Effects of cell swelling on intracellular calcium and membrane currents in bovine articular chondrocytes

Abstract

Chondrocytes experience a dynamic extracellular osmotic environment during normal joint loading when fluid is forced from the matrix, increasing the local proteoglycan concentration and therefore the ionic strength and osmolarity. To exist in such a challenging environment, chondrocytes must possess mechanisms by which cell volume can be regulated. In this study, we investigated the ability of bovine articular chondrocytes (BAC) to regulate cell volume during a hypo-osmotic challenge. We also examined the effect of hypo-osmotic stress on early signaling events including [Ca2+](i) and membrane currents. Changes in cell volume were measured by monitoring the fluorescence of calcein-loaded cells. [Ca2+](i) was quantified using fura-2, and membrane currents were recorded using patch clamp. BAC exhibited regulated volume decrease (RVD) when exposed to hypo-osmotic saline which was inhibited by Gd3+. Swelling stimulated [Ca2+](i) transients in BAC which were dependent on swelling magnitude. Gd3+, zero [Ca2+](o), and thapsigargin all attenuated the [Ca2+](i) response, suggesting roles for Ca2+ influx through stretch activated channels, and Ca2+ release from intracellular stores. Inward and outward membrane currents significantly increased during cell swelling and were inhibited by Gd3+. These results indicate that RVD in BAC may involve [Ca2+](i) and ion channel activation, both of which play pivotal roles in RVD in other cell types. These signaling pathways are also similar to those activated in chondrocytes subjected to other biophysical signals. It is possible, then, that these signaling events may also be involved in a mechanism by which mechanical loads are transduced into appropriate cellular responses by chondrocytes.

Mechanically induced calcium signaling in chondrocytes in situ

“…Results from previous studies suggest that calcium signaling in chondrocytes occurs via stretch activated calcium channels 14,43 and/or via stretch activated release of ATP. [44][45][46][47][48] In order to investigate these possible mechanotransduction pathways, we plan to perform studies in which stretch activated calcium channels are inhibited by gadolinium and intracellular calcium release is inhibited via thapsigargin. 30,44,45 Calcium signaling occurred faster and had higher amplitudes at 378C than at 218C, indicating that Ca 2þ signaling is temperature dependent, as has been found for other cell types.…”

“…[44][45][46][47][48] In order to investigate these possible mechanotransduction pathways, we plan to perform studies in which stretch activated calcium channels are inhibited by gadolinium and intracellular calcium release is inhibited via thapsigargin. 30,44,45 Calcium signaling occurred faster and had higher amplitudes at 378C than at 218C, indicating that Ca 2þ signaling is temperature dependent, as has been found for other cell types. 34,35 This finding may have significant functional implications as cartilage is an avascular tissue known to change temperature significantly when exposed to ambient temperature changes 49,50 which, according to our results, may affect the ability of chondrocytes to respond to mechanical stimuli.…”

Non‐enzymatic glycation of chondrocyte‐seeded collagen gels for cartilage tissue engineering

“…Hyperosmotic solutions affect gene expression, intracellular pH and calcium levels, cellular metabolism, as well as the physical and viscoelastic properties of chondrocytes. [41][42][43][44][45][46][47][48][49][50] Hyperosmotic stress has also recently been shown to lead to apoptotic cell death and plausible that the osmotic environment in the post-glycation processing method may be cytotoxic. 51 Figure 1.…”

Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading

“…5, the triggering of calcium transients in response to hypotonic loading may be related to the extent of volume/membrane expansion (17). Calcium is one candidate signaling molecule that has been shown to be important in chondrocyte mechanotransduction and can be monitored during stimulus application (2,13,38,39,47,62,64).…”

“…These studies have adopted manual solution changes or employed specialized chambers to achieve osmotic loading (24,64). Although sufficient for static or even low-frequency experiments, these methodologies are not practical for the study of higher-frequency osmotic loading, and may be complicated by the shear effects that accompany fluid application.…”