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T R Kleyman

Publications and source records attributed to T R Kleyman.

At least 55 records · Page 3Linked to original sources
PubMed1994

Regulation of mitochondrial cAMP-dependent protein kinase activity in yeast.

We have shown that transcription of the yeast (S. cerevisiae) mitochondrial (mt) genome is cAMP-sensitive, via a mt cAMP-dependent protein kinase (cAPK). In relation to that work, we examined whether the BCY 1 gene product functions as regulatory subunit for mt cAPK, as it does for the cytoplasmic enzyme. We demonstrate that mt protein extracts from a bcy 1 strain show no cAPK activity, whereas similar extracts from an otherwise isochromosomal BCY 1 strain show high levels of such activity. Partial purification of mt cAPK from each strain confirms this difference. Photoaffinity labeling with 8-N3[32P]cAMP and highly-purified mt protein extracts from the BCY 1 strain identifies one cAMP-binding protein (M(r) approximately 47000), while similar mt extracts from the bcy 1 strain lack all cAMP-binding proteins. These data suggest that BCY 1 regulates yeast mt cAPK, and that inactivation of BCY 1 removes that mt activity from cAMP control.

Affinity Labels↗
PubMed1993

Differential arrival of newly synthesized apical and basolateral plasma membrane proteins in the epithelial cell line A6.

The labeling of specific cell surface proteins with biotin was used to examine both protein distribution and delivery of newly synthesized proteins to the apical and basolateral cell surface in A6 cells. Steady-state metabolic labeling with [35S]methionine followed by specific cell surface biotinylation demonstrated polarization of membrane proteins. The delivery of newly synthesized proteins to the apical or basolateral cell surface was examined by metabolic labeling with [35S]methionine using a pulse-chase protocol in combination with specific cell surface biotinylation. Newly synthesized biotinylated proteins at the apical cell surface reached a maximum after a 5 min chase, and then fell over the remainder of a 2 hr chase. The bulk flow of newly synthesized proteins to the basolateral membrane slowly rose to a maximum after 90 min. The detergent Triton X-114 was used to examine delivery of hydrophilic and hydrophobic proteins to the cell surface. Delivery of both hydrophilic and hydrophobic proteins to the apical cell surface reached a maximum 5 to 10 min into the chase period. The arrival of hydrophilic proteins at the basolateral surface showed early delivery and a maximum peak delivery at 120 min into the chase period. In contrast, only an early peak of delivery of newly synthesized hydrophobic proteins to the basolateral membrane was observed.

Animals↗
PubMed1993

5-(N-Ethyl-N-isopropyl)amiloride sensitive Na+ currents in intact fetal distal lung epithelial cells.

To determine whether primary cultures of rat fetal distal lung epithelium (FDLE) possessed L-type Na+ channels on their plasma membrane we performed experiments with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and other amiloride analogs. Short-circuit current (Isc) was decreased by the apical application of amiloride and benzamil, but was unaffected by 10 microM dimethylamiloride (DMA). EIPA decreased Isc when added to either the apical or basal sides. Greatest effects were seen with bilateral EIPA, where half-maximal effects occurred in the micromolar range. Measurements of intracellular pH with the fluorescent dye BCECF demonstrated that DMA impaired (IC50 = 71 nM) the ability of FDLE to recover from intracellular acidification. Nystatin perforated patch clamp techniques showed that FDLE had nonrectifying Na+ currents but no detectable Cl- currents. The whole-cell currents were reversibly decreased by 20 microM concentrations of EIPA, benzamil, and amiloride but were unaffected by 20 microM DMA. These studies indicate that there are EIPA-sensitive Na+ conductances in intact FDLE and suggest the presence of L-type Na+ conductances on their apical membrane and EIPA-sensitive K+ channels on the basolateral membrane.

Adenine↗
PubMed1993

Distinct regulation of Na+ reabsorption and Cl- secretion by arginine vasopressin in the amphibian cell line A6.

The neurohypophysial peptide arginine vasopressin (AVP) increases Na+ absorption across A6 epithelia. In addition to the positive natriferic response, AVP increases net basolateral to apical Cl- flux. The time course of activation of electrogenic ion transport in A6 epithelia was examined by measuring transepithelial short-circuit current (ISC). Basolateral application of AVP (0.1 U/ml) or forskolin (10 microM) affects ISC in a biphasic manner. Shortly after addition of AVP, an early (transient) phase is observed in which ISC is rapidly stimulated, reaching a peak value at 1.4 +/- 0.1 min. A subsequent decrease in current is interrupted by a slower, late phase in which ISC reaches a peak 23 +/- 3 min after addition of AVP. The late increase in ISC is sustained over the remainder of the 40-min period of observation. The time course of ISC stimulation by forskolin is qualitatively similar. Replacement of external Cl- by aspartate lowers baseline transport nearly 40%, strongly blunts the early phase of ISC stimulation, and retains the late increase. Addition of amiloride (10 microM) to the apical bath before AVP or forskolin stimulation of ISC eliminates the late increase of ISC. Steady-state amiloride-insensitive ISC activated under these conditions was sensitive to apical application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (20 microM) and niflumic acid (100 microM). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (1 mM) was not an effective inhibitor of this current. Basolateral bumetanide (100 microM) inhibited baseline ISC and reduced both the peak transient and steady-state amiloride-insensitive ISC.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1993

Fetal lung epithelial cells contain two populations of amiloride-sensitive Na+ channels.

Active Na+ transport by the alveolar epithelium plays a major role in reabsorption of the fetal lung fluid after birth. We characterized the biochemical and physiological characteristics of Na+ conductive pathways in distal fetal lung epithelial (FLE) cells isolated from 20-day-old rat fetuses. We demonstrated that a polyclonal antibody to Na+ channel protein (NaAb) binds to the plasma membranes of FLE cells. In Western blot studies, this NaAb and an anti-idiotypic monoclonal antibody to the amiloride-binding subunit of the Na+ channel protein recognized 150- and 90-kDa polypeptides in plasma membrane vesicles of FLE. 22Na+ flux measurements across plasma membrane vesicles of FLE revealed the existence of electrogenic Na+ transport, which was twice as high as the corresponding adult value. One hundred micromolars of amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride inhibited 30, 40, and 70% of the electrogenic Na+ transport across plasma membrane vesicles of FLE cells, respectively. The half-maximum inhibition of electrogenic Na+ transport by these substances occurred between 0.3 and 1 microM. [3H]benzamil equilibrium binding studies in membrane vesicles of FLE cells revealed the existence of two binding sites that had dissociation constant values of 19 and 1,525 nM, respectively. These data indicate the presence of both high- and low-amiloride affinity Na+ conductive pathways (channels) in FLE cells.

Amiloride↗
PubMed1993

Transport defects of rabbit inner medullary collecting duct cells in obstructive nephropathy.

Urinary obstruction markedly reduces collecting duct Na+ reabsorption. To define the cellular mechanisms of this derangement in Na+ reabsorption in inner medullary collecting duct (IMCD) of obstructed kidneys, suspensions of intact IMCD cells and inner medulla plasma membranes (IMPM) were prepared from 24 h obstructed and untreated control kidneys. Oxygen consumption (QO2) studies revealed marked reductions in both amiloride-sensitive and ouabain-sensitive QO2 but not ouabain-insensitive QO2 in intact IMCD cells from obstructed, compared with control animals, indicating a reduction in oxygen-dependent transport activities of both the Na+ channel and the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Amiloride-sensitive conductive 22Na+ uptake in intact IMCD cells from obstructed kidneys was significantly decreased by 45% at 10 s, 30 s, and 1-5 min (10 s: 2.42 +/- 0.63 vs. 4.49 +/- 0.64 nmol Na+ flux/mg protein, n = 7, P < 0.05; 1 min: 4.65 +/- 0.7 vs. 8.27 +/- 0.98 nmol Na+ flux/mg protein, n = 7, P < 0.05), indicating decreased activity of amiloride-sensitive Na+ channels in these cells. However, immunoblots of IMPM with antibodies to Na+ channel proteins did not show significant differences in content of Na+ channel proteins between membranes from obstructed and control groups. Ouabain-sensitive Na(+)-K(+)-ATPase activity in IMPM of obstructed kidneys was also reduced (61.1 +/- 18.1 vs. 152.6 +/- 25.8 nmol ATP degradation.min-1.mg protein-1, n = 6, P < 0.02), and immunoblots with monoclonal antibodies against the alpha 1- and beta-subunits of rabbit Na(+)-K(+)-ATPase showed a 51 +/- 7% reduction of both subunits in IMPM from obstructed kidneys (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes.

An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.

Affinity Labels↗
PubMed1992

Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6.

The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone.

Aldosterone↗
PubMed1992

Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity.

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.

Amiloride↗
PubMed1992

Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia.

We used patch-clamp methods to examine the effects of depletion and readdition of aldosterone on single, highly selective, amiloride-blockable sodium channels in the A6 cell line. Single-channel characteristics changed little before 24 h of continuous aldosterone depletion, although there was some reduction in short-circuit current. Thereafter, apical sodium permeability, measured as product of channel number per patch and individual channel open probability (NPo), was reduced between five- and sevenfold, primarily due to a large decrease in channel mean open time. With about the same time course, short-circuit current also decreased approximately fivefold. Readdition of aldosterone to depleted cells produced an increase in NPo within 2 h, primarily through an increase in mean open time. After readdition, channel number per patch increased twofold compared with cells not hormone deprived, with a return to control levels between 24 and 48 h after continuous exposure. The increase in short-circuit current followed a similar time course. The primary effect of aldosterone appears to be modulation of the open time of channels continuously present in the apical membrane, rather than promotion of the appearance or disappearance of channels from the membrane. In particular, it cannot be demonstrated statistically that aldosterone removal reduces the number of channels per patch, and there may actually be up to a twofold increase after a long period of aldosterone depletion.

Aldosterone↗
PubMed1991

Characterization and cellular localization of the epithelial Na+ channel. Studies using an anti-Na+ channel antibody raised by an antiidiotypic route.

Amiloride-sensitive Na+ channels are expressed at the apical membrane of high resistance, Na+-transporting epithelial. The specific interaction of amiloride with this transport protein suggested the feasibility of raising anti-Na+ channel antibodies by an antiidiotypic approach designed to generate antibodies directed against the amiloride-binding domain on the channel. Antiidiotypic monoclonal antibody RA6.3 mimicked the effect of amiloride by inhibiting Na+ transport across A6 cell monolayers when applied to the apical cell surface. Inhibition of transport required pretreatment of the apical cell surface with trypsin in the presence of amiloride in order to enhance accessibility of the antibody to the amiloride-binding site. This antibody specifically immunoprecipitated a large 750,000-700,000 Da protein from [35S]methionine-labeled A6 cell cultures, which was resolved further under reducing conditions as a set of polypeptides with apparent molecular masses of 260,000-230,000, 180,000, 140,000-110,000, and 70,000 Da. The antibody recognized the 140,000-Da subunit, known to contain the amiloride-binding domain, on immunoblots of purified A6 cell Na+ channel. Immunoprecipitation of apical or basolateral plasma membrane proteins selectively labeled with 125I demonstrated that expression of the oligomeric Na+ channel was restricted to the apical plasma membrane. Immunocytochemical localization in A6 cultures revealed apical membrane as well as cytosolic immunoreactive sites. Immunostaining was also observed at or near the basolateral plasma membrane.

Amiloride↗
PubMed1991

Distinct epitopes on amiloride. II. Variably restricted epitopes defined by monoclonal anti-amiloride antibodies.

Specific regions of amiloride appear to participate in binding to receptors on amiloride-sensitive transport proteins. Previous studies characterizing epitopes on amiloride recognized by anti-amiloride antibodies have demonstrated that antibodies recognize specific domains on amiloride and that these epitopes are determined, in part, by the site on amiloride used to couple to carrier protein. The 3,5-diaminopyrazinyl and guanidinocarbonyl moieties were identified as distinct epitopes. Since Na(+)-selective transport proteins are sensitive to changes of the halide on the amiloride molecule, additional monoclonal anti-amiloride antibodies were raised to determine whether the C-6 halo group of amiloride could be identified as an important site for drug-antibody binding. The epitopes recognized by a series of three monoclonal antibodies raised against amiloride coupled to rabbit serum albumin through its C-5 NH2-group were defined. Two antibodies recognize extensive regions on the amiloride molecule, including both the acylguanidino and pyrazinyl groups. In addition, both antibodies are sensitive to changes in the C-6 halo group on amiloride. A third antibody was relatively insensitive to changes in the halide in the C-6 position of the pyrazine ring of amiloride and recognized a more restricted epitope on amiloride.

Amiloride↗
PubMed1990

Cation transport probes: the amiloride series.

The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, and of effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. We have reviewed the pharmacology of inhibition of these processes by amiloride an its analogs, as well as the use of amiloride analogs as potential probes for the characterization of ion transport systems.

Affinity Labels↗
PubMed1989

The cellular pool of Na+ channels in the amphibian cell line A6 is not altered by mineralocorticoids. Analysis using a new photoactive amiloride analog in combination with anti-amiloride antibodies.

An amiloride-sensitive Na+ channel is found in the apical plasma membrane of high resistance, Na+ transporting epithelia. We have developed a method for the identification of this channel based on the use of a new high affinity photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), and anti-amiloride antibodies to identify photolabeled polypeptides. NMBA specifically labels the putative Na+ channel in bovine kidney microsomes. A 130-kDa polypeptide is detected on immunoblots with anti-amiloride antibodies. NMBA is a potent inhibitor of Na+ transport in the established amphibian kidney epithelial cell line A6, and specifically labels a 130-kDa polypeptide. We utilized both NMBA photolabeling and [3H]benzamil binding in order to examine the cellular pool of putative channels following hormonal regulation of Na+ transport. This pool is not significantly altered by the mineralocorticoid agonist aldosterone or antagonist spironolactone, despite a 3.8-fold difference in transepithelial Na+ transport.

Affinity Labels↗
PubMed1989

Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.

Amiloride↗
PubMed1989

Distinct epitopes on amiloride.

Most Na(+)-selective transport proteins are inhibited by the drug amiloride. Studies using amiloride analogues suggest that specific regions of amiloride might participate in binding to receptors on these transport proteins. To determine whether certain domains of this drug are recognized as distinct epitopes, amiloride was coupled to albumin through either its C-5 NH2-group on the pyrazine ring or through a terminal NH2-group of the guanidino moiety, and antibodies were raised against these amiloride-albumin conjugates. Studies of antibody binding to amiloride analogues identified the 3,5-diaminopyrazinyl, the guanidinocarbonyl, and the C-6 halo moieties as distinct epitopes, although the antibodies required the presence of both the 3,5-diaminopyrazinyl as well as the guanidinocarbonyl moiety for binding.

Amiloride↗
PubMed1988

Amiloride and its analogs as tools in the study of ion transport.

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.

Amiloride↗