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A G Prat

Publications and source records attributed to A G Prat.

25 records · Page 2Linked to original sources
PubMed1993

The multidrug resistance (mdr1) gene product functions as an ATP channel.

The multidrug resistance (mdr1) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATP-conducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Vasopressin and protein kinase A activate G protein-sensitive epithelial Na+ channels.

To determine the molecular steps involved in the vasopressin-induced renal Na+ reabsorption, the patch-clamp technique was utilized to study the role of this hormone in the regulation of apical Na+ channels in renal epithelial A6 cells. Addition of arginine vasopressin (AVP) induced and/or enhanced Na+ channel activity within 5 min of addition under cell-attached conditions. The AVP-induced channel activity was a reflection of both an increase in the average apparent channel number (0.2-1.7) and the percent open time (2-56%). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, the adenosine 3',5'-cyclic monophosphate (cAMP) analogues, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, or forskolin elicited a comparable effect to that of AVP. The induced channels had similar properties to Na+ channels previously reported, including a channel conductance of 9 pS, Na(+)-to-K+ selectivity of 3-5:1, and high amiloride sensitivity. The cAMP-dependent protein kinase A (PKA) in the presence of ATP induced and/or enhanced Na+ channel activity in excised inside-out patches with a change in average apparent channel number and percent open probability similar to those observed with either AVP or cAMP analogues in intact cells. Addition of activated pertussis toxin (100 ng/ml) completely blocked the AVP- or PKA-induced Na+ channel activity in excised inside-out patches, whereas incubation of intact cells with the toxin completely prevented the effect of both activators. The data indicate that AVP mediates its effect through a cAMP-dependent pathway involving PKA activation whose target is the G protein pathway that regulates apical epithelial Na+ channel activity.

Amiloride↗
PubMed1993

Activation of epithelial Na+ channels by protein kinase A requires actin filaments.

We have recently demonstrated a novel role for "short" actin filaments, a distinct species of polymerized actin different from either monomeric (G-actin) or long actin filaments (F-actin), in the activation of epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) was investigated by patch-clamp techniques in A6 epithelial cells. In excised inside-out patches, addition of deoxyribonuclease I, which prevents actin polymerization, inhibited Na+ channel activation mediated by PKA. Disruption of endogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activation following the addition of actin to the cytosolic side of the patch. To assess the role of PKA on actin filament organization, actin was used as a substrate for the specific phosphorylation by the PKA. Actin was phosphorylated by PKA with an equilibrium stoichiometry of 2:1 mol PO4-actin monomer. Actin was phosphorylated in its monomeric form, but only poorly once polymerized. Furthermore, phosphorylated actin reduced the rate of actin polymerization. Thus actin allowed to polymerize for at least 1 h in the presence of PKA and ATP to obtain phosphorylated actin filaments induced Na+ channel activity in excised inside-out patches, in contrast to actin polymerized either in the absence of PKA or in the presence of PKA plus a PKA inhibitor (nonphosphorylated actin filaments). This was also confirmed by using purified phosphorylated G-actin incubated in a polymerizing buffer for at least 1 h at 37 degrees C. These data suggest that the form of actin required for Na+ channel activation (i.e., "short" actin filaments) may be favored by the phosphorylation of G-actin and may thus mediate or facilitate the activation of Na+ channels by PKA.

Actins↗
PubMed1991

Purification of cytochrome c peroxidase for monitoring H2O2 production.

One of the most precise methods of determining hydrogen peroxide (H2O2) formation by biological systems is based on measuring the rate of enzyme-substrate complex formation between H2O2 and cytochrome c peroxidase (CCP). The main problem with this method is that CCP is not commercially available and has to be prepared in the laboratory. We have modified some currently available methods for purifying a highly active preparation of CCP in about 4 d. It includes a batch extraction of protein using DEAE-sepharose followed by concentration either by lyophilization or by passing the extract through a small DEAE-sepharose column instead of by ultrafiltration. The concentrated preparation is passed through a Sephadex G-75 column and the final CCP crystallized against water. The final preparations had a purity index (PI, ratio of absorbance at 408 nm/280 nm, equivalent to heme/protein ratio) above 1.2. These changes make the overall procedure very simple, preserving enzyme activity and spectral properties. In addition, we point out that special care has to be taken to eliminate cytochrome c from crude CCP extracts. Cytochrome c not only introduces an artifact when determining PI, but is also may act as a hydrogen donor for CCP when monitoring H2O2 formation, thus decreasing the sensitivity of this method.

Cytochrome-c Peroxidase↗
PubMed1991

Actin filaments regulate epithelial Na+ channel activity.

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

Actins↗
PubMed1990

Ascorbate- and hemoglobin-dependent brain chemiluminescence.

It has been indicated recently that ascorbic acid is responsible for the hemoglobin-mediated oxidative damage to the central nervous system (Sadrzadeh & Eaton, J. Clin. Invest. 82:1510-1515, 1988). In this paper we describe the changes in chemiluminescence accompanying hemoglobin- and ascorbate-dependent oxidative injury to brain tissue. Addition of either hemoglobin (15 microM) or ascorbate (1 or 2 mM) to rat brain homogenates stimulated spontaneous chemiluminescence in a synergistic manner. This increase in chemiluminescence was inhibited by desferrioxamine indicating that free iron was involved in the reactions leading to lipid peroxidation. Preincubation with ascorbate oxidase inhibited both spontaneous and hemoglobin-dependent chemiluminescence, suggesting that ascorbate was required for the reactions leading to lipid peroxidation. Supplementation with aminotriazole (an irreversible inhibitor of the catalase-H2O2 complex) increased chemiluminescence in a time-dependent manner, as catalase reacted with accumulated H2O2, suggesting that ascorbic acid has a dual action being involved in the production of H2O2 and also maintaining Fe in the reduced state to catalyze a Fenton-like reaction. The excited species responsible for the chemiluminescence were partially characterized by adding specific fluorescent energy acceptors: dibromoanthracene (DBA) and diphenylanthracene (DPA). Both DBA and DPA stimulated chemiluminescence several-fold indicating that triplet and singlet species are responsible for the observed chemiluminescence. Excited singlet carbonyls (identified with DPA) may be produced during the collision of two ROO.. Singlet oxygen may also be generated during the same reaction. It decays to the triplet state (emitting chemiluminescence at 634 nm) and reacts with double bonds producing dioxetanes, which may breakdown generating triplet carbonyls (identified with DBA).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗