Mechanically Induced Calcium Waves in Articular Chondrocytes are Inhibited by Gadolinium and Amiloride

Abstract

Summary:Chondrocytes in articular cartilage utilize mechanical signals from their environment to regulate their metabolic activity. However, the sequence of events involved in thc transduction of mechanical signals to a biochemical signal is not fully undcrstood. It has been proposed that an increase in the concentration of intracellular calcium ion ([Ca2+Ii) is one of the earliest events in the process of cellular mechanical signal transduction. With use of fluorescent confocal microscopy, [Ca2+], was monitored in isolated articular chondrocytes subjectcd to controlled deformation with the edge of a glass micropipette. Mechanical stimulation resulted in an immediate and transienl increasc in [&*+Ii. The initiation of Ca2+ waves was abolished by removing Ca2+ from the extraccllular mcdia and was significantly inhibited by the presence of gadolinium ion (10 pM) or amiloride (I mM), which have previously been reported lo block mechanosensitive ion channels.Inhibitors of intracellular Ca2+ release (dantrolene and 8-dielhylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride) or cytoskeletal disrupting agents (cytochalasin D and colchicine) had no significant effect on the characteristics of the Ca2+ waves. These findings suggest that a possible mechanism of Ca2+ mobilization in this case is a self-reinforcing influx of Ca2+ from the extracellular media, initiated by a Caz+-permeable mechanosensitive ion channel. Our results indicate that a transient increase in intracellular Ca2+ concentration may be one of the earliest events involved in the response of chondrocytes to mechanical stress and support the hypothesis that deformation-induced Ca2+ waves are initiated through mechanosensitive ion channels.Articular cartilage is maintained in a balanced state of turnover by the chondrocyte cell population. These cells are highly sensitive to their biophysical environment and rely on mechanical signals together with genetic and other environmental factors to regulate their metabolic activity (29). Under physiological loading conditions, chondrocytes undergo significant changcs in shape due to deformation of the cartilage extracellular matrix (27,28,63), suggesting that cell deformation may serve as a signal in the process of mechanical signal transduction. The biological mechanisms by which such cellular deformation is transduced to an intracellular signal are still unclear but seem to involve the traditional intracellular second messenger pathways, such as production of cyclic adenosine

Mechanically induced calcium signaling in chondrocytes in situ

“…However, direct contact deformation of isolated chondrocytes through micropipette indentation resulted in virtually instantaneous calcium signaling suggesting that calcium signaling is not a downstream but a first response event (Table 1). 15,17,19 Considering these data together, we speculate that mechanical deformations induce intracellular calcium signaling, and that chondrocytes have a different response mechanism in the intact cartilage compared to cells embedded in in vitro agarose or monolayer constructs. Our findings suggest a direct relationship between tissue deformation and calcium signaling, and a pathway where calcium signaling may depend on matrix-cell interactions in the intact cartilage through cytoskeletal and trans-membrane proteins.…”

“…[10][11][12][13] Calcium is an ubiquitous second messenger which regulates many cellular processes including metabolism, proliferation, gene transcription, contraction, and mechanotransduction. 14 Previous studies have suggested that Ca 2þ signals form part of an intricate mechanotransduction pathway in chondrocytes: physical stimuli, such as compression, [15][16][17][18][19] fluid flow, [20][21][22] hydrostatic pressure, 23,24 and osmotic stress, [25][26][27][28][29] influence Ca 2þ signaling in chondrocytes. Therefore, insight into mechanically induced Ca 2þ signaling may provide novel understanding of chondrocyte mechanotransduction.…”

“…Previous research into compression induced articular cartilage mechanotransduction has been focused on mechanical loading of chondrocytes isolated from their native tissue and exposed to non-physiological boundary conditions; such as micropipette aspiration of isolated chondrocytes 15,17 and isolated chondrocytes seeded in agarose constructs. 16,18,30 However, these studies neglect the potential role of signaling pathways involving transmembrane proteins linked to the ECM, as well as the specialized peri-cellular matrices that affect chondrocyte mechanics, 31,32 and thus, the biological responses of chondrocytes to mechanical loading conditions.…”

The enhancement of periosteal chondrogenesis in organ culture by dynamic fluid pressure

“…Alternatively, cells could respond directly to pressur through specific mechanoreceptors. The presence of potential mechanoreceptor molecules, like integrins [35] and stretch activated ion channels [8] strengthen this line of thinking.…”

Two-Photon Excitation Laser Scanning Microscopy of Human, Porcine, and Rabbit Nasal Septal Cartilage

“…[1][2][3] However, CLSM has significant limitations, which include (1) the use of exogenous stains which are diffusion limited in distribution; (2) shallow penetration depth due to the shorter wavelengths; (3) photobleaching due to the high-intensity laser source; and (4) toxicity to living tissue (photothermal and photochemical injury), which precludes high-resolution vital imaging. While the use of ultraviolet (UV) sources in CLSM enables excitation of endogenous fluorophores, these fluorophores suffer the limitations listed above.…”