Primary astrocytes were used between 7 and 28 days and the results obtained were not dependent on the length of time the astrocytes were maintained in culture

Primary astrocytes were used between 7 and 28 days and the results obtained were not dependent on the length of time the astrocytes were maintained in culture. For a portion of the experiments, wild-type (Cx43+/+), heterozygous (Cx43+/?), and Cx43-null (Cx43?/?) astrocytes were obtained from CD57/Bl6 mice generated by crossing heterozygous mice with the null mutation (26) using procedures as described above. (inward rectifier (Kir), delayed rectifier (Kdr), and A-type (Ka) K+ channels). Astrocytes are also extensively coupled by gap junctions that aid in the spatial redistribution of K+ from areas of high [K+]to those of low [K+](1, 3,C6). This spatial buffering is achieved when a local increase in [K+]causes the K+ equilibrium potential (that might otherwise occur during K+ uptake is transient sequestration into intracellular stores. Mitochondria in close association with the plasmalemmal membrane have been shown to play an important role in internal Ca2+ and Na+ sequestration in a variety of cell types (7,C13) and the potential involvement of analogous mechanisms in internal K+ handling are MK-3102 starting to be explored. Several lines of evidence are consistent with a potential role for mitochondria in K+ sequestration. First, mitochondria are intimately associated with the plasma membrane (14, 15), placing them in an appropriate location to sequester K+ that enters a cell across the plasma membrane. Second, the mitochondrial inner membrane is endowed with a variety of K+ channels and transporters that contribute to the regulation of the inner mitochondrial transmembrane potential ((100 mm) (20). Finally, mitochondria in cardiomyocytes have been reported to act as sinks for K+(21). In this study we investigated the role of mitochondria in the uptake of K+by astrocytes. To avoid the difficulties and potential artifacts associated with isolated mitochondrial preparations (see Refs. 22 and 23) we assessed mitochondrial K+ uptake in intact astrocytes. We determined that mitochondrial KATP channels (mito-KATP) and potentially mitochondrial connexin43 (Cx43), a protein that is abundantly expressed in astrocytes and has recently been found to contribute to K+ uptake in isolated cardiac mitochondria (24), play a role in the temporary sequestration of K+ by astrocyte mitochondria. EXPERIMENTAL PROCEDURES Cortical Astrocyte Cultures Cortical astrocytes from neonatal (1C2 days) CD-1 wild-type mice were cultured and plated, unless otherwise indicated, onto poly-d-lysine-coated glass coverslips in 24-well plates and maintained as previously described (25). Primary astrocytes were used between 7 and 28 days and the results obtained were not dependent on the length of time the astrocytes were maintained in culture. For a portion of the experiments, wild-type (Cx43+/+), heterozygous (Cx43+/?), and Cx43-null (Cx43?/?) astrocytes were obtained from CD57/Bl6 mice generated by crossing heterozygous mice with the null mutation (26) using procedures as described above. Tissue from each newborn pup was genotyped by PCR using primers specific for the wild-type and the disrupted gene, as previously described (27). Solutions and Test Compounds The standard perfusion medium contained (in mm): 136.5 NaCl, 3 KCl, 1.5 NaH2PO4, 1.5 MgSO4, 10 d-glucose, 2 CaCl2, and 10 HEPES (titrated to pH 7.35 with 10 m NaOH). Bicarbonate-containing perfusion medium contained (in MK-3102 mm): 117.5 NaCl, 3 KCl, 1.5 NaH2PO4, 1.5 MgSO4, 10 d-glucose, 2 CaCl2, and 29 NaHCO3, and was equilibrated with 5% CO2 in air (pH7.35). Medium containing high [K+] or the K+ channel blocking mixture (3 mm BaCl2, 5 mm 4-aminopyridine (4-AP), and 1 mm TEA) were prepared by equimolar substitution for NaCl. In solutions containing BaCl2, NaH2PO4 was omitted and MgSO4 was replaced with MgCl2. Test compounds (bumetanide, carbenoxolone (CBX), carbonyl cyanide with PBFI were performed using the dual excitation ratio method. Fluorescence emissions 505 nm were captured by a 12-bit digital cooled CCD camera (Retiga EXi, QImaging, Burnaby, BC) from regions of interest placed on individual astrocytes. Raw emission intensity data at each excitation wavelength (340 and 380 nm; Lambda DG-5, Sutter Instrument Co., Novato, CA) were collected every 6 s, corrected for background fluorescence, and background-subtracted ratio pairs (were performed in non-PBFI-loaded cells and no changes in 340 and 380 nm emission signals or 340/380 ratio values were detected upon exposure to CCCP or high [K+]NADH) is unlikely to contribute to the changes in the PBFI-derived ratio values measured in the study (data not shown). Furthermore, CCCP responses were nearly abolished in gramicidin-permeabilized PBFI-loaded astrocytes (supplemental Fig. S1), indicating it is unlikely that the effect of CCCP to increase the PBFI signal represents an artifact. Open in a separate window FIGURE 3. Effects of changes in [K+]on CCCP-induced [K+]rises. = 3 mm) evoked a [K+]transient. Following the recovery of [K+]to near resting levels, [K+]was increased to 12.5 mm for 5 min, immediately after which CCCP was again applied at [K+]= 3 mm. The rise in [K+]evoked by the second application of.Physiol. (Kdr), and A-type (Ka) K+ channels). Astrocytes are also extensively coupled by gap junctions that aid in the spatial redistribution of K+ from areas of high [K+]to those of low [K+](1, 3,C6). This spatial buffering is achieved when a local increase in [K+]causes the K+ equilibrium potential (that might otherwise occur during K+ uptake is transient sequestration into intracellular stores. Mitochondria in close association with the plasmalemmal membrane have been shown to play an important role in internal Ca2+ and Na+ sequestration in a variety of cell types (7,C13) and the potential involvement of analogous mechanisms in internal K+ handling are starting to be explored. Several lines of evidence are consistent with a potential part for mitochondria in K+ sequestration. First, mitochondria are intimately associated with the plasma membrane (14, 15), placing them in an appropriate location to sequester K+ that enters a cell across the plasma membrane. Second, the mitochondrial inner membrane is definitely endowed with a variety of K+ channels and MK-3102 transporters that contribute to the rules of the inner mitochondrial transmembrane potential ((100 mm) (20). Finally, mitochondria in cardiomyocytes have been reported to act as sinks for K+(21). With this study we investigated the part of mitochondria in the uptake of K+by astrocytes. To avoid the difficulties and potential artifacts associated with isolated mitochondrial preparations (observe Refs. 22 and 23) we assessed mitochondrial K+ uptake in intact astrocytes. We identified that mitochondrial KATP channels (mito-KATP) and potentially mitochondrial connexin43 (Cx43), a protein that is abundantly indicated in astrocytes and has recently been found to contribute to K+ uptake in isolated cardiac mitochondria (24), play a role in the temporary sequestration of K+ by astrocyte mitochondria. EXPERIMENTAL Methods Cortical Astrocyte Ethnicities Cortical astrocytes from neonatal (1C2 days) CD-1 wild-type mice were cultured and plated, unless normally indicated, onto poly-d-lysine-coated glass coverslips in 24-well plates and managed as previously explained (25). Main astrocytes were used between 7 and 28 days and the results obtained were not dependent on the length of time the astrocytes Tmem26 were maintained in tradition. For a portion of the experiments, wild-type (Cx43+/+), heterozygous (Cx43+/?), and Cx43-null (Cx43?/?) astrocytes were obtained from CD57/Bl6 mice generated by crossing heterozygous mice with the null mutation (26) using methods as explained above. Cells from each newborn pup was genotyped by PCR using primers specific for the wild-type and the disrupted gene, as previously explained (27). Solutions and Test Compounds The standard perfusion medium contained (in mm): 136.5 NaCl, 3 KCl, 1.5 NaH2PO4, 1.5 MgSO4, 10 d-glucose, 2 CaCl2, and 10 HEPES (titrated to pH 7.35 with 10 m NaOH). Bicarbonate-containing perfusion medium contained (in mm): 117.5 NaCl, 3 KCl, 1.5 NaH2PO4, 1.5 MgSO4, 10 d-glucose, 2 CaCl2, and 29 NaHCO3, and was equilibrated with 5% CO2 in air (pH7.35). Medium comprising high [K+] or the K+ channel blocking combination (3 mm BaCl2, 5 mm 4-aminopyridine (4-AP), and 1 mm TEA) were prepared by equimolar substitution for NaCl. In solutions comprising BaCl2, NaH2PO4 was omitted and MgSO4 was replaced with MgCl2. Test compounds (bumetanide, carbenoxolone (CBX), carbonyl cyanide with PBFI were performed using the dual excitation percentage method. Fluorescence emissions 505 nm were captured by a 12-bit digital cooled CCD video camera (Retiga EXi, QImaging, Burnaby, BC) from regions of interest placed on individual astrocytes. Uncooked emission intensity data at each excitation wavelength (340 and 380 nm; Lambda DG-5, Sutter Instrument Co., Novato, CA) were collected every 6 s, corrected for background fluorescence, and background-subtracted.