Biomedical subjects

D J Benos

Publications and source records attributed to D J Benos.

At least 19 recordsLinked to original sources

PubMed1995

Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels.

We have previously described a protocol for the simultaneous isolation and reconstitution of a protein kinase A (PKA)-sensitive outwardly rectified chloride channel (ORCC) and the cystic fibrosis transmembrane conductance regulator (CFTR) from bovine tracheal epithelium. Immunoprecipitation of CFTR from this preparation prevented PKA activation of the ORCC, suggesting that CFTR regulated the ORCC and that this regulatory relationship was preserved throughout the purification procedure. We now report the purification of CFTR from bovine tracheal epithelia and the purification of a CFTR conduction mutant (G551D CFTR) from retrovirally transduced mouse L cells using a combination of alkali stripping, Triton-X extraction, and immunoaffinity chromatography. Immunopurified CFTR proteins were reconstituted in the absence and presence of ORCC. To test the hypothesis that only functional CFTR can support activation of ORCC by PKA and ATP, we used an inhibitory anti-CFTR505-511 peptide antibody or G551D CFTR. When anti-CFTR505-511 peptide antibodies were present prior to the addition of PKA and ATP, activation of both the ORCC and CFTR was prevented. If the antibody was added after activation of the ORCC and CFTR Cl- channels by PKA and ATP, only the CFTR Cl- channel was inhibited. When ORCC and G551D CFTR were co-incorporated into planar bilayers, only the ORCC was recorded and this channel could not be further activated by the addition of PKA and ATP. Thus, functional CFTR is required for activation of the ORCC by PKA and ATP. We also tested the hypothesis that PKA activation of ORCC was dependent on the extracellular presence of ATP. We added ATP on the presumed extracellular side of the lipid bilayer under conditions where it was not possible to activate the ORCC, i.e. in the presence of inhibitory anti-CFTR505-511 antibody or G551D CFTR. In both cases the ORCC regained PKA sensitivity. Moreover, the addition of hexokinase + glucose to the extracellular side prevented activation of the ORCCs by PKA and ATP in the presence of CFTR. These experiments confirm that both the presence of CFTR as well as the presence of ATP on the extracellular side is required for activation of the ORCC by PKA and ATP.

Adenosine Triphosphate↗

PubMed1995

Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.

We investigated the cellular and molecular events associated with the increase in sodium transport across the alveolar epithelium of rats exposed to hyperoxia (85% O2 for 7 days followed by 100% O2 for 4 days). Alveolar type II (ATII) cell RNA was isolated and probed with a cDNA for one of the rat colonic epithelial sodium channel subunits (alpha rENaC). The alpha rENaC mRNA (3.7-kb transcript) increased 3-fold in ATII cell RNA isolated from rats exposed to 85% O2 for 7 days and 6-fold after 4 days of subsequent exposure to 100% O2. In situ hybridization revealed increased expression of alpha rENaC mRNA transcripts in both airway and alveolar epithelial cells of hyperoxic rats. When immunostained with a polyclonal antibody to kidney sodium channel protein, ATII cells from hyperoxic rats exhibited a significant increase in the amount of immunogenic protein present in both the plasma membrane and the cytoplasm. When patched in the whole-cell mode, ATII cells from hyperoxic rats exhibited amiloride and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (EIPA)-sensitive currents that were 100% higher compared with those obtained from air-breathing rats. Single-channel sodium currents (mean conductance of 25 pS) were seen in ATII cells patched in both the inside-out and cell-attached modes. The number and open probability of these channels increased significantly during exposure to hyperoxia. Exposure to sublethal hyperoxia up-regulated both alpha rENaC mRNA and the functional expression of sodium channels in ATII cells.

Animals↗

PubMed1995

Cystic fibrosis transmembrane conductance regulator is required for protein kinase A activation of an outwardly rectified anion channel purified from bovine tracheal epithelia.

Our laboratory has developed a protocol for the isolation of a 140-kDa protein that forms an anion-selective channel when reconstituted into planar lipid bilayers. Polyclonal antibodies have been raised against the 38-kDa component of this purified protein. This channel has a linear current-voltage relationship and is not activated by protein kinase A (PKA) plus ATP. Using the same antibody and a modified purification protocol (eliminating the ion exchange chromatography steps), we isolated and reconstituted two other anion channels from tracheal membrane vesicles. In vitro phosphorylation of these isolated proteins by PKA and ATP revealed four bands migrating at 52, 85, 120, and 174 kDa. Immunoprecipitation experiments with anti-CFTR antibodies indicate that the 174-kDa phosphoprotein was CFTR. Upon incorporation of these isolated proteins into planar bilayers, an anion channel that exhibited a marked outward rectification in symmetrical Cl- solutions with a slope conductance of 82 pS at depolarizing voltages was observed. PKA and ATP increased channel activity but only from one side of the bilayer. However, channel activity was unaffected by addition of ATP alone from either side of the membrane. DIDS (100 microM) applied to the opposite side of the bilayer to which PKA and ATP act, blocked channel activity. A linear anion-selective channel with a conductance of 16 pS could be also resolved after inhibition of the outwardly rectified anion channel by DIDS in the presence of PKA and ATP. This small conductance channel was inhibited by 300 microM diphenylamine-2-carboxylic acid. Immunodepletion of the 174-kDa phosphoprotein from the preparation prevented activation of the 82-pS outwardly rectified anion channel by PKA and ATP. However, the PKA-dependent in vitro phosphorylation of the 52-, 85-, and 120-kDa phosphoproteins was unaffected by the absence of CFTR. Our results suggest a direct regulatory relationship between an outwardly rectified anion channel and CFTR.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Structure and function of amiloride-sensitive Na+ channels.

A new molecular biological epoch in amiloride-sensitive Na+ channel physiology has begun. With the application of these new techniques, undoubtedly a plethora of new information and new questions will be forthcoming. First and foremost, however, is the question of how many discrete amiloride-sensitive Na+ channels exist. This question is important not only for elucidating structure-function relationships, but also for developing strategies for pharmacological or, ultimately, genetic intervention in such diseases as obstructive nephropathy, Liddle's syndrome, or salt-sensitive hypertension where amiloride-sensitive Na+ channel dysfunction has been implicated [17, 62]. Epithelia Na+ channels purified from kidney are multimeric. However, it is not yet clear which subunits are regulatory and which participate directly as a part of the Na+ conducting core and what is the nature of the gate. The combination of electrophysiologic techniques such as patch clamp and the ability to study reconstituted channels in planar lipid bilayers along with molecular biology techniques to potentially manipulate the individual subunits should provide the answers to questions that have puzzled physiologists for decades. It seems clear that the robust versatility of the channel in responding to a wide range of differing and potentially synergistic regulatory inputs must be a function of its multimeric structure and relation to the cytoskeleton. Multiple mechanisms of regulation imply multiple regulatory sites. This hypothesis has been validated by the demonstration that enzymatic carboxyl methylation and phosphorylation have both individual and synergistic effects on the purified channel in planar lipid bilayers. Of the multiple mechanisms proposed for channel regulation, evidence is now available to support the ideas that channels may be activated (or inactivated) by direct modifications including phosphorylation and carboxyl methylation, by activation or association of regulatory proteins such as G proteins, and by recruitment from subapical membrane domains. The observation that channel gating is achieved primarily through regulation of open probability without alterations in conductance may simplify future understanding of the molecular events involved in gating once the regulatory sites have been identified. As more Na+ channels or Na+ channel subunits are cloned from different epithelia, it will become possible to piece together the puzzle of epithelial Na+ channels. It is interesting to observe that renal Na+ channel proteins contain a subunit which falls into the 70 kD range. This size protein is in the range reported for the aldosterone-induced proteins [12, 46, 153].(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗

PubMed1995

Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule.

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39 +/- 0.05 pA (9 patches). The conductance of these channels was 4.3 +/- 0.2 pS. The unitary current approached zero at a pipette voltage of -92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 microM amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines. We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.

Ambystoma↗

PubMed1995

Reconstitution of immunopurified alveolar type II cell Na+ channel protein into planar lipid bilayers.

Low-amiloride-affinity (L-type) Na+ channels have been functionally and immunologically localized to alveolar type II (ATII) cells. Purified rabbit ATII epithelial cells were isolated by elastase digestion and solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate. The solubilized proteins were purified by ion-exchange chromatography, followed by immunoaffinity purification over a column to which rabbit polyclonal antibodies raised against purified bovine renal Na+ channel protein were bound. The proteins eluted from the immunoaffinity column were assayed for specific binding of [3H]Br-benzamil and reconstituted into planar lipid bilayers. Sequential purification steps gave a final enrichment in specific [3H]Br-benzamil binding of > 2,000 compared with the homogenate. Single-channel currents of 25 pS were recorded from the immunopurified rabbit ATII cell protein. Addition of the catalytic subunit of protein kinase A (PKA) plus ATP to the presumed cytoplasmic side of the bilayer resulted in a significant increase in the single-channel open probability (Po), from 0.40 +/- 0.14 to 0.8 +/- 0.12, without altering single-channel conductance. The addition of amiloride or ethylisopropyl amiloride (EIPA) to the side opposite that in which PKA acts reduced Po with no change in single-channel conductance. Rabbit ATII Na+ channels in bilayers had an inhibitory constant for amiloride of 8 microM and 1 microM for EIPA. These data confirm the presence of L-type Na+ channels in adult mammalian ATII cells.

Adenosine Triphosphate↗

PubMed1995

HIV-gp120 activates large-conductance apamin-sensitive potassium channels in rat astrocytes.

Central nervous system (CNS) involvement usually occurs in individuals infected with human immunodeficiency virus type 1 (HIV-1). Evidence is now accumulating that neurons and astrocytes may be functionally compromised by exposure to viral components or cellular factors released from HIV-1-infected macrophages and/or microglia. We have previously reported that the HIV coat protein gp120 stimulates Na+/H+ exchange in primary cultured rat astrocytes, which, ultimately, results in the activation of a K+ conductance. In this report we characterize the electrophysiological and biophysical properties of the channels responsible for the gp120-induced increase in K+ conductance. These K+ channels had a relatively large unitary conductance (147 pS), were not gated by voltage, were sensitive to changes in H+ concentration at their cytosolic face, were specifically inhibited by apamin, and were insensitive to charybdotoxin and tetraethylammonium. The activation of these channels by gp120 is referable to cellular alkalinization subsequent to Na+/H+ exchange stimulation; gp120 failed to activate these K+ channels in the absence of external Na+ or in the presence of amiloride, an inhibitor of Na+/H+ exchange. Subsequent K+ loss from the astrocyte into the restricted extracellular space surrounding neurons can then lead to neuronal depolarization, activation of voltage-sensitive Ca2+ channels, and, eventually, cell death. Thus abnormal activation of astrocyte K+ channels by gp120 may contribute to the CNS pathophysiology associated with HIV-1 infection.

AIDS Dementia Complex↗

PubMed1995

A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers.

We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-bENaC protein 1) displayed voltage-independent Na+ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(i)amil approximately 150 nM], and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.

Amiloride↗

PubMed1995

Immunopurification and functional reconstitution of a Na+ channel complex from rat lymphocytes.

Patch-clamp experiments have demonstrated an amiloride-sensitive Na+ conductance in human B lymphoid cells. We measured whole cell currents in rat lymphocytes and observed a similar Na(+)-specific inward conductance. The presence of 400 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate in the bath significantly increased the inward current, and this adenosine 3',5'-cyclic monophosphate activation was abolished by 2 microM amiloride. We immunopurified a protein complex from rat lymphocyte membranes using an anti-bovine kidney Na+ channel antibody. The complex consisted of five distinct polypeptides with apparent M(r) values of 110,000, 92,000, 59,000, 48,000, and 42,000. This putative channel complex was incorporated into planar lipid bilayers, where we observed single Na+ channel activity that was blocked by amiloride in a concentration-dependent manner. The addition of protein kinase A and ATP to the "intracellular" solution elicited a twofold increase in channel activity. Reverse transcription-polymerase chain reaction analysis was used to determine if the rat lymphocytes express the message for the recently cloned Na+ channel of the rat colon (rENaC). Primers for the alpha-subunit of rENaC identified no message in the lymphocyte RNA, while primers for the beta-subunit of the clone produced low levels of the expected product. Thus it appears that a rENaC-like beta-subunit may be an essential component of the lymphocyte Na+ channel that was isolated. At the same time, this channel is different from those recently cloned in that it does not include an alpha-subunit homologous to that of rENaC.

Animals↗

PubMed1995

Cloning of a bovine renal epithelial Na+ channel subunit.

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.

Amino Acid Sequence↗

PubMed1994

Phosphorylation and activation of a bovine tracheal anion channel by Ca2+/calmodulin-dependent protein kinase II.

Secretion of Cl- by epithelial cells is fundamental to the processes of fluid and electrolyte transport by epithelia such as those of the airways, sweat-ducts, and gastrointestinal tract. In the present study, we show that a novel Cl- channel protein, immunoaffinity purified from bovine tracheal apical membrane vesicles, is sensitive to phosphorylation by Ca2+/calmodulin protein kinase II (CaMK II). The channel protein, which migrates with an M(r) of 140,000 under nonreducing conditions, is phosphorylated in vitro by CaMK II in a Ca(2+)- and calmodulin-dependent manner. When reconstituted into planar lipid bilayers, the protein behaves as an anion-selective, 4,4'-diisothiocyanostilbene- and dithiothreitol-sensitive channel. The open probability of this channel is significantly increased by Ca2+ alone but only at levels of Ca2+ (5-10 microM) that lie outside the physiological range. Addition of CaMK II to the presumptive cytoplasmic side of the bilayer in the presence of ATP and calmodulin dramatically increased the sensitivity of the channel to free Ca2+, shifting the dose-response curve for Ca(2+)-dependent channel activation to lower [Ca2+]i, the maximum increase in channel Po occurring between 0.6 and 1 microM. The addition of kinase in the absence of ATP or calmodulin or the addition of ATP or calmodulin in the absence of kinase was without effect on channel Po. Increasing [Ca2+] above 1 microM decreased channel mean current, causing a flickery block that was maximal at 2 microM. Increasing [Ca2+] as high as 10 microM in the presence of kinase did not further alter channel behavior. In contrast to CaMK II, the addition of the catalytic subunit of protein kinase A either alone or together with ATP had no effect on channel Po. These observations suggest that a novel Ca(2+)-sensitive anion channel isolated from bovine airway epithelium is regulated by CaMK II phosphorylation.

Adenosine Triphosphate↗

PubMed1994

Carboxyl methylation activates purified renal amiloride-sensitive Na+ channels in planar lipid bilayers.

The early increase in luminal membrane Na+ permeability by aldosterone in Na(+)-reabsorbing epithelia is attributed to an increase in the open probability (and number) of preexisting amiloride-sensitive Na+ channels. Carboxyl methylation reactions are involved, but the mechanism of action is unknown. We report that the 90-95-kDa polypeptide subunit of a purified renal Na+ channel protein can be specifically carboxymethylated and that this biochemical reaction, in the presence of guanosine 5'-3-O-(thio)triphosphate, leads directly to an increase in channel activity. Further, we show that protein kinase A-mediated phosphorylation can synergistically activate these channels. We suggest that renal Na+ channels have multiple biochemical regulatory inputs and that post-translational modifications underlie the increases in luminal membrane Na+ channel activity produced by aldosterone and vasopressin in Na(+)-reabsorbing epithelia.

Amiloride↗

PubMed1994

Amiloride-sensitive channels in marginal cells in the stria vascularis of the guinea pig cochlea.

We examined marginal cells in stria vascularis for the presence of amiloride-sensitive Na+ channels, a possible pathway for maintaining a low Na+ concentration in the endolymph. Whole-cell voltage-clamp experiment shows that amiloride at 1 microM concentration reversibly reduces inward current more than outward current. Immunogold-labeling method shows that the luminal and lateral membrane have antigenic sites for these antibodies. These observations indicate the presence of amiloride-sensitive channels in the marginal cell. If amiloride-sensitive channels in the luminal membrane are highly selective to Na+, they could be an efficient pathway for Na+ uptake from the endolymph. In the basolateral membrane, amiloride-sensitive Na+ channels may make a relatively small contribution to the unusual resting potential.

Amiloride↗

PubMed1994

Cytokines and HIV envelope glycoprotein gp120 stimulate Na+/H+ exchange in astrocytes.

The pathogenesis of the human immunodeficiency virus (HIV)-associated cognitive/motor complex, or acquired immunodeficiency syndrome (AIDS) dementia complex, is unknown, but it afflicts over 50% of all patients infected with HIV-1. Because neurons are not directly infected with HIV-1, the causes of neuronal dysfunction are undoubtedly indirect. We investigated the role of the astrocyte in the development of AIDS dementia complex, focusing on cytokine and HIV-1 gp120 stimulation of Na+/H+ exchange (NHE) activity of primary rat astrocytes. Our results show that the cytokines tumor necrosis factor-alpha, interferon (IFN)-gamma, and interleukin (IL)-1 beta (all found to be elevated in the central nervous system of AIDS patients), can stimulate Na+/H+ exchange, but that transforming growth factor-beta, IL-2, and IL-6 do not. IFN-gamma and gp120-induced activation of Na+/H+ exchange appears to be mediated through activation of tyrosine kinase (TK), because TK inhibitors block the action of IFN-gamma and gp120. Additionally, gp120 induces tyrosine phosphorylation of two proteins (approximately 90 and 130 kDa), which is also inhibited by TK inhibitors. The predominant NHE isoform present in rat astrocytes is NHE-1; however, other isoforms are also present. We conclude that Na+/H+ exchange of rat astrocytes can be differentially stimulated by cytokines and HIV-1 gp120. We hypothesize that the resultant increase in intracellular pH with its concomitant changes in astrocyte membrane permeability properties produces an imbalance in the K+ and glutamate microenvironment of the neurons, leading to a rise in intraneuronal Ca2+ and eventual neuronal dysfunction and/or demise.

Amiloride↗

PubMed1994

Protein kinase A phosphorylation and G protein regulation of purified renal Na+ channels in planar bilayer membranes.

Purified bovine renal epithelial Na+ channels incorporated into planar lipid bilayer membranes were used to evaluate the biophysical consequences of its phosphorylation by protein kinase A (PKA). We also studied the effects of pertussis toxin-induced ADP-ribosylation on single channel activity of nonphosphorylated and PKA-phosphorylated channels. PKA-induced phosphorylation resulted in a significant increase in single channel open probability (Po) with no change in single channel conductance, as well as increased the probability of multiple channel openings in the bilayer. Further, PKA conferred a voltage sensitivity to channel gating without affecting open channel conduction properties. PKA-phosphorylated Na+ channels were inhibited by subsequent ADP-ribosylation with pertussis toxin (PTX). Addition of guanosine 5'-3-O-(thio)triphosphate reversed this inhibition. However, exposure of nonphosphorylated Na+ channels to PTX increased channel open probability by a factor of 3-5. These results demonstrate that a cAMP-dependent pathway is an important regulatory element for amiloride-sensitive Na+ channels and that the effects of PTX-induced ADP-ribosylation of the channel-associated Gi protein on function depend upon the previous phosphorylation state of the protein.

Adenosine Triphosphate↗

PubMed1994

Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex.

Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex, presents as a subcortical dementia with cognitive, motor, and behavioral disturbances and is unique to HIV-1 infection. The pathogenesis of this syndrome is poorly understood but is believed to involve interactions among virally infected macrophages/microglia, astrocytes, and neurons. In this study, we show that exposure of primary rat and human astrocytes to heat-activated HIV-1 virions, or to eukaryotically expressed HIV-1 and HIV-2 envelope glycoproteins (gp120) stimulates amiloride-sensitive Na+/H+ antiport, potassium conductance, and glutamate efflux. These effects are blocked specifically by amiloride, an inhibitor of Na+/H+ antiport and by the selective removal of gp120 with immobilized monoclonal antibody. As a result of modulation of astrocytic function by gp120, the ensuing neuronal depolarization and glutamate exposure could activate both voltage-gated and N-methyl-D-aspartate-regulated Ca2+ channels, leading to increases in intraneuronal Ca2+ and neuronal death. These findings implicate the astrocyte directly in the pathogenesis of AIDS dementia complex.

AIDS Dementia Complex↗

PubMed1994

gp120-mediated alterations in astrocyte ion transport.

The pathogenesis of the acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) is unknown. However, recent work indicates that neurons and astrocytes are functionally compromised by exposure to viral components or cellular factors released from HIV-1-infected macrophages/microglia. We show that exposure of primary cultured rat astrocytes to the major HIV envelope glycoprotein gp120 results in alterations of ion and solute transport that may contribute to neuronal cell injury.

AIDS Dementia Complex↗

PubMed1994

Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells.

The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for the trans-Golgi network (TGN). In preconfluent monolayers or unpolarized HT-29 cells, BFA had no effect on CFTR staining, which remained perinuclear. Mature, Golgi-processed CFTR protein was isolated from both polarized and unpolarized colonocytes. Thus the mechanism for polarization-dependent apical membrane CFTR targeting and the acquisition of cAMP-dependent Cl- secretion lies at or beyond the late Golgi-TGN in epithelial cells.

Brefeldin A↗