Changes in intracellular calcium (Ca 2þ ) concentration, also known as Ca 2þ signaling, have been widely studied in articular cartilage chondrocytes to investigate pathways of mechanotransduction. Various physical stimuli can generate an influx of Ca 2þ into the cell, which in turn is thought to trigger a range of metabolic and signaling processes. In contrast to most studies, the approach used in this study allows for continuous real time recording of calcium signals in chondrocytes in their native environment. Therefore, interactions of cells with the extracellular matrix (ECM) are fully accounted for. Calcium signaling was quantified for dynamic loading conditions and at different temperatures. Peak magnitudes of calcium signals were greater and of shorter duration at 378C than at 218C. Furthermore, Ca 2þ signals were involved in a greater percentage of cells in the dynamic compared to the relaxation phases of loading. In contrast to the time-delayed signaling observed in isolated chondrocytes seeded in agarose gel, Ca 2þ signaling in situ is virtually instantaneous in response to dynamic loading. These differences between in situ and in vitro cell signaling responses might provide crucial insight into the role of the ECM in providing pathways of mechanotransduction in the intact cartilage that are absent in isolated cells seeded in gel constructs.

SUMMARYThe intracellular ionic distribution in uncleaved and cleaving Ambystoma eggs was investigated by analysing the influx of 3H,0, by determining the total content of Na+, K+ and Cl-in extracts of eggs at different stages by both flame spectrophotometry and ion-selective microelectrodes, and by the continuous measurement of the Na+, K+ and Cl-activities (uh, uk and a&) using intracellular ion-selective microelectrodes. The electrical membrane potential (E,) and membrane resistance (R,) were measured continuously in uncleaved and normally cleaving eggs as well as in eggs cleaving after removal of the vitelline membrane. The latter eggs expose their newly formed cleavage membrane to the external medium. Ionic permeability of the cell membrane before and during cleavage was analysed by a statistical comparison of the experimentally determined relationship between E,,, and the ionic gradients across the cell membrane with those predicted theoretically from a constant field equation in dependence on the relative permeability, through insertion of the measured intracellular ion activities.3H,0 influx revealed the existence of a single intracellular water compartment (3.06 @l/egg) and a low water permeability (5.35 x 10M5 cm set-I). Na +, K+ and Cl-concentrations were constant at 54.1, 72.1 and 73.1 mM respectively, while nk,, uk and u&r were constant at 5.8, 51.8 and 59.7 mM respectively. It was concluded that all Cl-ions are in solution, while 12.5 % of all K+ and 86 % of all Na+ is bound. The uncleaved egg showed a positive E,,, of ca 40 mV and a specific membrane resistance of 39 kOhm cm'. E,,, could be described by a constant field equation with a permeability ratio P K /P N,==O.O73. Shortly after the onset of first cleavage, E,,, rapidly decreased concomitant with a rise in R, (68.5 kOhm cm2). This was interpreted as a drop in Nat permeability. During the cleavage process E, progressively hyperpolarized and R, decreased due to the insertion of a small fraction (3.3 %) of the newly formed intercellular membrane into the cleavage furrow. This new membrane had a low specific resistance (0.69 kOhm cm"). Both in normally cleaving eggs and in eggs cleaving in the absence of the vitelline membrane E,,, behaved according to the constant field equation, PNa/PK being 0.69 and 0.39, respectively. The differences with other amphibian eggs were discussed.In a series of previous reports [IA] we have small electrical membrane potential-E,-presented a detailed description of the changes (-10 mV) and a high specific membrane rein electrical membrane properties, cell mem-sistance (74 kOhm cm2), while any permbrane permeability, and intracellular distriselectivity was absent. Starting at 6-8 min bution of Na+, Kf and Cl-ions during the after the onset of first cleavage, E, hyperfirst cleavage of eggs of the amphibian polarized from -10 to -20 mV and the Xenopus laevis. The uncleaved egg showed a electrical membrane resistance-R,,-de- creased from 850 to 500 kOhm. The intracellular Na+, K+ and Cl-concentrations 59, 87, 63 mM/l ...