Hypertonic Shock

Abstract: Hyperosmotic stress can be encountered by the kidney and the skin, as well as during treatment of acute brain damage. It can lead to cell cycle arrest or apoptosis. Exactly how mammalian cells dete...

Abstract: The evolution of the volume, the Na+ and K+ contents and the glycerol and ATP contents were investigated after subjectingDunaliella tertiolecta cells to hypertonic shocks. It was found that the variations in the glycerol and the ion contents superimpose as the cell regulates its volume. Hypertonic shock induces a rapid increase (some minutes) in the Na+ influx and Na+ content followed by a decrease until a new steady value is reached after 30 min of cell transfer. The regulatory mechanism extruding Na+ out of the cells was dependent on the presence of K− or Rb− ions in the external medium. A transient pumping of K+ ions was found after subjecting the cells to a hypertonic shock. This increase in K+ content resulted from the transient increase in the K+ influxes. The K+ pumping mechanism was blocked by the absence of Ca++ and Mg++ ions in the external medium and was inhibited by DCCD, FCCP and DCMU, whereas ouabain, cyanide and PCMBS were ineffective. The increase in K+ content was observed if the hypertonic shock was induced by the addition of NaCl, glycerol or choline chloride. These results are interpreted on the basis of two distinct mechanisms: a Na−/K− exchange pump and a Na+ independent K+ pump. These ionic transfer mechanisms would participate in the osmoregulation ofDunaliella cells and would be of importance, particularly during the onset of the osmotic shock when glycerol synthesis is incomplete.

Abstract: Salmonid fish sperm motility is known to be suppressed in millimolar concentrations of extracellular K(+), and dilution of K(+) upon spawning triggers cAMP-dependent signaling for motility initiation. In a previous study, however, we demonstrated that suspending sperm in a 10% glycerol solution and subsequent dilution into a low-osmotic solution induced motility independently of extracellular K(+) and cAMP. In the present study, we further investigated the glycerol-induced motility mechanism. We found that treatment with solutions consisting of organic or inorganic ions, as well as glycerol, induced sperm motility in an osmolarity-dependent manner. Elimination of intracellular Ca(2+) by BAPTA-AM significantly inhibited glycerol-treated sperm motility, whereas removal of extracellular Ca(2+) by EGTA did not. Monitoring intracellular Ca(2+), using fluo-4, revealed that intracellular Ca(2+) increased when sperm were suspended in hypertonic solutions, and a subsequent dilution into a hypotonic solution led to a decrease in intracellular Ca(2+) concomitant with motility initiation. In addition, upon dilution of sperm into a hypertonic glycerol solution prior to demembranation, the motility of demembranated sperm was reactivated in the absence of cAMP. The motility recovery suggests that completion of axonemal maturation occurred during exposure to a hypertonic environment. As a result, it is likely that glycerol treatment of sperm undergoing hypertonic shock causes mobilization of intracellular Ca(2+) from the intracellular Ca(2+) store and also causes maturation of axonemal proteins for motility initiation. The subsequent dilution into a hypotonic solution induces a decrease in intracellular Ca(2+) and flagellar movement. This novel mechanism of sperm motility initiation seems to act in a salvaging manner for the well-known K(+)-dependent pathway.

Abstract: ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mm). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.