Gap junctions and fluid flow response in MC3T3-E1 cells

In thecurrent study, we examined the role of gap junctions in oscillatoryfluid flow-induced changes in intracellular Ca²⁺concentration and prostaglandin release in osteoblastic cells. Thiswork was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Ourresults demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E₂ (PGE₂) release, whereas cellswith diminished gap junctional communication do not. Furthermore, wefound that cytosolic Ca²⁺ (Ca) responsewas unaltered by the disruption in gap junctional communication and wasnot significantly different among the cell lines. Thus our resultssuggest that gap junctions contribute to the PGE₂ but notto the Ca response to oscillatory fluid flow. Thesefindings implicate gap junctional intercellular communication (GJIC) inbone cell ensemble responsiveness to oscillatory fluid flow and suggestthat gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

     

PGE2 reduces arachidonic acid release in murine podocytes: evidence for an autocrine feedback loop

Increased glomerularprostaglandin E₂ (PGE₂) production isassociated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. Weinvestigated the signaling pathways that regulate the synthesis andactions of PGE₂ in glomerular podocytes. To study itsactions, we assessed the ability of PGE₂ to regulate theproduction of its own precursor, arachidonic acid (AA), in a mousepodocyte cell line. PGE₂ dose-dependently reduced phorbolester (PMA)-mediated AA release. Inhibition of PMA-stimulated AArelease by PGE₂ was found to be cAMP/PKA-dependent, becausePGE₂ significantly increased levels of this secondmessenger, whereas the inhibitory actions of PGE₂ werereversed by PKA inhibition and reproduced by the cAMP-elevating agentsforskolin and IBMX. PGE₂ synthesis in this podocyte cellline increased fourfold at 60 min in response to PMA, coinciding withupregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However,PGE₂ synthesis was significantly reduced by COX-1-selectiveinhibition, yet to a lesser extent by COX-2-selective inhibition. Ourfindings suggest that PMA-stimulated PGE₂ synthesis inmouse podocytes requires both basal COX-1 activity and induced COX-2expression, and that PGE₂ reduces PMA-stimulated AA releasein a cAMP/PKA-dependent manner. Such an autocrine regulatory loop mighthave important consequences for podocyte and glomerular function in thecontext of renal diseases involving PGE₂ synthesis.

     

Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton

Disruption of microfilaments in human umbilical vein endothelialcells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA)resulted in a 3.3- to 5.7-fold increase in total synthesis ofprostaglandin E₂ (PGE₂) and a 3.4- to 6.5-foldincrease in prostacyclin (PGI₂) compared with controlcells. Disruption of the microtubule network with nocodazole orcolchicine increased synthesis of PGE₂ 1.7- to 1.9-fold andPGI₂ 1.9- to 2.0-fold compared with control cells.Interestingly, however, increased release of PGE₂ andPGI₂ from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold morePGE₂ and 3.8-fold more PGI₂ released from HUVECcompared with control cells; latA treatment resulted in 17.7-fold more PGE₂ and 11.2-fold more PGI₂ released comparedwith control cells. Both increased synthesis and release ofprostaglandins in response to all drug treatments were completelyinhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2).Disruption of either microfilaments using cytD or latA or ofmicrotubules using nocodazole or colchicine resulted in a significantincrease in COX-2 protein levels, suggesting that the increasedsynthesis of prostaglandins in response to drug treatments may resultfrom increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest thatthe cytoskeleton plays an important role in maintenance of endothelialbarrier function by regulating prostaglandin synthesis and release from HUVEC.

     

Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells

Polyunsaturated fatty acids (PUFAs) as well as oestrogen (E2) and parathyroid hormone (PTH) affect bone cells. The aim of the study was to determine whether arachidonic acid (AA), E2, and PTH increase prostaglandin E₂ (PGE₂) synthesis in MG-63 and MC3T3-E1 osteoblastic cells and the level of mediation by COX-1 and COX-2. PGE₂ levels were determined in the conditioned culture media of MG-63 and MC3T3-E1 osteoblasts after exposure to AA, PTH and E2. Cells were pre-incubated in some experiments with the unselective COX inhibitor indomethacin or the COX-2 specific blocker NS-398. Indirect immunofluorescence was performed on MG-63 cells to detect the presence and location of the two enzymes involved. AA increased PGE₂ secretion in both cell lines; production by MC3T3-E1 cells, however, was significantly higher than that of MG-63 cells. This could be due to autoamplification via the EP₁ subtype of PGE receptors in mouse MC3T3-E1 osteoblasts. Both COX-1 and COX-2 affected the regulation of PGE₂ synthesis in MG-63 cells. E2 had no effect on PGE₂ secretion in both cell lines, while PTH caused a slight increase in PGE₂ synthesis in the MG-63 cell line.     

COX-2 expression and cell cycle progression in human fibroblasts

Cyclooxygenase-2 (COX-2) is continuously expressed in mostcancerous cells where it appears to modulate cellular proliferation andapoptosis. However, little is known about the contribution oftransient COX-2 induction to cell cycle progression or programmed celldeath in primary cells. In this study we determined whether COX-2regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E₂(PGE₂) were not detected in quiescent cells but wereexpressed during the G₀/G₁ phase of the cellcycle induced by serum. Inhibition of COX-2 did not alter G₀/G₁ to S phase transition or induceapoptosis at concentrations that diminished PGE₂.Addition of interleukin-1 to serum enhanced COX-2 expression andPGE₂ synthesis over that by serum alone but had no effecton the progression of these cells into S phase. Furthermore,platelet-derived growth factor drove the G₀ fibroblasts into the cell cycle without inducing detectable levels of COX-2 orPGE₂. Collectively, these data show that transient COX-2expression in primary human fibroblasts does not influence cell cycle progression.

     

Prostaglandin E2 is crucial in the response of podocytes to fluid flow shear stress

Podocytes play a key role in maintaining and modulating the filtration barrier of the glomerulus. Because of their location, podocytes are exposed to mechanical strain in the form of fluid flow shear stress (FFSS). Several human diseases are characterized by glomerular hyperfiltration, such as diabetes mellitus and hypertension. The response of podocytes to FFSS at physiological or pathological levels is not known. We exposed cultured podocytes to FFSS, and studied changes in actin cytoskeleton, prostaglandin E₂ (PGE₂) production and expression of cyclooxygenase-1 and–2 (COX-1, COX-2). FFSS caused a reduction in transversal F-actin stress filaments and the appearance of cortical actin network in the early recovery period. Cells exhibited a pattern similar to control state by 24 h following FFSS without significant loss of podocytes or apoptosis. FFSS caused increased levels of PGE₂ as early as 30 min after onset of shear stress, levels that increased over time. PGE₂ production by podocytes at post-2 h and post-24 h was also significantly increased compared to control cells (p < 0.039 and 0.012, respectively). Intracellular PGE₂ synthesis and expression of COX-2 was increased at post-2 h following FFSS. The expression of COX-1 mRNA was unchanged. We conclude that podocytes are sensitive and responsive to FFSS, exhibiting morphological and physiological changes. We believe that PGE₂ plays an important role in mechanoperception in podocytes.     

Mechanical Stimulation of Gap Junctions in Bone Osteocytes is Mediated by Prostaglandin E2

Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factors) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin E₂ (PGE₂) release and increase cyclooxygenase 2 (COX-2) expression. When PGE₂ was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE₂. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.     

5-lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B₄ (LTB₄) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E₂ (PGE₂) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0–120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB₄ signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB₄. Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE₂ reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro. leukotriene B₄; prostaglandin E₂; spreading; migration; bridges     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction

We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y(2) puringeric receptors. However, the molecular mechanisms by which fluid flow initiates cellular responses are still unclear. Accumulating evidence suggests that lipid rafts, one of the important membrane structural components, may play an important role in transducing extracellular fluid shear stress to intracellular responses. Due to the limitations of current techniques, there is no direct approach to study the role of lipid rafts in transmitting fluid shear stress. In this study, we targeted two important membrane components associated with lipid rafts, cholesterol, and glycosylphosphatidylinositol-anchored proteins (GPI-anchored proteins), to disrupt the integrity of cell membrane structures. We first demonstrated that membrane cholesterol depletion with the treatment of methyl-β-cyclodextrin inhibits oscillatory fluid flow induced intracellular calcium mobilization and ERK1/2 phosphorylation in MC3T3-E1 osteoblastic cells. Secondly, we used a novel approach to decrease the levels of GPI-anchored proteins on cell membranes by overexpressing glycosylphosphatidylinositol-specific phospholipase D in MC3T3-E1 osteoblastic cells. This resulted in significant inhibition of intracellular calcium mobilization and ERK1/2 phosphorylation in response to oscillatory fluid flow. Finally, we demonstrated that cholesterol depletion inhibited oscillatory fluid flow induced ATP releases, which were responsible for the activation of calcium signaling pathways in MC3T3-E1 osteoblastic cells. Our findings suggest that cholesterol and GPI-anchored proteins, two membrane structural components related to lipid rafts, may play an important role in osteoblastic cell mechanotransduction.     

Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca(2+)i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m(2) at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca(2+)i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca(2+)i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway.     

F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca(2+) signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca(2+) signaling.     

Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca²⁺]_i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca²⁺]_i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca²⁺]_i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca²⁺]_i, which was sensitive to antioxidants and removal of external Ca²⁺, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca²⁺-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca²⁺ uptake. This suggests that hydrogen peroxide promotes Ca²⁺ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca²⁺ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca²⁺]_i and altered cholecystokinin-evoked [Ca²⁺]_i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca²⁺]_i also upregulated the activity of the plasma membrane Ca²⁺-ATPase in a Ca²⁺-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca²⁺-ATPase. This may be important in facilitating "Ca²⁺ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump     

Differential subcellular localization of COX-2 in macrophages phagocytosing heat-killed Mycobacterium bovis BCG

Cyclooxygenase-2 (COX-2)-mediated prostaglandin E₂ (PGE₂) biosynthesis by macrophages downregulates microbicidal activities in innate and acquired immune responses against intracellular bacteria. Previous studies in mice showed that intraperitoneal administration of heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) resulted in induction of splenic PGE₂-releasing macrophages in 7–14 days. In contrast, HK-BCG induced catalytically inactive COX-2 at relatively high levels in the macrophages within 1 day. In the present study, we found that COX-2 was localized subcellularly in the nuclear envelope (NE) 7 and 14 days after HK-BCG treatment, whereas COX-2 was dissociated from the NE 1 day after treatment. At 1 day after treatment, the majority of COX-2-positive macrophages had phagocytosed HK-BCG. In contrast, no intracellular HK-BCG was detected 7 and 14 days after treatment in COX-2-positive macrophages, where COX-2 was associated with the NE. However, when macrophages phagocytosed HK-BCG in vitro, all COX-2 was associated with the NE. Thus the administration of HK-BCG induces the biphasic COX-2 expression of an NE-dissociated catalytically inactive or an NE-associated catalytically active form in splenic macrophages. The catalytically inactive COX-2-positive macrophages develop microbicidal activities effectively, since they lack PGE₂ biosynthesis. nuclear envelope; autoimmune disease; prostaglandin E₂     

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

Mechanical stimulation of bone induces new bone formation invivo and increases the metabolic activity and gene expression ofosteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission ofmechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts causedreorganization of actin filaments into contractile stress fibers andinvolved recruitment of₁-integrins and -actinin tofocal adhesions. Fluid shear also increased expression of two proteinslinked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and theearly response gene product c-fos. Inhibition of actin stress fiberdevelopment by treatment of cells with cytochalasin D, by expression ofa dominant negative form of the small GTPase Rho, or by microinjectioninto cells of a proteolytic fragment of -actinin that inhibits-actinin-mediated anchoring of actin filaments to integrins at theplasma membrane each blocked fluid-shear-induced gene expression inosteoblasts. We conclude that fluid shear-induced mechanical signalingin osteoblasts leads to increased expression of COX-2 and c-Fos througha mechanism that involves reorganization of the actin cytoskeleton.Thus Rho-mediated stress fiber formation and the -actinin-dependentanchorage of stress fibers to integrins in focal adhesions may promotefluid shear-induced metabolic changes in bone cells.

     

Impaired Expression of Prostaglandin E2 (PGE2) Synthesis and Degradation Enzymes during Differentiation of Immortalized Urothelial Cells from Patients with Interstitial Cystitis/Painful Bladder Syndrome

Purpose

The differentiated superficial cells of the urothelium restrict urine flow into the bladder wall. We have demonstrated that urothelial cells isolated from bladders of patients with interstitial cystitis/painful bladder syndrome (IC/PBS) fail to release PGE₂ in response to tryptase. This study examines the expression of PGE₂ synthesis and degradation enzymes in urothelial cells during differentiation.

Materials and Methods

We measured immunoprotein expression of cyclooxygenase-2 (COX-2), prostaglandin E₂ synthase (PGES) and 15-hydroxyprostaglandin dehydrogenase (PGDH) in human urothelial cells and in immortalized urothelial cells isolated from the bladders of IC/PBS patients or normal subjects during stratification and differentiation produced by increased calcium and fetal bovine serum (Ca/FBS) in the culture medium for 1, 3 and 7 days.

Results

PGES immunoprotein expression increased during differentiation in normal and IC/PBS urothelial cells. COX-2 expression also increased in cells from normal patients following differentiation. Remarkably, no COX-2 expression was detectable in urothelial cells isolated from 3 out of 4 IC/PBS patients. PGDH immunoprotein expression decreased in normal cells after 1 and 3 days of Ca/FBS addition, but returned to normal after 7 days. PGDH expression was unchanged during differentiation at 1 and 3 days, but was more than 2-fold higher at 7 days compared to day 0 in the IC/PBS cells. Urothelial cells isolated from IC/PBS patients demonstrated no PGE₂ release in response to tryptase under any of the experimental conditions studied.

Conclusions

Taken together, our results indicate that PGE₂ release is compromised during stratification and differentiation in IC/PBS urothelium and may contribute to impaired barrier function.     

Influence of increased mechanical loading by hypergravity on the microtubule cytoskeleton and prostaglandin E2 release in primary osteoblasts

Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesized that bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, and then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded using hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E₂ (PGE₂). In immature osteoblasts, a stimulus of 10x gravity (10 g) for 3 h increased PGE₂ 2.5-fold and decreased microtubule network height 1.12-fold without affecting cell viability. Hypergravity (3 h) caused dose-dependent (5–50 g) increases in PGE₂ (5.3-fold at 50 g) and decreases (1.26-fold at 50 g) in microtubule network height. PGE₂ release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading, which appears to be blunted in mature osteoblasts. mechanotransduction; differentiation; bone     

PAR1-dependent and independent increases in COX-2 and PGE2 in human colonic myofibroblasts stimulated by thrombin

Subepithelial myofibroblast-derivedprostaglandin E₂ (PGE₂) regulatesepithelial chloride secretion in the intestine. Thrombin is elevated ininflammatory conditions of the bowel. Therefore, we sought to determinea role for thrombin in regulating PGE₂ synthesis by colonicmyofibroblasts. Incubation of cultured CCD-18Co colonic myofibroblastswith thrombin, the proteinase-activated receptor 1 (PAR₁)-activating peptide (Cit-NH₂), andpeptides corresponding to 2 noncatalytic regions of thrombin (TP367 andTP508) for 18 h increased both cyclooxygenase (COX)-2 expression(immunocytochemistry) and PGE₂ synthesis (enzymeimmunoassay). Inhibition of thrombin byD-Phe-Pro-Arg-chloromethylketone (PPACK) did not significantly reducePGE₂ synthesis, which remained elevated compared withcontrol. We also investigated the basic fibroblast growth factor (bFGF) dependence of thrombin-induced PGE₂ elevations. Recombinanthuman bFGF concentration dependently increased PGE₂synthesis, and a bFGF neutralizing antibody inhibited PGE₂synthesis induced by TP367 and TP508 (~40%) and by thrombin(~20%) (but not Cit-NH₂). Thrombin, therefore,upregulates COX-2-derived PGE₂ synthesis by both catalyticcleavage of PAR₁ and bFGF-dependent noncatalytic activity.This presents a novel mechanism by which intestinal myofibroblastsmight regulate epithelial chloride secretion.

     

Reversible histochemical modifications of endoplasmic reticulum following arginine vasopressin stimulation of granular cells of toad bladder

The endoplasmic reticulum is generally absent from schematic representations of transport phenomena, although it shows a well-organized network in most transport epithelial cells. In order to examine the correlation between this organelle and cellular activity, bladders of Bufo marinus were studied under different experimental conditions and fixed by immersion in glutaraldehyde, followed by OsO₄ impregnation for 3 days. Normal granular and mitochondria-rich cells showed a rich cytoplasmic network of canaliculi, well-impregnated by osmium deposits. Following a 2 to 15-min stimulation (serosal bath) with arginine vasopressin, the V₂ receptor agonist dD-arginine-vasopressin or cyclic AMP (cAMP), the staining of endoplasmic reticulum in granular cells disappeared. After washing out of the hormone or the agonist, impregnation of the endoplasmic reticulum could be observed once again. Arginine vasopressin did not modify the impregnation of endoplasmic reticulum of either mitochondria-rich or basal cells. Our data indicate a correlation between the reactivity of endoplasmic reticulum to osmium, and a cAMP-dependent effect of arginine vasopressin through its V₂ receptors. Incubation of toad bladders carried out with agents interfering with cellular calcium (calcium ionophores, high or low bath calcium) or with calcium release from the endoplasmic reticulum (TMB-8, thapsigargin) suggested that an early step in the cAMP-dependent effect of arginine vasopressin must involve the release of intracellular calcium from the endoplasmic reticulum. However, calcium ATPases in this organelle do not seem to participate in the hormonal effect. The reversible loss of osmium impregnation induced by arginine vasopressin may represent protein changes in the endoplasmic reticulum accompanying a cAMP-dependent calcium release, from the organelle.     

Ceramide triggers intracellular calcium release via the IP3 receptor in Xenopus laevis oocytes

Ceramide, a product of sphingomyelin turnover, is a lipid secondmessenger that mediates diverse signaling pathways, including thoseleading to cell cycle arrest and differentiation. The mechanism(s) bywhich ceramide signals downstream events have not been fully elucidated. Here we show that, in Xenopuslaevis oocytes, ceramide-induced maturation isassociated with the release of intracellular calcium stores. Ceramidecaused a dose-dependent elevation in the second messenger inositol1,4,5-trisphosphate (IP₃) viaactivation of G_q/11 andphospholipase C-X. Elevation ofIP₃, in turn, activated theIP₃ receptor calcium releasechannel on the endoplasmic reticulum, resulting in a rise incytoplasmic calcium. Thus our study demonstrates that cross talkbetween the ceramide and phosphoinositide signaling pathways modulatesintracellular calcium homeostasis.