC, significantly reduced the flow-induced PGE2 response in OCY. This indicates that PLC, or rather the breakdown products of phosphatidylinositol trisphosphate IP3 and DAG, mediate the PGE2 response of OCY to fluid flow, in agreement with earlier studies in bone cells (45).
DAG and Ca2+ are known activators of PKC (36). Furthermore, studies in osteoblasts have shown the involvement of PKC in flow-induced PGE2 production (46). This, taken together with our results, indicates that activation of PKC might be an intermediate step in the flow-induced PGE2 production in OCY. Inhibition of PKC by Hypericin strongly inhibited the flow-induced PGE2 response in OCY, supporting the hypothesis that PKC is involved in the response of OCY to mechanical loading.
The mechanisms by which fluid shear stress activates GdCl3-sensitive channels might be direct activation via membrane perturbation, resulting from fluid shear forces, or force transmission through the cytoskel-etal components, which may be physically linked to ion channnels. The integrin-cytoskeleton complexes are thought to be primary mechanotransduction sites, transmitting stress applied to the cell surface into the cellular compartment (23). The cytoskeleton is physically linked to many cellular components, and some of them may be involved in the flow-induced PG production. These include ion channels (49), PKC (42), and the actin filament-linked protein profilin, which binds to phosphatidylinositol biphosphate, thereby regulating PLC activity (17). Therefore, disrupting the cytoskeleton may modulate the functions of these and other enzymes and might also alter the permeability of ion channels located in the membrane, including mechanically stimulated channels. This notion is supported by studies involving endothelial cells that have been mechanically stimulated in the presence of actin filament-disrupting agents. Cytochalasin B completely abolished the load-induced intracellular Ca2+ elevations in these cells (13), suggesting that disruption of the cytoskeleton might deter cytoskeleton-mediated calcium entry into the cell, which could eventually lead to PG synthesis. Our results indicate that the disruption of the actin cytoskeleton with cytochalasin B caused a significant reduction in the flow-induced PGE2 response in OCY, suggesting that the actin filaments mediate the PGE2 response of OCY to PFF.
Mechanosensitive channels, together with PLC, are primary sites for mechanotransduction in cells (12). Because both are involved in the load-induced intracellular Ca2+ elevations (leading to PG synthesis) resulting from extracellular Ca2+ entry and IP3-mediated Ca2+ releases, their combined inhibitory effect was expected to completely abolish the PFF-induced PGE2 response. However, combination resulted in only a 60% decreased PFF-induced PGE2 response. The explanation for this probably lies in the fact that increase in PLC activity depends on an increase of intracellular Ca2+ (12, 14). One may presume that inhibition of Ca2+ entry by GdCl3 also inhibited the expression of stress-activated PLC (12).
Thus, it is reasonable to believe that Ca2+ entering via a mechanosensitive channel contributes to the stimulation of PLC. Experiments measuring PLC activity (IP3 production) in cells exposed to mechanical stimulation (e.g., PFF) in the presence of blockers of mechanosensitive channels might offer us further insight into this matter.
One necessary step in the PG synthesis is substrate AA generation, which is converted to PGs (including PGE2) by the action of cyclooxygenase. This AA can be generated via at least two known pathways (53) involving PLA2 and the combined action of PLC and DAG-lipase. Our results indicate that PLA2 and also the combination of the action of PLC and DAG-lipase are involved in the AA generation and, subsequently, in the acute PGE2 response of OCY to PFF. These data corroborate a recent study showing that cPLA2 is rapidly activated (within 2 min) in endothelial cells exposed to fluid flow (40).
In conclusion, several signaling pathways are involved in the acute PGE2 response of isolated OCY to PFF. We propose the following model of the signal transduction processes by which OCY respond to fluid flow (Fig. 7). Cellular deformation as a result of flow-induced shear stresses leads to actin-dependent opening of a Ca2+-permeable, gadolinium-sensitive ion channel. This subsequent increase in Ca2+ near the inner plasma membrane leads, in part, to the activation of PLC, which generates secondary Ca2+ responses through IP3-mediated release of intracellular calcium from internal stores (e.g., endoplasmic reticulum). DAG, a coproduct of the action of PLC together with Ca2+, activates PKC, which in its turn enhances the PLA2 sensitivity to activation by calcium, leading eventually to increases of AA release. This increase in substrate, perhaps together with the activation of cyclooxygenase by load-related calcium-induced nitric oxide (50), results in enhanced PG synthesis.
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ACKNOWLEDGEMENTS |
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This study was supported by the Dutch Organisation for Scientific Research (NWO Grant 903-41-144).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P. J. Nijweide, Dept. of Molecular Cell Biology, Leiden Univ. Medical Centre, PO Box 2156, 2301 CD Leiden, The Netherlands.
Received 8 June 1998; accepted in final form 29 September 1998.
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