Stretch-activated Calcium Channels Research Articles

Piezo1 Channel Activation Reverses Pulmonary Artery Vasoconstriction in an Early Rat Model of Pulmonary Hypertension: The Role of Ca2+ Influx and Akt-eNOS Pathway.

In intrapulmonary arteries (IPAs), mechanical forces due to blood flow control vessel tone, and these forces change during pulmonary hypertension (PH). Piezo1, a stretch-activated calcium channel, is a sensor of mechanical stress present in both endothelial cells (ECs) and smooth muscle cells (SMCs). The present study investigated the role of Piezo1 on IPA in the chronic hypoxia model of PH. Rats were raised in chronically hypoxic conditions for 1 (1W-CH, early stage) or 3 weeks (3W-CH, late-stage) of PH or in normoxic conditions (Nx). Immunofluorescence labeling and patch-clamping revealed the presence of Piezo1 in both ECs and SMCs. The Piezo1 agonist, Yoda1, induced an IPA contraction in Nx and 3W-CH. Conversely, Yoda1 induced an endothelial nitric oxide (eNOS) dependent relaxation in 1W-CH. In ECs, the Yoda1-mediated intracellular calcium concentration ([Ca2+]i) increase was greater in 1W-CH as compared to Nx. Yoda1 induced an EC hyperpolarization in 1W-CH. The eNOS levels were increased in 1W-CH IPA compared to Nx or 3W-CH PH and Yoda1 activated phosphorylation of Akt (Ser473) and eNOS (Ser1177). Thus, we demonstrated that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i, endothelial-dependent hyperpolarization, and Akt-eNOS pathway activation in the early stage of PH.

TRPV4 and TRPC1 channels mediate the response to tensile strain in mouse Müller cells

Müller glia, a pillar of metabolic, volume regulatory and immune/inflammatory signaling in the mammalian retina, are among the earliest responders to mechanical stressors in the eye. Ocular trauma, edema, detachment and glaucoma evoke early inflammatory activation of Müller cells yet the identity of their mechanotransducers and signaling mechanisms downstream remains unknown. Here, we investigate expression of genes that encode putative stretch-activated calcium channels (SACs) in mouse Müller cells and study their responses to dynamical tensile loading in cells loaded with a calcium indicator dye. Transcript levels in purified glia were Trpc1>Piezo1>Trpv2>Trpv4>>Trpv1>Trpa1. Cyclic radial deformation of matrix-coated substrates produced dose-dependent increases in [Ca2+]i that were suppressed by the TRPV4 channel antagonist HC-067047 and by ablation of the Trpv4 gene. Stretch-evoked calcium responses were also reduced by knockdown and pharmacological inhibition of TRPC1 channels whereas the TRPV2 inhibitor tranilast had no effect. These data demonstrate that Müller cells are intrinsically mechanosensitive, with the response to tensile loading mediated through synergistic activation of TRPV4 and TRPC1 channels. Coupling between mechanical stress and Müller Ca2+ homeostasis has treatment implications, since many neuronal injury paradigms in the retina involve calcium dysregulation associated with inflammatory and immune signaling.

Blocking Cell Extrusion Prevents Bronchoconstriction-Induced Airway Epithelial Damage and Inflammation

Asthma is a common disease characterized by airway constriction, excess mucus, and inflammation. We previously showed that cell crowding drives steady-state epithelial cell extrusion and cell death. Using ex vivo lung slices, we show that excessive crowding from methacholine-induced bronchoconstriction causes extensive cell extrusion leading to destruction of the airway epithelial barrier. In live mice, bronchoconstriction-induced extrusion caused airway epithelia damage and inflammation in secondary and tertiary airways. In primary airways, bronchoconstriction triggered mucus secretion by a mechanism reminiscent of extrusion. Reversing bronchoconstriction with albuterol failed to ameliorate these symptoms, whereas inhibiting the stretch-activated calcium channel, Piezo1, prevented extrusion, inflammation, and mucus secretion. Our findings propose a new etiology for asthma where the mechanical crowding from bronchoconstriction causes so much extrusion that it compromises the barrier, triggering subsequent inflammation. Furthermore, inhibiting extrusion, potentially with gadolinium, an inexpensive Piezo1 inhibitor, may prevent the asthma inflammatory cycle.

Mechanical Ventilation Induces Desensitization of Lung Axl Tyrosine Kinase Receptors.

Lower tidal volumes are increasingly used in acute respiratory distress syndrome, but mortality has changed little in the last 20 yr. Therefore, in addition to ventilator settings, it is important to target molecular mediators of injury. Sepsis and other inflammatory states increase circulating concentrations of Gas6, a ligand for the antiinflammatory receptor Axl, and of a soluble decoy form of Axl. We investigated the effects of lung stretch on Axl signaling. We used a mouse model of early injury from high tidal volume and assessed the effects of inhibiting Axl on in vivo lung injury (using an antagonist R428, n = 4/group). We further determined the effects of stretch on Axl activation using in vitro lung endothelial cells. High tidal volume caused mild injury (compliance decreased 6%) as intended, and shedding of the Axl receptor (soluble Axl in bronchoalveolar fluid increased 77%). The Axl antagonist R428 blocked the principal downstream Axl target (suppressor of cytokine signaling 3 [SOCS3]) but did not worsen lung physiology or inflammation. Cyclic stretch in vitro caused Axl to become insensitive to activation by its agonist, Gas6. Finally, in vitro Axl responses were rescued by blocking stretch-activated calcium channels (using guanidinium chloride [GdCl3]), and the calcium ionophore ionomycin replicated the effect of stretch. These data suggest that lung endothelial cell overdistention activates ion channels, and the resultant influx of Ca inactivates Axl. Downstream inactivation of Axl by stretch was not anticipated; preventing this would be required to exploit Axl receptors in reducing lung injury.

Fate of tenogenic differentiation potential of human bone marrow stromal cells by uniaxial stretching affected by stretch-activated calcium channel agonist gadolinium.

The role for mechanical stimulation in the control of cell fate has been previously proposed, suggesting that there may be a role of mechanical conditioning in directing mesenchymal stromal cells (MSCs) towards specific lineage for tissue engineering applications. Although previous studies have reported that calcium signalling is involved in regulating many cellular processes in many cell types, its role in managing cellular responses to tensile loading (mechanotransduction) of MSCs has not been fully elucidated. In order to establish this, we disrupted calcium signalling by blocking stretch-activated calcium channel (SACC) in human MSCs (hMSCs) in vitro. Passaged-2 hMSCs were exposed to cyclic tensile loading (1 Hz + 8% for 6, 24, 48, and 72 hours) in the presence of the SACC blocker, gadolinium. Analyses include image observations of immunochemistry and immunofluorescence staining from extracellular matrix (ECM) production, and measuring related tenogenic and apoptosis gene marker expression. Uniaxial tensile loading increased the expression of tenogenic markers and ECM production. However, exposure to strain in the presence of 20 μM gadolinium reduced the induction of almost all tenogenic markers and ECM staining, suggesting that SACC acts as a mechanosensor in strain-induced hMSC tenogenic differentiation process. Although cell death was observed in prolonged stretching, it did not appear to be apoptosis mediated. In conclusion, the knowledge gained in this study by elucidating the role of calcium in MSC mechanotransduction processes, and that in prolonged stretching results in non-apoptosis mediated cell death may be potential useful for regenerative medicine applications.

Effects of direct current electric fields on lung cancer cell electrotaxis in a PMMA-based microfluidic device.

Tumor metastasis is the primary cause of cancer death. Numerous studies have demonstrated the electrotactic responses of various cancer cell types, and suggested its potential implications in metastasis. In this study, we used a microfluidic device to emulate endogenous direct current electric field (dcEF) environment, and studied the electrotactic migration of non-small cell lung cancer cell lines (H460, HCC827, H1299, and H1975) and the underlying mechanisms. These cell lines exhibited greatly different response in applied dcEFs (2-6V/cm). While H460 cells (large cell carcinoma) showed slight migration toward cathode, H1299 cells (large cell carcinoma) showed increased motility and dcEF-dependent anodal migration with cell reorientation. H1975 cells (adenocarcinoma) showed dcEF-dependent cathodal migration with increased motility, and HCC827 cells (adenocarcinoma) responded positively in migration speed and reorientation but minimally in migrating directions to dcEF. Activation of MAPK and PI3K signaling pathways was found to be associated with the realignment and directed migration of lung cancer cells. In addition, both Ca2+ influx through activated stretch-activated calcium channels (SACCs) (but not voltage-gated calcium channels, VGCCs) and Ca2+ release from intracellular storage were involved in lung cancer cell electrotactic responses. The results demonstrated that the microfluidic device provided astable and controllable microenvironment for cell electrotaxis study, and revealed that the electrotactic responses of lung cancer cells were heterogeneous and cell-type dependent, and multiple signals contributed to lung cancer cells electrotaxis.

P2Y2 purinergic receptors are highly expressed in cardiac and diaphragm muscles of mdx mice, and their expression is decreased by suramin.

In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin leads to increased calcium influx and muscle necrosis. Patients suffer progressive muscle loss, and cardiomyopathy is an important determinant of morbidity. P2 purinergic receptors participate in the increased calcium levels in dystrophic skeletal muscles. In this study, we evaluated whether P2 receptors are involved in cardiomyopathy in mdx mice at later stages of the disease. Western blotting revealed that P2Y2 receptor levels were upregulated (54%) in dystrophic heart compared with a normal heart. Suramin reduced the levels of P2Y2 to almost normal values. Suramin also decreased heart necrosis (reduced CK-MB) and the expression of the stretch-activated calcium channel TRPC1. This study suggests that P2Y2 may participate in cardiomyopathy in mdx mice. P2-selective drugs with specific actions in the dystrophic heart may ameliorate cardiomyopathy in dystrophinopathies. Muscle Nerve 55: 116-121, 2017.

The role of calcium signalling in the chondrogenic response of mesenchymal stem cells to hydrostatic pressure.

The object of this study was to elucidate the role of Ca++ signalling in the chondrogenic response of mesenchymal stem cells (MSCs) to hydrostatic pressure (HP). MSCs were seeded into agarose hydrogels, subjected to HP or kept in free swelling conditions, and were cultured either with or without pharmacological inhibitors of Ca++ mobility and downstream targets. Chelating free Ca++, inhibiting voltage-gated calcium channels, and depleting intracellular calcium storessuppressed the beneficial effect of HP on chondrogenesis, indicating that Ca++ mobility may play an important role in the mechanotransduction of HP. However, inhibition of stretch-activated calcium channels in the current experiment yielded similar results to the control group, suggesting that mechanotransduction of HP is distinct from loads that generate cell deformations. Inhibition of the downstream targets calmodulin, calmodulin kinase II, and calcineurin all knocked down the effect of HP on chondrogenesis, implicating these targets in MSCs response to HP. All of the pharmacological inhibitors that abolished the chondrogenic response to HP also maintained a punctate vimentin organisation in the presence of HP, as opposed to the mechanoresponsive groups where the vimentin structure became more diffuse. These results suggest that Ca++ signalling may transduce HP via vimentin adaptation to loading.

Regulation of vectorial supply of vesicles to the hyphal tip determines thigmotropism in Neurospora crassa

Thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. The thigmotropic response of the model fungus Neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. We show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of N. crassa. Deletion of N. crassa genes encoding the formin, BNI-1, and the Rho-GTPase, CDC-42, an activator of BNI-1 in yeast, CDC-24, its guanine nucleotide exchange factor (GEF), and BEM-1, a scaffold protein in the same pathway, were all shown to significantly decrease the thigmotropic response. In contrast, deletion of genes encoding the cell end-marker protein, TEA-1, and KIP-1, the kinesin responsible for the localisation of TEA-1, significantly increased the thigmotropic response. These results suggest a mechanism of thigmotropism involving vesicle delivery to the hyphal tip via the actin cytoskeleton and microtubules. Neurospora crassa thigmotropic response differed subtly from that of Candida albicans where the stretch-activated calcium channel, Mid1, has been linked with thigmotropic behaviour. The MID-1 deficient mutant of N. crassa (Δmid-1) and the effects of calcium depletion were examined here but no change in the thigmotropic response was observed. However, SPRAY, a putative calcium channel protein, was shown to be required for N. crassa thigmotropism. We propose that the thigmotropic response is a result of changes in the polarity machinery at the hyphal tip which are thought to be downstream effects of calcium signalling pathways triggered by mechanical stress at the tip.

High Concentration of Gadolinium Ion Modifying Isolated Rice Mitochondrial Biogenesis

Mitochondria play an important role in plant growth and development, cooperating with the endoplasmic reticulum and nucleus. Gadolinium, one of the rare earth elements, is an inhibitor of stretch-activated calcium channels located on the endoplasmic reticulum and plasma membrane and has no effect on nuclear calcium variation in plant cells. We analyzed the effects of Gd3+ on mitochondria function by monitoring mitochondrial swelling, changes of membrane fluidity, and transmembrane potential collapse and by observing mitochondrial ultrastructure. We found that high concentration of Gd3+ induces rice mitochondrial dysfunction through mitochondrial permeability transition (MPT). The protection of DTT and EDTA demonstrate that Gd3+ blocks the inner membrane ion channel through thiol chelation.

Neuroprotective effect of gadolinium: a stretch-activated calcium channel blocker in mouse model of ischemia–reperfusion injury

The present study was designed to investigate the potential of gadolinium, a stretch-activated calcium channel blocker in ischemic reperfusion (I/R)-induced brain injury in mice. Bilateral carotid artery occlusion of 12min followed by reperfusion for 24h was given to induce cerebral injury in male Swiss mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was assessed using Morris water maze test and motor incoordination was evaluated using rota-rod, lateral push, and inclined beam walking tests. In addition, total calcium, thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH), and acetylcholinesterase (AChE) activity were also estimated in brain tissue. I/R injury produced a significant increase in cerebral infarct size. A significant loss of memory along with impairment of motor performance was also noted. Furthermore, I/R injury also produced a significant increase in levels of TBARS, total calcium, AChE activity, and a decrease in GSH levels. Pretreatment of gadolinium significantly attenuated I/R-induced infarct size, behavioral and biochemical changes. On the basis of the present findings, we can suggest that opening of stretch-activated calcium channel may play a critical role in ischemic reperfusion-induced brain injury and that gadolinium has neuroprotective potential in I/R-induced injury.

Osteoblasts detect pericellular calcium concentration increase via neomycin-sensitive voltage gated calcium channels

The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.

Intracellular Ca2+ accumulation is strain-dependent and correlates with apoptosis in aortic valve fibroblasts

Aortic valve (AV) disease is often characterized by the formation of calcific nodules within AV leaflets that alter functional biomechanics. In vitro, formation of these nodules is associated with osteogenic differentiation and/or increased contraction and apoptosis of AV interstitial cells (AVICs), leading to growth of calcium phosphate crystal structures. In several other cell types, increased intracellular Ca2+ has been shown to be an important part in activation of osteogenic differentiability. However, elevated intracellular Ca2+ is known to mediate cell contraction, and has also been shown to lead to apoptosis in many cell types. Therefore, a rise in intracellular Ca2+ may precede cellular changes that lead to calcification, and fibroblasts similar to AVICs have been shown to exhibit increases in intracellular Ca2+ in response to mechanical strain. In this study, we hypothesized that strain induces intracellular Ca2+ accumulation through stretch-activated calcium channels. We were also interested in assessing possible correlations between intracellular Ca2+ increases and apoptosis in AVICs. To test our hypothesis, cultured porcine AVICs were used to assess correlates between strain, intracellular Ca2+, and apoptosis. Ca2+ sensitive fluorescent dyes were utilized to measure real-time intracellular Ca2+ changes in strained AVICs. Ca2+ changes were then correlated with AVIC apoptosis using flow cytometric Annexin V apoptosis assays. These data indicate that strain-dependent accumulation of intracellular Ca2+ is correlated with apoptosis in AVICs. We believe that these findings indicate early mechanotransductive events that may initiate AV calcification pathways.

Calcium Signaling in Live Cells on Elastic Gels under Mechanical Vibration at Subcellular Levels

A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels.

Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice

In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin is related to enhanced calcium influx and muscle degeneration. Stretch-activated channels (SACs) might be directly involved in the pathology of DMD, and transient receptor potential cation channels have been proposed as likely candidates of SACs. We investigated the levels of transient receptor potential canonical channel 1 (TRPC1) and the effects of streptomycin, a SAC blocker, in muscles showing different degrees of the dystrophic phenotype. Mdx mice (18 days old, n = 16) received daily intraperitoneal injections of streptomycin (182 mg/kg body wt) for 18 days, followed by removal of the diaphragm, sternomastoid (STN), biceps brachii, and tibialis anterior muscles. Control mdx mice (n = 37) were injected with saline. Western blot analysis showed higher levels of TRPC1 in diaphragm muscle compared with STN and limb muscles. Streptomycin reduced creatine kinase and prevented exercise-induced increases of total calcium and Evans blue dye uptake in diaphragm and in STN muscles. It is suggested that different levels of the stretch-activated calcium channel protein TRPC1 may contribute to the different degrees of the dystrophic phenotype seen in mdx mice. Early treatment designed to regulate the activity of these channels may ameliorate the progression of dystrophy in the most affected muscle, the diaphragm.

Cortical Oscillations as a Model for Studying Cytoskeleton Regulation During Cell Spreading

Cell spreading and attachment are integral to multiple physiological processes including wound healing, immune cell-antigen recognition, and tumor cell metastasis. We have discovered that Swiss 3T3 fibroblasts and CHO cells undergo periodic oscillations of the cell body during cell spreading that last from .5 hours after attachment to 1.5 hours and longer. The amplitude and duration of the oscillating phenotype are increased when microtubules are depolymerized. Previously we developed a mechanochemical ODE model describing a possible negative feedback from actomyosin based contractility to stretch-activated calcium channels in propagating cell oscillations. Cortical oscillations provide an ideal model for studying cytoskeleton regulation because the oscillation mechanism is easily quantifiable through the relative phase, amplitude, and period of native oscillations vs. those that have experienced perturbations. Further, we examine the spatiotemporal distribution of [Ca2+i] during cell oscillations. We propose that the interplay of the calcium and Rho A pathways both contribute to the propagation of cortical oscillations, with high levels of active Rho A replacing the need for highly dynamic calcium signaling.

Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence

The putative Claviceps purpurea homologue of the Saccharomyces cerevisiae stretch-activated calcium ion channel Mid1 was investigated for its role in vegetative growth, differentiation and pathogenicity on rye (Secale cereale). Gene replacement mutants of Cl. purpurea mid1 were not affected in polar growth and branching in axenic culture but showed a significantly reduced growth rate. The growth defect could not be complemented by Ca(2+) supplementation, in contrast to mid1 mutants in yeast, but the altered sensitivity of the mutants to changes in external and internal Ca(2+) concentrations indicates some role of Mid1 in Ca(2+) homeostasis. The major effect of mid1 deletion, however, was the complete loss of virulence: infected rye plants showed no disease symptoms at all. Detailed analyses of in vitro-infected rye ovaries demonstrated that the Deltamid1 mutants had multiple apical branches and were unable to infect the host tissue, suggesting that Mid1 is essential for generating the necessary mechanical force for penetration. This is believed to be the first report of an essential role for a Mid1 homologue in the virulence of a plant-pathogenic fungus.

T.P.2.08 The stretch-activated calcium channel candidate proteins TRPC1 and 3 present a distinctly different expression pattern in mdx mice

T.P.2.08 The stretch-activated calcium channel candidate proteins TRPC1 and 3 present a distinctly different expression pattern in mdx mice

TRPV4 Channels Mediate Cyclic Strain–Induced Endothelial Cell Reorientation Through Integrin-to-Integrin Signaling

Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response; however, the molecular basis remains unknown. Here, we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 (transient receptor potential vanilloid 4) ion channels that, in turn, stimulate phosphatidylinositol 3-kinase-dependent activation and binding of additional beta1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knock down of TRPV4 channels using specific small interfering RNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent "integrin-to-integrin" signaling mechanism mediated by force-induced activation of mechanically gated TRPV4 ion channels on the cell surface.

NMDA receptor expression and activity in osteoarthritic human articular chondrocytes

Classical neuronal signalling molecules such as substance P and glutamate have been identified in cartilage and have roles in regulation of chondrocyte function. This study looks at expression and activity of the ionotropic glutamate NMDA (N-methyl-D-aspartic acid) receptor (NMDAR) in human osteoarthritic (OA) chondrocytes. Chondrocytes were obtained from human knee joint arthroplasty specimens. NMDAR subunits and PSD-95 (postsynaptic density protein 95) expression were analysed by reverse transcription-polymerase chain reaction and Western blotting. Activity of NMDAR was assayed by radioactive calcium(45) uptake and changes in membrane potential in the presence and absence of NMDA and NMDAR antagonists and blockade of cell membrane ion channels. NMDAR 1, 2A, 2B and PSD-95 were detected in human OA chondrocytes whereas NR2B was absent from normal chondrocytes. NMDA induced calcium flux into OA chondrocytes and cell membrane depolarisation. These responses were blocked by NMDAR antagonists, removal of extracellular calcium, inhibition of nNOS (neuronal nitric oxide synthase) activity and uncoupling of NMDAR from PSD-95. Blockade of sodium channels by tetrodotoxin resulted in NMDA-induced membrane hyperpolarisation which was, in turn inhibited by apamin, a blocker of SK channels. NMDA-induced changes in cell membrane potential were not affected by l-type and stretch activated calcium channel inhibitors. Human OA and normal articular chondrocytes differ in the expression of NMDAR subunits. In OA chondrocytes NMDAR signalling requires extracellular calcium, association with PSD-95, and nNOS activity. Downstream signalling results in activation of tetrodotoxin sensitive sodium channels and SK channels, a response that differs from that of normal chondrocytes suggesting altered activity of NMDAR in OA.