Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways

Protease-activated receptors (PARs), newlyidentified members of G protein-coupled receptors, are widelydistributed in the brain. Thrombin evokes multiple cellular responsesin a large variety of cells by activating PAR-1, -3, and -4. Incultured rat astrocytes we investigated the signaling pathway ofthrombin- and PAR-activating peptide (PAR-AP)-induced cellproliferation. Our results show that PAR activation stimulatesproliferation of astrocytes through the ERK pathway. Thrombinstimulates ERK1/2 phosphorylation in a time- andconcentration-dependent manner. This effect can be fully mimicked by aspecific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP.PAR-2-AP can induce a moderate ERK1/2 activation as well.Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 viatwo branches: 1) the PTX-sensitive Gprotein/(-subunits)-phosphatidylinositol 3-kinase branch, and2) the G_q-PLC-(InsP₃receptor)/Ca²⁺-PKC pathway. Thrombin- or PAR-1-AP-inducedERK activation is partially blocked by a selective EGF receptorinhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptoris unlikely for ERK1/2 activation and is certainly not involved inPAR-1-induced proliferation. The metalloproteinase mechanism involvingtransactivation of the EGF receptor by released heparin-binding EGF wasexcluded. EGF receptor activation was detected by the receptorautophosphorylation site, tyrosine 1068. Our data suggest thatthrombin-induced mitogenic action in astrocytes occurs independently ofEGF receptor transphosphorylation.