A major hallmark of apoptosis is normotonic shrinkage of cells. Here, we studied the relation between apoptotic cell shrinkage and apoptotic cell death. Induction of the apoptotic volume decrease (AVD) under normotonic conditions was found to be coupled to facilitation of the regulatory volume decrease (RVD), which is known to be attained by parallel operation of Cl ⁻ and K ⁺ channels, under hypotonic conditions. Both the AVD induction and the RVD facilitation were found to precede cytochrome c release, caspase-3 activation, DNA laddering, and ultrastructural alterations in three cell types after apoptotic insults with two distinct apoptosis inducers. Also, the AVD was not prevented by a broad-spectrum caspase inhibitor. When the AVD induction and the RVD facilitation were prevented by blocking volume-regulatory Cl ⁻ or K ⁺ channels, these cells did not show succeeding apoptotic biochemical and morphological events and were rescued from death. Thus, it is concluded that the AVD, which is caused by disordered cell volume regulation, is an early prerequisite to apoptotic events leading to cell death.

Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl] _L ) and transmit signals to the mesangial cell/afferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch–clamp studies of macula densa cells, we identified an [NaCl] _L -sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 μM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl] _L , by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca ²⁺ concentration to extracellular application of ATP (EC ₅₀ 0.8 μM). Importantly, we also found increases in cytosolic Ca ²⁺ concentration with elevations in [NaCl] _L , when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y ₂ receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

Apoptotic volume decrease is a pivotal event triggering a cell to undergo apoptosis and is induced by ionic effluxes resulting mainly from increased K⁺and Cl^-conductances. Here, we demonstrate that in human epithelia HeLa cells both mitochondrion- and death receptor-mediated apoptosis inducers [staurosporine and Fas ligand or tumor necrosis factor (TNF)-α] rapidly activate Cl^-currents that show properties phenotypical of volume-sensitive outwardly rectifying Cl^-channel currents, including outward rectification, voltage-dependent inactivation gating at large positive potentials, inhibition by osmotic shrinkage, sensitivity to classic Cl^-channel blockers, and dependence on cytosolic ATP. Staurosporine, but not Fas ligand or TNF-α, rapidly (within 30 min) increased the intracellular level of reactive oxygen species (ROS). A ROS scavenger and an NAD(P)H oxidase inhibitor blocked the current activation by staurosporine but not by Fas ligand or TNF-α. A ROS scavenger also inhibited apoptotic volume decrease, caspase-3 activation, and apoptotic cell death induced by staurosporine. Thus, it is concluded that an apoptosis-triggering anion conductance is carried by the volume-sensitive outwardly rectifying Cl^-channel and that the channel activation on apoptotic stimulation with staurosporine, but not with Fas ligand or TNF-α, is mediated by ROS.

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg²⁺- and Gd³⁺-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg²⁺ and Gd³⁺. The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca²⁺ was removed or Gd³⁺ was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca²⁺ influx pathway, it plays an important role in cell volume regulation.