leupeptins and benzamil

Cytosolic Ca(2+) concentration ([Ca(2+)](i)) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage-gated Ca(2+) channels restores cytosolic Ca(2+) levels and reduces this neuronal death (Snider et al. 2002). We now show that this reduction in [Ca(2+)](i) is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca(2+) influx such as activation of voltage-gated Ca(2+) channels or stimulation of Ca(2+) entry via the plasma membrane Na-Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage-gated Ca(2+) channels and a less robust increase in [Ca(2+)](i) after depolarization. Levels of endoplasmic reticulum Ca(2+) were reduced and capacitative Ca(2+) entry was impaired early in the cell death process. Mitochondrial Ca(2+) was slightly increased. Preventing the transfer of Ca(2+) from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca(2+) uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca(2+,) possibly mediated by dysfunction of voltage-gated Ca(2+) channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Amiloride; Animals; Astrocytes; Biophysics; Calcium; Calcium Channel Blockers; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytosol; Dose-Response Relationship, Drug; Electric Stimulation; Embryo, Mammalian; Endoplasmic Reticulum; Excitatory Amino Acid Agonists; Lactones; Leupeptins; Membrane Potentials; Mice; Neocortex; Neurons; Patch-Clamp Techniques; Proteasome Inhibitors; Sesquiterpenes; Time Factors

Transepithelial Cl(-) secretion in polarized renal A6 cells is composed of two steps: (1) Cl(-) entry step across the basolateral membrane mediated by Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and (2) Cl(-) releasing step across the apical membrane via cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We estimated CFTR Cl(-) channel activity and transcellular Cl(-) secretion by measuring 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB, a blocker of CFTR Cl(-) channel)-sensitive transepithelial conductance (Gt) and short-circuit current (Isc), respectively. Pretreatment with 1 microM insulin for 24 h had no effects on NPPB-sensitive Gt or Isc. On the other hand, in A6 cells treated with carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132; 100 microM for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Isc with no effects on NPPB-sensitive Gt. Genistein (100 microM) induced sustained increases in NPPB-sensitive Gt and Isc, which were diminished by brefeldin A (a blocker of protein translocation to Golgi apparatus from endoplasmic reticulum). Co-application of insulin and genistein synergically stimulated the NPPB-sensitive Isc without any effects on NPPB-sensitive Gt. These observations suggest that: (1) insertion and endocytosis of NKCC are stimulated by insulin, (2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, (3) genistein stimulates insertion of both CFTR Cl(-) channel into the apical membrane and NKCC into the basolateral membrane, and (4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane.

Topics: Amiloride; Animals; Biological Transport; Brefeldin A; Cell Line; Cell Membrane; Chloride Channels; Drug Synergism; Endocytosis; Endoplasmic Reticulum; Epithelium; Genistein; Golgi Apparatus; Insulin; Ions; Kidney; Leupeptins; Nitrobenzoates; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Temperature; Time Factors; Xenopus laevis