Gene transfer by lipofection in rabbit and human secretory epithelial cells.

Lipofection, a recently-developed method for gene transfer, was tested in secretory epithelial cells. Lipofection facilitated both transient DNA transfection with plasmids containing the chloramphenicol acetyltransferase gene and stable transfection with a plasmid containing the neomycin resistance gene, which confers resistance to the antibiotic G418 (Geneticin). Gene transfer occurred efficiently in a rabbit kidney medullary thick ascending limb cell line and in primary cultures of rabbit tracheal epithelial cells.

Membrane stretch: a physiological stimulator of Ca2+-activated K+ channels in thick ascending limb.

The effects of membrane stretch on Ca2+-activated (maxi) K+ channels were examined in the apical membrane of cultured medullary thick ascending limb (MTAL) cells. Using cell-attached patchclamp technique, we found that negative pressure (-33 +/- 5 cmH2O) applied to the patch membrane increased fractional open probability (NPo) from 0.3 +/- 0.

Ca2+-activated K+ channels from cultured renal medullary thick ascending limb cells: effects of pH.

Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb cells (MTAL) using the patch-clamp technique. The purpose was to determine the effect of acidic pH on channel properties in excised patches of apical cell membrane. At pH 7.

Techniques for isolation of brush-border and basolateral membrane vesicles from dog kidney cortex.

Two methods are reported for renal membrane preparation from the dog kidney cortex. One method is a simultaneous preparation of brush-border (BBMV) and basolateral (BLMV) membranes. Using readily available laboratory equipment, differential centrifugation produced a supernatant which was treated with Mg2+.

Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase.

Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C.

Steroid hormones: modulators of Na+ absorption and Cl- secretion in cultured tracheal epithelia.

Cultures of tracheal epithelial cells from adult nonpregnant female rabbits maintained a different balance of Na+ absorption and Cl- secretion from cells of pregnant animals and fetal cultures. Cells of nonpregnant rabbits had a large Na+ absorptive current (11 microA/cm2 or 53% of baseline short circuit current) and a small Cl- secretory current (2.4 microA/cm2 or 11%).

Isomeric yohimbine alkaloids block calcium-activated K+ channels in medullary thick ascending limb cells of rabbit kidney.

The alpha2-adrenergic antagonist yohimbine (YOH) and the closely related isomers corynanthine (COR) and rauwolscine (RAU) caused brief interruptions in current characteristic of a fast blocker Ca2+-activated K+ channels in cultured medullary thick ascending limb (MTAL) cells. The apparent dissociation constants (Kapp) for COR, YOH, and RAU, respectively, at the intracellular face of the channel in the presence of 200 mM K+ are 45 +/- 1, 98 +/- 2, and 310 +/- 33 microM. The Kapp for COR on the extracellular side also in the presence of 200 mM K+ was much greater at 1.

The amphibian diluting segment.

Diluting segments function to reabsorb NaCl and to reduce the osmolality of tubule fluid. These segments in amphibians are important in the conservation of NaCl. The diluting segment of mammals, the thick ascending limb, besides being an important site for the reabsorption of NaCl, supplies the energy that enables the kidney to excrete a concentrated urine.

Ion transport in cultured fetal and adult rabbit tracheal epithelia.

Transepithelial potential difference (Vte), short circuit current (Isc), and transepithelial resistance (Rte) were measured to assess Na+ and Cl- transport in cultured tracheal epithelial cells from fetal and adult rabbits. Cultured fetal and adult cells developed differentiated morphology as determined by electron microscopy. Base-line Vte, Isc, and Rte were equivalent in late gestation (28-30 days) and adult cultures.

Cell volume regulation by the thin descending limb of Henle's loop.

Thin descending limb cells from Henle's loop (from the inner strip of the outer medulla of long loops) were studied with optical and video techniques to identify the mechanisms of ion transport and cell volume regulation. Increasing the K+ concentration in the basolateral solution from 5 to 90 mM caused the cells to swell. This K+-induced swelling was inhibited by exposure of the basolateral membrane to 9 mM Ba2+ and was abolished by removing Cl- from the perfusion solutions.

Blocking agents of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells.

Ca2+-activated K+ channels with estimated single channel conductances of 127 +/- 2 pS were identified in the apical cell membrane of clone A3 of cultured medullary thick ascending limb (MTAL) cells. Both Ba2+ and the scorpion toxin, charybdotoxin (CTX), are slow blockers of the channels. An application of 0.

Ca2+-activated K+ channels in cultured medullary thick ascending limb cells.

The conductive properties of a clone of medullary thick ascending limb (MTAL) cells (GRB-MAL1) were assessed using conventional microelectrodes and the patch clamp technique. The apical cell membrane potential (Va) of MTAL cells was -46 +/- 3 mV. Addition of Ba2+ (1 mM) to the apical solution induced a 22 +/- 2 mV depolarization of Va, whereas furosemide hyperpolarized Va by -5 +/- 1 mV.

Volume regulation in the early proximal tubule of the Necturus kidney.

The ability of early proximal tubule cells of the Necturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes. Necturus proximal tubule cells regulate volume in both hyper- and hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO3- dependent and is associated with a decrease in the relative K+ conductance of the basolateral cell membrane and a decrease in the resistance ratio, Ra/Rbl.

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide.

Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca2+ activation site was modified using N-bromoacetamide (NBA). 1 mM NBA in the bath solution, at 2.

Functional heterogeneity in the early distal tubule of the Amphiuma kidney: evidence for two modes of Cl- and K+ transport across the basolateral cell membrane.

Measurements of cell volume and basolateral cell membrane potential difference (Vbl) were made simultaneously and on the same cell to study chloride and potassium transport across the basolateral cell membrane of individual cells from the Amphiuma early distal tubule or diluting segment. Two distinct types of responses of Vbl are evident when either chloride is lowered or potassium is raised in the basolateral solution. In one cell type (high basolateral conductance, HBC), these maneuvers cause large depolarizations in Vbl, suggesting that the basolateral cell membrane has significant partial conductances to both chloride and to potassium.

Forskolin and antidiuretic hormone stimulate a Ca2+-activated K+ channel in cultured kidney cells.

Single channels in the apical cell membrane of primary cultured chick kidney cells were studied using the patch clamp technique. Cell-attached recordings revealed the presence of a 107 +/- 6 pS channel that increased fractional open time upon depolarization. Experiments with inside-out excised patches indicated that the channel is K+ selective, Ca2+ activated, and inhibited by Ba2+.

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney.

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane.

Resistance properties of the diluting segment of Amphiuma kidney: influence of potassium adaptation.

Chronic exposure to high potassium stimulates K+-secretory mechanisms in the diluting segment of the amphibian kidney (K+ adaptation). Since K+ net flux depends critically on the passive cell membrane permeabilities for K+ ions, cable analysis and K+-concentration step changes were applied in this nephron segment to assess the individual resistances of the epithelium and the K+ conductance of the luminal cell membrane. Experiments were performed in the isolated, doubly-perfused kidney of both control and K+-adapted Amphiuma.

Electrical effects of potassium and bicarbonate on proximal tubule cells of Necturus.

The effects of stepwise concentration changes of K+ and HCO3- in the basolateral solution on the basolateral membrane potential (Vbl) of proximal tubule cells of the doubly-perfused Necturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes in Vb1 after alterations in external K+ concentration from 1.0 to 2.

Regulation of the basolateral potassium conductance of the Necturus proximal tubule.

Two methods, the measurement of the response of the basolateral membrane potential (Vbl) of proximal tubule cells of Necturus to step changes in basolateral K+ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (GK) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K+ activity (aiK) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0 mM K+ basolateral solution for 15 min followed by 45 min of 1 mM K+ solution resulted in a fall in basolateral potassium (apparent) transference number (tK), Vbl and aiK.

Anthracene-9-carboxylic acid inhibits renal chloride reabsorption.

From previous studies, it is known that in the diluting segment, C1- -ions are transported from the tubule lumen into the cell together with Na+ and K+ via a furosemide-sensitive cotransport system. This carrier-mediated process, located in the luminal cell membrane, is driven by the steep "downhill" Na+ gradient (directed from lumen to cell) which is maintained by the ouabain-sensitive Na+/K+-pump at the peritubular cell membrane. C1- -ions are accumulated within the cell cytosol and are supposed to leave the cell by a C1- -conductive pathway.

Potassium transport in the early distal tubule of Amphiuma kidney. Effects of potassium adaptation.

Studies were performed to investigate potassium transport in early distal tubule of the doubly-perfused kidney of Amphiuma under control conditions and following K-adaptation. Double barreled K-sensitive microelectrodes were used in stationary microperfusion experiments. Net K-flux was evaluated along with measurements of both cell membrane potential and cell K activity.

The effect of furosemide on luminal sodium, chloride and potassium transport in the early distal tubule of Amphiuma kidney. Effects of potassium adaptation.

Previous experiments in the early distal tubule of the doubly perfused kidney of Amphiuma demonstrated net reabsorption of potassium (K) which is reversed to net K secretion after K adaptation. Furthermore, it is known that this particular segment exhibits extensive chloride (Cl) net reabsorption which depends on the presence of sodium (Na) and which is inhibited by furosemide. In order to test for a possible interrelationship between NaCl and K transport, K activity in lumen and cell, transepithelial electrical potential difference, peritubular cell membrane potentials and volume reabsorption were measured in control animals and after K adaptation, in presence and absence of furosemide.