Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes

Abstract

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.

Fig. 1

Effect of Ni²⁺ on whole-cell currents in bovine articular chondrocytes. A: Typical current traces in the absence (i) and in the presence (ii) of 5 mM Ni²⁺ and the Ni²⁺-sensitive current obtained (iii) by subtracting (ii) from (i). B: I-V curve derived from the data in A.

Fig. 2

Effect of [Ca²⁺]_p on the Ni²⁺-sensitive currents in bovine articular chondroytes. A: Typical I-V curves obtained at [Ca²⁺]_p of 0, 102, 334 and 540 nM as indicated. B: Relationship between [Ca²⁺]_p and the outward Ni²⁺-sensitive current measured at +80 mV. C: Relationship between [Ca²⁺]_p and the inward Ni²⁺-sensitive current measured at −120 mV. Data points represent mean ± SEM (n = 4).

Fig. 3

Effect of [Ca²⁺]_o on the Ni²⁺-sensitive currents in bovine articular chondroytes. A: Typical I-V curves obtained at [Ca²⁺]_o of 0, 1, 2 and 5 mM as indicated. B: Relationship between [Ca²⁺]_o and the outward Ni²⁺-sensitive current measured at +80 mV. C: Relationship between [Ca²⁺]_o and the inward Ni²⁺-sensitive current measured at −120 mV. Data points represent mean ± SEM (n = 4).

Fig. 4

Effect of [Na⁺]_p on the Ni²⁺-sensitive currents in bovine articular chondroytes. A: Typical I-V curves obtained at [Na⁺]_p of 0, 10, 20 and 40 mM as indicated. B: Relationship between [Na⁺]_p and the outward Ni²⁺-sensitive current measured at +80 mV. C: Relationship between [Na⁺]_p and the inward Ni²⁺-sensitive current measured at −120 mV. Data points represent mean ± SEM (n = 4).

Fig. 5

Effect of [Na⁺]_o on the Ni²⁺-sensitive currents in bovine articular chondroytes. A: Typical I-V curves obtained at [Na⁺]_o of 0, 75 and 140 mM as indicated. B. Relationship between [Na⁺]_o and the outward Ni²⁺-sensitive current measured at +80 mV. C: Typical I-V curves obtained at [Na⁺]_o of 0, 50, 100 and 140 mM as indicated, using a [Na⁺]_p of 5 mM. The latter enabled inward Ni²⁺-sensitive currents at negative potentials to be measured, which was not possible when using the standard [Na⁺]_p of 20 mM. D. Relationship between [Na⁺]_o and the inward Ni²⁺-sensitive current measured at −120 mV. Data points represent mean ± SEM (n = 4).