Regulatory Volume Decrease Process Research Articles

Initiation of RVD response in human platelets: mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A2.

Platelets revert hypotonic-induced swelling by the process of regulatory volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A3. In this work, we investigated the mechanical-biochemical transduction that leads to hepoxilin A3 formation. We demonstrate that this process is mediated by pertussis-toxin-sensitive G protein, which activates Ca(2+)-insensitive phospholipase A2, and the sequential release of arachidonic acid. This conclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A2 inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 microM, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca2+ depletion by EGTA (up to 10 mM) does not affect RVD; (iii) intracellular Ca2+ depletion by BAPTA-AM (100 microM) inhibits RVD but not hepoxilin A3 formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP beta S (1 microM) and pertussis toxin (1 ng/ml). This inhibition is overcome by addition of arachidonic acid or hypotonic cell-free eluate that contains hepoxilin A3; (v) NaF, 1 mM, induces hepoxilin A3 formation, tested by the RVD reconstitution assay; and (vii) GDP beta S inhibits hepoxilin A3 formation associated with flow. Therefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A3 formation in human platelets.

Dietary n-3 and n-6 fatty acids are equipotent in stimulating volume regulation in Ehrlich ascites tumor cells.

This investigation addresses whether enriching cellular phospholipids with n-3 or n-6 fatty acids affects the process of regulatory volume decrease (RVD) in murine Ehrlich ascites tumor cells. Two weeks of dietary n-3-rich fish oil (7.5%, wt/wt) increased the ratio of eicosapentaenoic acid to arachidonic acid in cellular phospholipids compared with an olive oil control diet. Cells grown in mice fed on fish oil had an accelerated RVD response after hypotonic exposure, indicating that the volume-induced K+ conductance was increased. The fish oil diet furthermore resulted in an increased Cl- conductance during RVD, demonstrated as an increased initial rate of cell shrinkage after addition of K+ ionophore to the swollen cells. The initial rate of volume recovery correlated positively with the sum of eicosanoid precursors (arachidonic acid plus eicosapentaenoic acid) (P = 0.007). Diet supplemented with n-6 fatty acids resulted in an enhanced RVD response as well. RVD was inhibited by anti-calmodulin drugs, and exogenous leukotriene D5 and leukotriene D4 were equipotent in attenuating this inhibition. We conclude that dietary polyunsaturated fatty acids result in a more effective RVD response because of an increase in the volume-induced Cl- and K+ conductances. We propose that this is caused by an enhanced volume-induced leukotriene synthesis due to an increase in eicosanoid precursor availability.

Volume-Regulatory Ion Channels in Epithelial Cells

Patch-clamp studies showed that the regulatory volume decrease process is closely associated with functions of K+, Cl-, and Ca2+-permeable channels in epithelial cells. Na+-permeable channels in the regulatory volume increase process may also be involved, as deduced from whole cell recordings using intestinal epithelial cells.

Ca2+ sensitivity of volume‐regulatory K+ and Cl‐ channels in cultured human epithelial cells.

1. During exposure to a hypotonic solution, cultured human epithelial cells (Intestine 407) exhibited a regulatory volume decrease (RVD) after initial osmotic swelling. 2. The volume readjustment was slowed by elevating the extracellular K+ concentration and facilitated by reducing the extracellular Cl- concentration. Not only putative K+ channel blockers, quinine and Ba2+, but also a stilbene derivative Cl- channel blocker (SITS) inhibited the RVD. 3. The volume recovery of hypoosmotically swollen cells was very much suppressed by the deprivation of extracellular Ca2+ ions or by chelation of cytosolic Ca2+ ions with Quin-2 loaded within the cells. 4. Biphasic membrane potential changes were associated with the RVD process at low extracellular K+ and Cl- concentrations. The initial hyperpolarizing response was inhibited by quinine and Ba2+, whereas the late depolarizing response was inhibited by SITS. The deprivation of extracellular Ca2+ inhibited the initial hyperpolarizing phase but not the late depolarizing phase. 5. Two-microelectrode voltage clamp studies showed that the initial hyperpolarization and late depolarization were associated with quinine-sensitive outward currents and SITS-sensitive inward currents, respectively. The reversal potentials estimated from the current-voltage curves were about -80 mV for the initial response and -27 mV for the late response. Tenfold changes in the K+ and Cl- concentrations shifted these reversal potentials by 50 mV for the initial response and by 42 mV for the late response. 6. Under whole-cell recordings, similar current changes were observed in the cells exposed to a hypotonic solution, when the intracellular Ca2+ ions were moderately buffered with 1 mM-EGTA in the dialysing solution filled in a patch pipette. When most Ca2+ ions were chelated with 10 mM-EGTA in the pipette solution, the initial outward current as well as the corresponding hyperpolarization was suppressed, but the late current associated with the depolarizing phase was preserved. 7. Intracellular Ca2+ injections induced an increase in the quinine-sensitive K+ conductance but failed to activate the Cl- conductance. 8. It is concluded that both K+ and Cl- channels are involved in the regulatory volume decrease, and that the former channel is exclusively activated by elevation of the cytosolic Ca2+ concentration in the epithelial cells.