Progressive disruption of the plasma membrane during renal proximal tubule cellular injury.

The goal of this study was to examine the progression of plasma membrane disruption during cell injury using rabbit renal proximal tubules (RPT). The results demonstrated that the plasma membrane became permeable to larger and larger molecules as anoxia proceeded. At least three distinctive phases of membrane disruption were differentiated during anoxia. In phases 1, 2, and 3, plasma membranes became permeable to propidium iodide (PI, molecular weight = 668), 3 kDa dextrans, and 70 kDa dextrans or lactate dehydrogenase (LDH, molecular weight = 140 kDa), respectively. Phase 1 was reversible by reoxygenation but not prevented by the glycine. Phase 2 was inhibited by glycine. Phase 3 was inhibited by several membrane-permeable homobifunctional crosslinkers, dimethyl-pimelimidate (DMP), ethylene-glycolbis(succinimidylsuccinate), and dithiobis(succinimidylpropionate), but not by the membrane-impermeable crosslinker dithiobis(sulfosuccinimidylpropionate). In addition, DMP decreased RPT LDH release produced by mitochondrial inhibition (antimycin A), an oxidant (t-butylhydroperoxide) and a nephrotoxicant that is metabolized to an electrophile (tetrafluoroethyl-l-cysteine). These results identify (1) different phases of plasma membrane damage with increasing permeability during cell injury, (2) the reversibility of phase 1, (3) the relative site of action of the cytoprotectant glycine (prevents phase 2), and (4) the protective effects of chemical crosslinkers in RPT cell death produced by different toxicants.

Distribution of epithelial ankyrin (Ank3) spliceoforms in renal proximal and distal tubules.

In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3(200/215). To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3(200/215) but no Ank3(170) or Ank3(120). Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3(200/215). These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3(200/215) and Na-K-ATPase abundance measured a lower Ank3(200/215)-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1 cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3(200/215) suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.

Role of water and electrolyte influxes in anoxic plasma membrane disruption.

The role of water and electrolyte influxes in anoxia-induced plasma membrane disruption was investigated using rabbit proximal tubule suspension. The results indicated that normal proximal tubule (PT) cells have a great capacity for expanding cell volume in response to water influx, whereas anoxia increases the susceptibility to water influx-induced disruption, and this was attenuated by glycine. However, resistance of anoxic plasma membranes to water influx-induced stress is not lost, although their mechanical strength was diminished, compared with normoxic membranes. Anoxic membranes did not disrupt under an intra-to-extracellular osmotic difference as great as 150 mosM. Potentiating or attenuating water influx by incubating PT cells in hypotonic or hypertonic medium, respectively, during anoxia, did not affect anoxia-induced membrane disruption. After the transmembrane electrolyte concentration gradient was eliminated by a "intracellular" buffer or by permeabilizing the plasma membrane to molecules <4 kDa using alpha-toxin, anoxia still caused further membrane disruption that was prevented by glycine or low pH. These results demonstrate that 1) water or net electrolyte influxes are probably not a primary cause for anoxia-induced membrane disruption and 2) glycine could prevent the plasma membrane disruption during anoxia independently from its effect on transmembrane electrolyte or water influxes. The present data support a biochemical rather than a mechanical alteration of the plasma membrane as the underlying cause of membrane disruption during anoxia.

Unopposed phosphatase action initiates ezrin dysfunction: a potential mechanism for anoxic injury.

Because extensive kinase inhibition during anoxia has previously been reported, we investigated the role of kinase inhibition in anoxic cell injury by studying the effects of kinase inhibitors on a membrane-microvillar cytoskeleton linker protein, ezrin, in rabbit renal proximal tubules. Like anoxia, kinase inhibitors caused ezrin dephosphorylation in a dose-dependent manner under normoxia. The kinase inhibitor chelerythrine also induced ezrin dissociation from the cytoskeleton, i.e., causing it to lose its membrane-cytoskeleton linker function. Blockage of kinase inhibitor-induced ezrin dephosphorylation by a phosphatase inhibitor, calyculin A, ameliorated ezrin dissociation. Stimulation of the kinase during anoxia did not improve ezrin phosphorylation, suggesting that anoxia-induced kinase inhibition might be due to the lack of the substrate ATP. Finally, in vitro study of ezrin phosphatase revealed no increase in its activity during anoxia, suggesting the principal role of kinase inhibition in the loss of the linker function of ezrin during anoxia. Our results provide, for the first time at the molecular level, a mechanistic insight into anoxic cell injury caused by unopposed phosphatase action.

Parathyroid hormone inhibits Na(+)-K(+)-ATPase through Gq/G11 and the calcium-independent phospholipase A2.

Previous studies from this laboratory have demonstrated that the 3-34 analog of parathyroid hormone (PTH) causes a 15-30% inhibition of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity in rat renal proximal tubules through the generation of an increase in intracellular arachidonic acid, followed by its conversion to 20-hydroxyeicosatetraenoic acid (20-HETE) [C. P. Ribeiro and L. J. Mandel. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F209-F216, 1992; and C. P. Ribeiro, G. Dubay, J. R. Falk, and L. J. Mandel. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F497-F505, 1994]. The present study also uses proximal tubule suspensions to further elucidate this signaling pathway. Guanosine 5'-O-(2-thiodiphosphate), 500 microM, an inhibitor of heterotrimeric GTP-binding proteins (G proteins), and an anti-Gq/G11 antibody (1:500) both blocked the inhibition of the Na(+)-K(+)-ATPase by PTH-(3-34). Furthermore, a 42-kDa protein was identified in proximal tubules by the anti-Gq/G11 antibody (1:1,000). Bromoenol lactone (BEL), 1 microM, a suicide inhibitor of the calcium-independent 40-kDa phospholipase A2 (PLA2), prevented PTH-(3-34) inhibition of the Na(+)-K(+)-ATPase, unless exogenous 10 microM 20-HETE was added. In addition, BEL blocked the PTH-(3-34)-induced increase in arachidonic acid release in the proximal tubules. We conclude that a member of the Gq family and the calcium-independent 40-kDa PLA2 participate in the PTH-(3-34) signaling pathway in rat proximal tubules by mediating the steps between the binding of PTH-(3-34) to its receptor and the subsequent generation of arachidonic acid.

Loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption in renal cells.

The goal of this study was to determine whether anoxic membrane disruption is initiated by loss of cytoskeletal support in rabbit renal proximal tubules (PT). We specifically tested 1) whether cytoskeletal perturbation affects membrane integrity under normoxia, 2) whether cytoskeletal perturbation potentiates anoxic membrane damage, and 3) whether the membrane protection by glycine depends on cytoskeletal integrity. Cytoskeletal perturbation was achieved with 10 microM cytochalasin D (CD) because it selectively disturbs F-actin organization and has similar effects as anoxia on the cytoskeleton of PT. During normoxia, CD caused decreased basal F-actin content, microvillar breakdown, and membrane-cytoskeleton dissociation, as revealed by the use of laser tweezers. However, membrane integrity was not altered by CD, as monitored by lactate dehydrogenase release. CD pretreatment of PT did not potentiate anoxic membrane damage. Finally, plasma membrane protection by glycine during anoxia remained in CD-pretreated PT despite loss of cytoskeletal support. These results demonstrate that loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption.

Loss of plasma membrane structural support in ATP-depleted renal epithelia.

Renal ischemia induces cytoskeletal alterations, membrane perturbations, including bleb formation, and ultimately membrane lysis. The mechanisms that underlie these alterations are largely unknown. Through the use of isolated rat renal proximal tubule fragments and calibrated micropipette techniques, two potential mechanisms for membrane bleb formation during ATP depletion were examined: 1) decreased cytoskeletal retention of the plasma membrane and 2) increased intracellular pressure. Under control conditions, the pressure required to pull the membrane from the underlying cellular matrix was 73 +/- 10 kdyn/cm2. After 30 min of ATP depletion, this pressure was diminished by >95% and blebs began to emerge from the basal membrane. The intracellular pressure within these blebbed cells was only 0.08 +/- 0.02 kdyn/cm2. These observations indicate that, during ATP depletion, the strength of membrane retention diminished until the relatively low intracellular pressure was capable of driving membrane bleb formation. Cytochalasin D, which disrupts the actin cytoskeleton, decreased the strength of membrane retention by 65 +/- 7%. This suggests that, during ATP depletion, alterations of the actin cytoskeleton may mediate the loss of membrane retention.

Hypoxia-induced amphiphiles inhibit renal Na+, K(+)-ATPase.

We have characterized the effects of hypoxia on carnitine metabolism in proximal tubules. Hypoxia for 10 minutes resulted in a significant increase in the mass of long chain acylcarnitines (LCAC) (control 53 +/- 20 vs. hypoxia 118 +/- 38 pmol. mg-1 protein). Since LCAC are proximal metabolites in the beta-oxidation of fatty acids, these data suggest that inhibition of fatty acid oxidation occurs during hypoxia in the proximal tubule. In addition to LCAC accumulation, hypoxia resulted in a significant increase in the mass of lysoplasmenylcholine LPLasCho (control 62 +/- 15 pmol/mg vs. 20 min hypoxia 146 +/- 21 pmol/mg protein, N = 4) and also in increases in the mass of monoacyl LPC (control 122 +/- 24 pmol/mg protein vs. 283 +/- 35 pmol/mg protein after 40 min of hypoxia). We tested the possibility that these compounds that accumulate during hypoxia could inhibit proximal tubule Na+, K(+)-ATPase. LPC, LPlasC, and LCAC inhibited proximal tubule nystatin-stimulated oxygen consumption (QO2) and proximal tubule Na+, K(+)-ATPase activity in a dose-dependent manner. In addition, LPC, LPlasC, and LCAC directly inhibited' (65%, 80%, and 60%, respectively) Na+, K(+)-ATPase activity purified from kidney cortex at similar concentrations at which they accumulate during hypoxia (above 25 microM). The present data suggest that amphiphile accumulation may have a potential pathophysiologic role in the proximal tubule during renal ischemia.

Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells.

ATP-depletion in renal cultured cells has been used as a model for studying various cytoskeletal and functional alterations induced by renal ischemia. This communication explores the reversibility of these effects utilizing a novel method [1] that depleted ATP (ATP-D) to 2% of control within 30 minutes and caused complete recovery (REC) of ATP in one hour. Under confocal microscopy, ATP-D (30 min) caused thinning of F-actin from the microvilli, cortical region, and basal stress fibers, with the concurrent appearance of intracellular F-actin patches. These changes were more pronounced after 60 minutes of ATP-D. One hour of REC following 30 minutes of ATP-D produced complete recovery of F-actin in each region of the cell. However, after 60 minutes of ATP-D, a heterogeneous F-actin recovery pattern was observed: almost complete recovery of the apical ring and microvilli, thinned cortical actin with occasional breaks along the basolateral membrane, and a dramatic reduction in basal stress fiber density. The time course of cortical actin and actin ring disruption and recovery coincided with a drop recovery in the transepithelial resistance and the cytoskeletal dissociation and reassociation of the Na,K-ATPase. Additionally, the microvilli retracted into the cells during ATP-D, a process that was reversed during REC. Triton extraction and confocal microscopy demonstrated that villin remained closely associated with microvillar actin during both ATP-D and REC. These distinctive regional differences in the responses of F-actin to ATP depletion and repletion in cultured renal epithelial cells may help to clarify some of the differential tubular responses to ischemia and reperfusion in the kidney.

Fluctuating shear stress effects on stress fiber architecture and energy metabolism of cultured renal cells.

The project investigates the relationship between the external shear force and the actin cytoskeleton along with the metabolic changes occurring inside the cells due to this force. Anchorage-dependent Madin Darby canine kidney (MDCK) cells were placed in spinner flasks with paddle-type stirrers agitated at 20 rpm, where they experienced shear stress fluctuations from 0.02 to 0.27 dyn/cm2 in magnitude. Following fixation, permeabilization, and staining with rhodamine-phalloidin, the relative amounts and distribution of F-actin stress fibers in the 1 micron basal layer of the cells were visualized by confocal microscopy. These structures disappeared after 12-15 h of exposure to shear stress. Previous results showed that the stress fibers disappear, leading to loss of epithelial attachment, after only 1 h of starvation-induced energy depletion. Therefore, in this study, the energy metabolism of the cells was established by measuring adenosine triphosphate (ATP) levels at different time intervals. No statistical difference in ATP content was found between the shear-stressed cells and the controls, showing that shear stresses cause cytoskeletal reorganization by a mechanism other than ATP depletion.

Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury.

Disruption of the renal proximal tubule (PT) brush border is a prominent early event during ischemic injury to the kidney. The molecular basis for this event is unknown. Within the brush border, ezrin may normally link the cytoskeleton to the cell plasma membrane. Anoxia causes ezrin to dissociate from the cytoskeleton and also causes many cell proteins to become dephosphorylated in renal PTs. This study examines the hypothesis that ezrin dephosphorylation accompanies and may mediate the anoxic disruption of the rabbit renal PT. During normoxia, 73 +/- 3% of the cytoskeleton-associated (Triton-insoluble) ezrin was phosphorylated, but 88 +/- 6% of dissociated (Triton-soluble) ezrin was dephosphorylated. Phosphorylation was on serine/threonine resides, since ezrin was not detectable by an antibody against phosphotyrosine. After 60 min of anoxia, phosphorylation of total intracellular ezrin significantly decreased from 72 +/- 2% to 21 +/- 9%, and ezrin associated with the cytoskeleton decreased from 91 +/- 2% to 58 +/- 2%. Calyculin A (1 microM), the serine/threonine phosphatase inhibitor, inhibited the dephosphorylation of ezrin during anoxia by 57% and also blocked the dissociation of ezrin from the cytoskeleton by 53%. Our results demonstrate that (i) the association of ezrin with the renal microvillar cytoskeleton is correlated with phosphorylation of ezrin serine/threonine residues and (ii) anoxia may cause disruption of the renal brush border by dephosphorylating ezrin and thereby dissociating the brush border membrane from the cytoskeleton.

ATP depletion: a novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton.

The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine kidney (MDCK) and JTC cell lines. In prior studies, it was shown that ATP depletion uncouples the gate and fence functions of the tight junction. This paper extends these observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and confocal fluorescence microscopy in combination with computer-aided three-dimensional reconstruction. Marked regional differences in the sensitivity to the effects of ATP depletion were observed in the actin cytoskeleton. Actin depolymerization appears to first affect the cortical actin network running along the apical basal axis of the cell. The next actin network that is disrupted is the stress fibers found at the basal surface of the cell. Finally, the actin ring at the level of the zonulae occludens and adherens is compromised. The breakup of the actin ring correlates with ultrastructural changes in tight junction strands and the loss of the tight junction's role as a molecular fence. During the process of actin network dissolution, polymerized actin aggregates form in the cytoplasm. The changes in the junctional complexes and the potential to reverse the ATP depletion suggest that this may be a useful method to study junctional complex formation and its relationship to the actin cytoskeletal network.

ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K(+)-ATPase and E-cadherin.

MDCK and JTC cells were subjected to ATP depletion by treating the cells with 10 microM antimycin A and 10 mM 2-deoxyglucose. As visualized by confocal fluorescence microscopy, E-cadherin and Na+,K(+)-ATPase were rapidly internalized following depletion of the intracellular ATP stores. The time course of internalization was similar to the depolymerization of the cortical actin network and dissolution of the actin ring (see companion paper, this volume, pp. 3301-3313). Cell surface biotinylation was used to assay the amount of surface-accessible E-cadherin and Na+,K(+)-ATPase during ATP depletion. At 30 minutes of ATP depletion, 74% and 69% of E-cadherin and Na+,K(+)-ATPase were internalized, respectively, in MDCK cells. By 60 minutes of ATP depletion, internalization increased to 95% and 89%, respectively. The redistribution of both plasma membrane proteins was not microtubule dependent. Similar results were observed in JTC cells. Total biotinylated protein decreased by 67% and 82%, after 30 minutes and 60 minutes of ATP depletion, respectively. The E-cadherin internalization strongly suggests that disruption of adherens junctions occurred following ATP depletion. These results, along with the previously described loss of tight junction integrity, suggest that ATP depletion may be a useful method to study the assembly and disassembly of junctional complexes in epithelial cells.

Cytoskeletal regulation of membrane transport events.

Recent advances in our understanding of the function of various components of the cytoskeleton indicate that, besides serving a structural role, the membrane skeleton may regulate the activity or number of transport proteins in the cell membrane. Abundant evidence indicates that individual proteins of the cytoskeletal system bind directly to transport proteins, resulting in the three-dimensional organization of a structure termed the membrane skeleton. This interaction between cytoskeleton and transporters can most readily be interpreted to serve a structural role, keeping the transporters in specific domains. However, because disruption or alteration of the cytoskeletal organization can lead to changes in transport, the interaction may play a key role in regulating transporter activity. Elements of the cytoskeleton also interact with components of second messenger systems. Thus, the cytoskeleton could play a regulatory role by altering the availability of signal transducers or by being an integral part of the signal transduction system.

Decreased protein phosphorylation induced by anoxia in proximal renal tubules.

Anoxia-induced depletion of cellular ATP may affect the degree of protein phosphorylation due to kinase inhibition. In this study, protein phosphorylation was measured in rabbit kidney proximal tubules under normoxic or anoxic conditions in a medium containing 32P. During the first 20 min of normoxia, phosphate incorporation was linear, averaging 17 +/- 5 pmol.mg protein-1.min-1 and was 70% inhibited by the protein kinase C inhibitor chelerythrine chloride. Phosphorylation measurements initiated simultaneously with anoxic conditions (95% N2-5% CO2) significantly reduced the initial rate to 58% of control, saturating after 15 min, and reaching 28 +/- 5% of the normoxic value after 60 min of incubation. The phosphatase inhibitor calyculin A did not affect the initial rate of phosphate incorporation by anoxic tubules but increased phosphate incorporation at 60 min to 43 +/- 4% of normoxia. Addition of 32P after 15 min of anoxia abolished phosphate incorporation, demonstrating that kinase activity was completely inhibited. Cellular phosphate uptake was measured and found not to be rate limiting for phosphorylation. Chelerythrine chloride increased lactate dehydrogenase (LDH) release during normoxia, and calyculin A decreased anoxia-induced LDH release, suggesting that protein phosphorylation events may control plasma membrane permeability.

Cytoskeletal dissociation of ezrin during renal anoxia: role in microvillar injury.

The association/dissociation of ezrin, a microvillar membrane-cytoskeleton linker, was studied to search for the initial step leading to anoxia-induced brush-border breakdown in a rabbit proximal tubule suspension. Electron microscopy studies display time-dependent damage to the microvilli during anoxia; immunoblots demonstrate the dissociation of ezrin from the cytoskeleton, reflected by the significant decrease in Triton X-100-insoluble ezrin from control (91%) to 39% after 30 min. Simultaneously, Triton X-100-soluble and extracellular ezrin increased with no change in total ezrin, Triton X-100 solubility of actin, or total intracellular protein. Parallel immunocytochemistry studies show diffusion of ezrin from the brush border, where ezrin is highly colocalized with F-actin during normoxia into the cytoplasm. Thirty minutes of reoxygenation following 30 min of anoxia causes recovery of the microvillar structure and reassociation of ezrin to the cytoskeleton and the brush border. Application of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (4 mM) or inhibition of intracellular calpain or calcineurin do not prevent the dissociation of ezrin during anoxia. We conclude that ezrin-cytoskeletal dissociation may initiate microvillar breakdown during anoxia via calcium-independent mechanisms.

Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures.

Cultured renal cells provide a highly reproducible and malleable model to study cellular responses to metabolic perturbations. Nevertheless, there is currently no good method to achieve metabolic inhibition and complete recovery in cultured cells. This study describes a specific method for reversibly inhibiting both glycolytic and oxidative metabolism. Glycolysis was inhibited by removing all glycolytic substrates, and mitochondrial respiration was inhibited with rotenone, a site I inhibitor of the electron transport chain. Within 30 min, ATP values were decreased by 98%. Glycolysis was restored through the reintroduction of glucose. Oxidative metabolism was restored by the addition of heptanoate, a short odd-chain fatty acid, which supplies reducing equivalents to site II of the electron transport chain. Employing Madin-Darby canine kidney and LLC-PK1 cell lines, this protocol caused the immediate and complete recovery of mitochondrial respiration and, by 60 min, the complete recovery of cellular ATP levels. Application of this protocol should allow the investigation of the cellular effects and alterations that occur within cells recovering from sublethal energy depletion.

Parathyroid hormone inhibits Na(+)-K(+)-ATPase through a cytochrome P-450 pathway.

We have previously shown that parathyroid hormone (PTH)-(1-34) or its analogue PTH-(3-34) inhibits proximal tubule (PT) Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity independently of adenosine 3',5'-cyclic monophosphate generation. The present study used PT suspensions to investigate the signaling pathway responsible for this hormonal action. PTH-(1-34) and PTH-(3-34) significantly increased the release of arachidonic acid (AA) compared with control tubules, suggesting activation of phospholipase A2 (PLA2). AA, 10(-6) M, mimicked the inhibition of the pump by 10(-8) M PTH-(3-34), and together were not additive. Eicosatetraynoic acid, 3 microM, a general inhibitor of AA metabolism, blocked the PTH action. Indomethacin, 10 microM, an inhibitor of AA-dependent cyclooxygenase, did not prevent the PTH action, but 2 microM 7-ethoxyresorufin, a cytochrome P-450 inhibitor, prevented the PTH effect. 20-Hydroxyeicosatetraenoic acid (20-HETE), the main product of P-450 metabolism in PT, inhibited Na(+)-K(+)-ATPase activity to the same extent as 10(-8) M PTH-(3-34), was not additive with PTH, and was maximally inhibitory at 10(-7) M. To further investigate the signaling pathway responsible for PTH-activated PLA2, we tested the effect of PTH on cytoplasmic free Ca2+ ([Ca2+]i). PTH-(1-34), 10(-7) M, did not affect [Ca2+]i, although 10(-8) M angiotensin II promoted a Ca2+ transient. Treatment of PT with pertussis toxin (PTX) did not prevent the PTH action.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced Na+ transport in an air-liquid interface culture system.

Use of the air-liquid interface culture technique has produced improved morphological differentiation of rodent, canine, and human tracheal epithelia. We have investigated the effect of this culture technique on ion transport activities of cultured canine bronchial epithelia. These cells were isolated from excised airways by enzymatic digestion and plated on permeable collagen membrane substrates. All cultures were maintained utilizing standard culture techniques, by bathing both apical and basolateral sides with hormone supplemented, serum-free media until confluent (days 4-6). Half of the cultures were converted to air-liquid interface cultures (ALIC) by gentle aspiration of the apical medium and half were continued under standard technique culture (STC) conditions. After three additional days, preparations cultured under both conditions were mounted in modified Ussing chambers where bioelectric properties were measured under short-circuit conditions. Mean short-circuit current (Isc) was significantly greater in ALIC (-91.3 +/- 7.84 microA/cm2) than in STC (-54.8 +/- 5.03 microA/cm2). The sodium channel blocker, amiloride, reduced Isc by 68.4 +/- 5.0% in STC and by 84.8 +/- 3.0% in ALIC. 22Na and 36Cl fluxes confirmed the presence of enhanced sodium absorption in ALIC when compared with STC. The depth of the apical fluid, measured by microelectrodes during ALIC, was approximately 15 microns. Studies of cellular metabolism demonstrated a shift in metabolism from an anaerobic to an oxidative pattern in ALIC. This change in the pattern of metabolism suggests that the ALIC technique enhanced sodium transport in canine bronchial epithelia by increasing oxygen delivery to the epithelium.

Degradation of spectrin and ankyrin in the ischemic rat kidney.

This study investigates ischemia-induced degradation of the spectrin-based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na(+)-K(+)-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.