Oscillating fluid flow regulates cytosolic calcium concentration in bovine articular chondrocytes.

Mechanical loading is a well-known regulator of cartilage metabolism. This suggests that a loading-induced physical signal regulates chondrocyte behavior. Previous studies have focused on the effects of steady fluid flow on chondrocytes. In contrast to steady flow, loading induced fluid flow occurs in an oscillatory pattern and includes a reversal of flow direction with each loading event. In this study we examined the hypothesis that oscillating fluid flow increases cytosolic Ca2+ concentration ([Ca2+]i) in bovine articular chondrocytes (BAC) in a frequency-dependent manner and that the presence of serum affects this response. The aims of our study were to examine (1) whether BAC respond to physiologic oscillating fluid flow in vitro and compare these results to steady fluid flow, (2) the effect of fetal bovine serum on fluid flow responsiveness of BAC and (3) whether the response of BAC to fluid flow is flow rate and/or frequency dependent. [Ca2+]i was quantified using the fluorescent dye fura-2. BAC were exposed to steady, 0.5, 1, or 5 Hz sinusoidal oscillating fluid flow at five different flow rates in a parallel plate flow chamber. Our findings demonstrate that BAC respond to oscillating fluid flow with an increase in [Ca2+]i (p > 0.05), and furthermore, chondrocyte responsiveness to fluid flow increases with peak flow rate (p < 0.0001) and decreases with increasing frequencies (p < 0.0001). Finally, the presence of serum in the media potentiated the responsiveness of BAC to fluid flow (p < 0.0001). Our results suggest an important role for mechanical load-induced oscillating fluid flow in chondrocyte mechanotransduction.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.     

Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.     

Angiotensin II increases cytosolic calcium and stimulates catecholamine release in cultured bovine adrenomedullary cells

In bovine adrenomedullary cells in primary culture, angiotensin II (AII) elicited virtually immediate, dose-related increments in cytosolic calcium [( Ca++]i) measured by the Quin 2 technique and stimulated approximately proportional secretion of norepinephrine, epinephrine, and dopamine measured by liquid chromatography with electrochemical detection. Peak responses of [Ca++]i to AII were similar to peak responses to nicotine or KCl. Pre-treatment with verapamil or washing the cells in calcium-free medium attenuated the stimulatory effect of AII on [Ca++]i. Pre-treatment with nicotine, which temporarily inactivates cholinergic receptor-activated calcium channels, did not affect [Ca++]i responses to AII. The results indicate functional effects of AII on cultured chromaffin cells. The mechanism of cellular activation by AII appears to include increases in [Ca++]i due to opening of membrane calcium channels which may be unrelated to cholinergic receptor-operated calcium channels.     

Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

Increases in cytosolic calcium, but not fluid flow, affect aggrecan mRNA levels in articular chondrocytes

Fluctuations in intracellular free calcium concentration ([Ca2+]i) is thought to be one mechanism by which cells transduce mechanical signals into biological responses. Primary cultures of bovine articular chondrocytes (BAC) respond to oscillating fluid flow with a transient rise in [Ca2+]i. However, specific down-stream effects of [Ca2+]i on gene expression and phenotype in BAC remain to be defined. The present work was designed to determine whether [Ca2+]i mobilization regulates aggrecan mRNA levels. [Ca2+]i was transiently elevated by exposing BAC to the [Ca2+]-specific ionophore, ionomycin. The results show that ionomycin increases [Ca2+]i in a dose-dependent fashion. Semi-quantitative real time (RT)-PCR was used to study the effects of increased [Ca2+]i on steady state levels of aggrecan mRNA. Four hours after a brief exposure to 1.5 microM ionomycin, BAC displayed a nearly four-fold decrease in aggrecan mRNA levels compared to control cells. This effect of ionomycin on aggrecan mRNA was no longer evident 6 or 10 h later. Despite previous observations that oscillating fluid flow elicits increased [Ca2+]i in BAC, it did not affect aggrecan mRNA levels. Taken together, these data suggest that ionomycin-induced [Ca2+]i fluctuations regulate aggrecan mRNA levels, but that flow induced [Ca2+]i fluctuations do not.     

Stimulation of cytosolic free calcium and inositol phosphates by prostaglandins in cultured rat mesangial cells

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF2 alpha, PGE2 and the thromboxane A2/endoperoxide analogue U-46619 rapidly increased cytosolic free Ca2+, measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca2+, indicating release from internal stores. The thromboxane A2-receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI2 analogue Iloprost had no effect on cytosolic Ca2+. PGF2 alpha, PGE2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.     

Decrease in cytosolic free Ca2+ and enhanced proteoglycan synthesis induced by cartilage derived growth factors in cultured chondrocytes

Cartilage-derived growth factors, enhance proteoglycan synthesis in cultured chick-embryo chondrocytes, and have almost no effect on cell proliferation. Addition of cartilage derived growth factors to cartilage cells loaded with the fluorescent Ca2+ indicator quin 2, caused a rapid, concentration dependent decrease in cytoplasmic free Ca2+. This decrease persisted also in Ca2+-free medium, indicating that it is not mediated by a decrease in the passive permeability of cell membrane to Ca2+. Addition of the Ca2+ ionophore A23187, with or without cartilage derived factors, caused an increase in cytoplasmic free Ca2+ together with inhibition of proteoglycan synthesis and enhanced cell proliferation. The results may indicate that whereas cell proliferation in chondrocytes is signaled by an increase in cytoplasmic Ca2+ ([Ca2+]in), proteoglycan synthesis is signaled by a decrease in [Ca2+]in. The data lead to suggesting a mechanism for antagonistic regulation of cell proliferation and the expression of the differentiated state.     

Interactions between inositol phosphates and cytosolic free calcium following bradykinin stimulation in cultured human skin fibroblasts

The inositol triphosphate (IP3) that results from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is generally accepted to be responsible for the mobilization of intracellular calcium. However, some studies suggest that low concentrations of agonists elevate cytosolic free calcium concentration ([Ca2+]i) without IP3 formation. Thus, in the present studies, a comparison of the temporal response of inositol phosphates (IP3, IP2 and IP) and [Ca2+]i to a wide range of bradykinin concentrations was used to examine the relation of these two signal transduction events in cultured human skin fibroblasts (GM3652). In addition, the effects of alterations in internal or external calcium on the response of these second messengers to bradykinin were determined. Bradykinin stimulated accumulation of inositol phosphates and a rise of [Ca2+]i in a time- and dose-dependent manner. Decreasing the bradykinin concentration from 1 microM to 0.1 microM increased the time until the IP3 peak, and when the bradykinin concentration was reduced to 0.01 microM IP3 was not detected. [Ca2+]i was examined under parallel conditions. As the bradykinin concentration was reduced from 1 microM to 0.01 microM, the time to reach the peak of [Ca2+]i increased progressively, but the magnitude of the peak was unaltered. These two second messengers were variably dependent on external calcium. Although the bradykinin-stimulated initial spike of [Ca2+]i did not depend on extracellular calcium, the subsequent sustained levels of [Ca2+]i were abolished in calcium free medium. The bradykinin-stimulated inositol phosphate formation was not dependent on the extracellular calcium nor on the elevation of [Ca2+]i that was produced with Br-A23187. These results demonstrate that bradykinin-induced IP3 formation can be independent of [Ca2+]i and of external calcium, whereas changes in [Ca2+]i are partially dependent on external calcium.     

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.     

Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells

To determine whether increases in the cytosolic free Ca2+ concentration ([Ca2+]i) accompany agonist-stimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca2+]i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca2+]i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca2+]i had been reduced by loading cells with quin2 in medium containing low [Ca2+]. Ionomycin increased [Ca2+]i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca2+]i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca2+]i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+]i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca2+]i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca2+]i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca2+]i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca2+]i than does TPA-stimulated secretion.     

Folate deficiency and homocysteine induce toxicity in cultured dorsal root ganglion neurons via cytosolic calcium accumulation

Folate deficiency induces neurotoxicity by multiple routes, including increasing cytosolic calcium and oxidative stress via increasing levels of the neurotoxin homocysteine (HC), and inducing mitochondrial and DNA damage. Because some of these neurotoxic effects overlap with those observed in motor neuron disease, we examined the impact of folate deprivation on dorsal root ganglion (DRG) neurons in culture. Folate deprivation for 2 h increased cytosolic calcium and reactive oxygen species (ROS) and impaired mitochondrial function. Treatment with nimodipine [an L voltage-sensitive calcium channel (LVSCC) antagonist], MK-801 (an NMDA channel antagonist) and thapsigarin (an inhibitor of efflux of calcium from internal stores) indicated that folate deprivation initially induced calcium influx via the LVSCC, with subsequent additional calcium derived from NMDA channels and internal stores. These compounds also reduced ROS and mitochondrial degeneration, indicating that calcium influx contributed to these phenomena. Calcium influx was prevented by co-treatment with 3-deaza-adenosine, which inhibits HC formation, indicating that HC mediated increased cytosolic calcium following folate deprivation. Nimodipine, MK-801 and thapsigargin had similar effects following direct treatment with HC as they did following folate deprivation. These findings support the idea that folate deprivation and HC treatment can compromise the health of DRG neurons by perturbing calcium homeostasis.     

Transient increase of cytosolic free calcium in cultured human vascular endothelial cells by platelet-activating factor

The effect of platelet-activating factor (PAF-acether) on cytosolic free calcium, [Ca2+]i, in adherent human vascular endothelial cells in culture was directly determined using a new fluorescent calcium indicator, fura-2. It was found that PAF-acether but not lyso PAF-acether induced a rapid and transient increase in [Ca2+]i in endothelial cells. Restimulation with PAF-acether after the first challenge did not cause further response, while the cells were able to respond to thrombin. In the absence of extracellular calcium, PAF-acether evoked a similar transient increase, suggesting that PAF-acether raises [Ca2+]i mainly by discharging calcium from intracellular pools. PAF-acether-induced rise in [Ca2+]i was completely blocked by a specific antagonist, BN 52021. These results suggest the receptor-mediated increase in [Ca2+]i as an early event in PAF-acether activation of human vascular endothelial cells.     

UDP-glucuronate carboxy-lyase in cultured chondrocytes.

UDP-glucuronate carboxy-lyase has been demonstrated in chick chondrocytes in tissue culture. It occurs in the particulate fraction, and its activity is stimulated by exogenous NAD. The enzyme is allosterically activated by UDP-glucuronate and inhibited by UDP-xylose, n Values of 2.8 indicate positive cooperativity of at least three interacting sites on the enzyme. These data suggest that UDP-xylose concentration in chondrocytes is regulated by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. Activity levels of the enzyme during growth of the cells peak towards mid-log phase and decline thereafter, closely paralleling levels of chondroitin sulfate glycosyltransferases determined previously (Schwartz, N. B. (1976) J. Biol. Chem. 251, 3346-3351). Thus, it appears that during chondrocyte development a common mechanism governs induction of glycosyltransferases and of UDP-glucuronate carboxy-lyase.     

Apoptosis staining in cultured pseudoachondroplasia chondrocytes

Pseudoachondroplasia (PSACH) is a skeletal dysplasia caused by a mutation in cartilage oligomeric matrix protein (COMP), a glycoprotein of normal cartilage matrix. PSACH chondrocytes have a distinctive phenotype with enlarged rER cisternae containing COMP, aggrecan, type IX collagen, and chaperone proteins. Ultrastructural studies suggested that this accumulation compromises cell function, hastening cell death, and consequently reducing the number of cells in the growth plate contributing to linear bone growth. Using the alginate bead system, we cultured control and PSACH chondrocytes for twenty weeks and one year to determine the effect of the mutation on size and number of cartilage nodules; and the presence of apoptotic cell death (TUNEL assay). At 20 weeks, beads containing PSACH or control chondrocytes did not differ in size and number of cartilage nodules or number of TUNEL-positive cells. After one year, nodule number, size and percent cartilage per bead were significantly less in PSACH nodules, and the number of cells staining positive for apoptosis was significantly greater than in controls (71.8% vs. 44.6%). The increase in apoptosis in PSACH nodules correlates with a decrease in growth of cartilage, supporting our hypothesis that death of damaged cells contributes to the growth plate defects in PSACH.     

Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.     

Effect of superoxide and lipid peroxide on cytosolic free calcium concentration in cultured pig aortic endothelial cells

The effect of reactive oxygen on cytosolic free calcium concentration [( Ca++]i) in pig aortic endothelial cells (ECs) was studied. Linoleate hydroperoxide (LHO) and superoxide radicals generated from xanthine with xanthine oxidase (X-XO) were used as sources of reactive oxygen. [Ca++]i in ECs was measured with quin 2 and the value for quiescent ECs was 112 +/- 11 nM. Both LHO and X-XO increased [Ca++]i in a dose-dependent manner without accompanying the significant cellular damage. Nifedipine suppressed the increase in [Ca++]i provoked by LHO and X-XO. Thus, the biological effects of reactive oxygen might be mediated, at least in part, by the activation of voltage-dependent calcium channels in ECs.