Biomedical subjects

M F Romero

Publications and source records attributed to M F Romero.

36 records · Page 2Linked to original sources

PubMed1998

Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes.

It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2 permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM HCO3- to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 x 10(-4) pH units/s in control oocytes and 13.3 x 10(-4) pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 x 10(-4) pH units/s). Ethoxzolamide (ETX; 10 microM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 x 10(-4) pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were approximately 40% faster than in the water/CA oocytes (75 x 10(-4) pH units/s), and ETX reduced the rates substantially (14.7 x 10(-4) pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux, it would remain to be determined whether this CO2 movement is quantitatively important.

Animals↗

PubMed1998

Cloning and functional expression of rNBC, an electrogenic Na(+)-HCO3- cotransporter from rat kidney.

We have recently cloned the renal electrogenic Na(+)-bicarbonate contransporter of the salamander Ambystoma tigrinum (aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409-413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na(+)-bicarbonate cotransporter. NBC constitutes the major route for HCO3- reabsorption and assists in Na+ reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in lambda gt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3'-untranslated region of approximately 500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular mass of 116 kDa. Its deduced amino acid sequence is 86% identical to that of aNBC. Comparison of both the aNBC and rNBC sequences to the GenBank database reveals a low level of amino acid identity (approximately 30%) to the AE family of Cl-/HCO3- exchangers. Injection of rNBC cRNA into Xenopus oocytes leads to expression of an electrogenic Na(+)-HCO3- contransporter that is qualitatively similar to that of aNBC but at a much lower level. Placement of the rNBC cDNA into the context of a Xenopus expression vector produces a substantial increase in rNBC expression. Addition of 1.5% CO2/10 mM HCO3- elicits a hyperpolarization of > 50 mV and a rapid decrease of intracellular pH (pHi), followed by an increase in pHi. Subsequent removal of Na+ in the presence of CO2/HCO3- causes a depolarization of > 50 mV and a concomitant decrease of pHi. Thus rNBC is in the same newly identified family of Na(+)-linked HCO3- transporters as is aNBC.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of a mammalian proton-coupled metal-ion transporter.

Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases. Despite their pivotal physiological roles, however, there is no molecular information on how metal ions are actively absorbed by mammalian cells. We have now identified a new metal-ion transporter in the rat, DCT1, which has an unusually broad substrate range that includes Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+. DCT1 mediates active transport that is proton-coupled and depends on the cell membrane potential. It is a 561-amino-acid protein with 12 putative membrane-spanning domains and is ubiquitously expressed, most notably in the proximal duodenum. DCT1 is upregulated by dietary iron deficiency, and may represent a key mediator of intestinal iron absorption. DCT1 is a member of the 'natural-resistance-associated macrophage protein' (Nramp) family and thus its properties provide insight into how these proteins confer resistance to pathogens.

Amino Acid Sequence↗

PubMed1997

The electrogenic Na/HCO3 cotransporter.

The electrogenic Na/HCO3 cotransporter (symporter) is the major HCO3- transporter of the renal proximal tubule (PiT), located at the basolateral membrane (BLM), and also plays a noteworthy role in Na+ reabsorption. HCO3 transporters are important for regulation of intracellular pH (pHi) in most cells and also thereby regulate blood pH. This electrogenic Na/HCO3 cotransporter was first discovered using perfused Ambystoma tigrinum (salamander) renal, proximal tubules. This novel cotransporter mediates the movement of one Na+ ion with several HCO3- ions, making it electrogenic, is blocked by stilbene compounds, but does not depend on intra- or extracellular Cl-. This and similar cotransporters have been found in a number of tissues and cell types. Recently, we used Xenopus-laevis oocytes to expression clone the salamander renal electrogenic Na Bicarbonate Cotransporter (NBC). Using microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi), we followed oocyte expression after injecting poly (A)+, fractioned poly (A)+, or cRNA. All experimental solutions contained 100 microM ouabain to block the Na+/K+ pump. Our expression assay was to apply 1.5% CO2/10 mM HCO3- (pH 7.5), allow pHi to stabilize from the CO2-induced acidification, and then remove bath Na+. Removing bath Na+ from native oocytes and water-injected controls, hyperpolarized the oocytes by approximately 5 mV and had no effect on pHi. However, for oocytes injected with poly (A)+ RNA, removing Na+ transiently depolarized the cell by approximately 10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. Electrophoretic fractionation of the poly (A)+ RNA, enriched the expression signal. From the optimal expression-fraction, we constructed a size-selected cDNA library in pSPORT1. Screening our Ambystoma library yielded a single clone (aNBC). We could detect expression 3 days after injection of NBC cRNA. In aNBC-expressing oocytes, adding CO2/HCO3-elicited a large (> 50mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Na+ removal in CO2/HCO3-depolarized the cell by > 40mV and decreased pHi, aNBC encodes a protein of 1035 amino acids with several putative membrane-spanning domains, and has a low level of amino-acid homology (approximately 30% to the AE family of Cl-HCO3 exchangers. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters and, together with the AE family, defines a new superfamily of HCO3- transporters. Using aNBC to screen a rat-kidney cDNA library, we identified a full-length cDNA clone (rNBC), rNBC encodes a protein of 1035 amino acids, is 86% identical to aNBC, and can be functionally expressed in oocytes.

Acid-Base Equilibrium↗

PubMed1997

Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter.

Bicarbonate transporters are the principal regulators of pH in animal cells, and play a vital role in acid-base movement in the stomach, pancreas, intestine, kidney, reproductive system and central nervous system. The functional family of HCO3- transporters includes Cl- -HCO3- exchangers, three Na+/HCO3- cotransporters, a K+/HCO3- cotransporter, and a Na+-driven Cl- -HCO3- exchanger. Molecular information is sparse on HCO3- transporters, apart from Cl- -HCO3- exchangers ('anion exchangers'), whose complementary DNAs were cloned several years ago. Attempts to clone other HCO3- transporters, based on binding of inhibitors, protein purification or homology with anion exchangers, have so far been unsuccessful. Here we monitor the intracellular pH and membrane voltage in Xenopus oocytes to follow the expression of the most electrogenic transporter known: the renal 1:3 electrogenic Na+/HCO3- cotransporter from the salamander Ambystoma tigrinum. We now report the successful cloning and characterization of a cDNA encoding a cation-coupled HCO3- transporter. The encoded protein is 1,035 amino acids long with several potential membrane-spanning domains. We show that when it is expressed in Xenopus oocytes, this protein is electrogenic, Na+ and HCO3- dependent, and blocked by the anion-transport inhibitor DIDS, and conclude that it is the renal electrogenic sodium bicarbonate cotransporter (NBC).

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

PubMed1997

Symmetry of H+ binding to the intra- and extracellular side of the H+-coupled oligopeptide cotransporter PepT1.

Ion-coupled solute transporters exhibit pre-steady-tate currents that resemble those of voltage-dependent ion channels. These currents were assumed to be mostly due to binding and dissociation of the coupling ion near the extracellular transporter surface. Little attention was given to analogous events that may occur at the intracellular surface. To address this issue, we performed voltage clamp studies of Xenopus oocytes expressing the intestinal H+-coupled peptide cotransporter PepT1 and recorded the dependence of transient charge movements in the absence of peptide substrate on changing intra- (pHi) and extracellular pH (pHo). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pHi and pHo. At a pHo of 7.0 and a holding potential (Vh) of -50 mV, the charge movements were mostly inwardly directed, whereas reduction of pHo to below 7.0 resulted in outwardly directed charge movements. When pHi was reduced, inwardly directed charge movements were observed. The data on the voltage dependence of the transient charge movements were fitted by the Boltzmann equation, yielding an apparent valence of 0.65 +/- 0.03 (n = 7). The midpoint voltage (V0.5) of the charge distribution shifted linearly as a function of pHi and pHo. Our results indicate that, as a first approximation, the magnitude and polarity of the transient charge movements depend upon the prevailing H+ electrochemical gradient. We propose that PepT1 has a single proton binding site that is symmetrically accessible from both sides of the membrane and that decreasing the H+ chemical potential (DeltamuH) or increasing the membrane potential (Vm) shifts this binding site from an outwardly to an inwardly facing occluded state. This concept constitutes an important extension of previous kinetic models of ion-coupled solute transporters by including a more detailed description of intracellular events.

Animals↗

PubMed1997

Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1.

1. The intestinal H(+)-coupled peptide transporter PepT1, displays a broad substrate specificity and accepts most charged and neutral di- and tripeptides. To study the proton-to-peptide stoichiometry and the dependence of the kinetic parameters on extracellular pH (pHo), rabbit PepT1 was expressed in Xenopus laevis oocytes and used for uptake studies of radiolabelled neutral and charged dipeptides, voltage-clamp analysis and intracellular pH measurements. 2. PepT1 did not display the substrate-gated anion conductances that have been found to be characteristic of members of the Na(+)- and H(+)-coupled high-affinity glutamate transporter family. In conjunction with previous data on the ion dependence of PepT1, it can therefore be concluded that peptide-evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic and basic dipeptides induced intracellular acidification. The rate of acidification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton-substrate coupling ratios of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respectively. 4. Maximal transport of the neutral and charged dipeptides Gly-Leu, Gly-Glu, Gly-Lys and Ala-Lys occurred at pHo 5.5, 5.2, 6.2 and 5.8, respectively. The Imax values were relatively pHo independent but the apparent affinity (Km(app) values for these peptides were shown to be highly pHo dependent. 5. Our data show that at physiological pH (pHo 5.5-6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly-Glu to a lower pH value suggests that acidic dipeptides are transported in the protonated form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a side-chain on the transporter molecule (e.g. protonation of a histidine group). These considerations have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.

Animals↗

PubMed1997

The renal electrogenic Na+:HCO-3 cotransporter.

The electrogenic Na+:HCO3- cotransporter (symporter) is the major transporter for HCO3- reabsorption across the basolateral membrane of the renal proximal tubule and also contributes significantly to Na+ reabsorption. We expression-cloned the salamander renal electrogenic Na+:Bicarbonate Cotransporter (NBC) in Xenopus laevis oocytes. After injecting poly(A)+ RNA, fractionated poly(A)+ RNA or cRNA, we used microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi) All solutions contained ouabain to block the Na+/K+ pump (P-ATPase). After applying 1.5% CO2/10 mmol l-1 HCO3- (pH 7.5) and allowing pHi to stabilize from the CO2-induced acidification, we removed Na+. In native oocytes or water-injected controls, removing Na+ hyperpolarized the cell by -5 mV and had no effect on pHi. In oocytes injected with poly(A)+ RNA, removing Na+ transiently depolarized the cell by -10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. We enriched the signal by electrophoretic fractionation of the poly(A)+ RNA, and constructed a size-selected cDNA library in pSPORT1 using the optimal fraction. Screening the Ambystoma library yielded a single clone (aNBC). Expression was first obvious 3 days after injection of NBC cRNA. Adding CO2/HCO3- induced a large (> 50 mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Subsequent Na+ removal depolarized the cell by more than 40 mV and decreased pHi. aNBC is a full-length clone with a start Met and a poly(A)+ tail; it encodes a protein with 1025 amino acids and several putative membrane-spanning domains. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters. We used aNBC to screen a rat kidney cDNA library, and identified a full-length cDNA clone (rNBC) that encodes a protein of 1035 amino acids. rNBC is 86% identical to aNBC and can be functionally expressed in oocytes.

Ambystoma↗

PubMed1995

Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter.

Active ion-coupled glutamate transport is of critical importance for excitatory synaptic transmission, normal cellular function, and epithelial amino acid metabolism. We previously reported the cloning of the rabbit intestinal high affinity glutamate transporter EAAC1 (Kanai, Y., and Hediger, M. A. (1992) Nature 360, 467-471), which is expressed in numerous tissues including intestine, kidney, liver, heart, and brain. Here, we report a detailed stoichiometric and kinetic analysis of EAAC1 expressed in Xenopus laevis oocytes. Uptake studies of 22Na+ and [14C]glutamate, in combination with measurements of intracellular pH with pH microelectrodes gave a glutamate to charge ratio of 1:1, a glutamate to Na+ ratio of 1:2, and a OH-/H+ to charge ratio of 1:1. Since transport is K+ dependent it can be concluded that EAAC1-mediated glutamate transport is coupled to the cotransport of 2 Na+ ions, the countertransport of one K+ ion and either the countertransport of one OH- ion or the cotransport of 1 H+ ion. We further demonstrate that under conditions where the electrochemical gradients for these ions are disrupted, EAAC1 runs in reverse, a transport mode which is of pathologic importance. 22Na+ uptake studies revealed that there is a low level of Na+ uptake in the absence of extracellular glutamate which appears to be analogous to the Na+ leak observed for the intestinal Na+/glucose cotransporter SGLT1. In voltage clamp studies, reducing extracellular Na+ from 100 to 10 mM strongly increased K0.5L-glutamate and decreased I(max). The data indicate that Na+ binding at the extracellular transporter surface becomes rate-limiting. Studies addressing the cooperativity of the substrate-binding sites indicate that there are two distinct Na(+)-binding sites with different affinities and that Na+ binding is modulated by extracellular glutamate. A hypothetical ordered kinetic transport model for EAAC1 is discussed.

ATP-Binding Cassette Transporters↗

PubMed1994

Expression cloning of a mammalian proton-coupled oligopeptide transporter.

In mammals, active transport of organic solutes across plasma membranes was thought to be primarily driven by the Na+ gradient. Here we report the cloning and functional characterization of a H(+)-coupled transporter of oligopeptides and peptide-derived antibiotics from rabbit small intestine. This new protein, named PepT1, displays an unusually broad substrate specificity. PepT1-mediated uptake is electrogenic, independent of extracellular Na+, K+ and Cl-, and of membrane potential. PepT1 messenger RNA was found in intestine, kidney and liver and in small amounts in brain. In the intestine, the PepT1 pathway constitutes a major mechanism for absorption of the products of protein digestion. To our knowledge, the PepT1 primary structure is the first reported for a proton-coupled organic solute transporter in vertebrates and represents an interesting evolutionary link between prokaryotic H(+)-coupled and vertebrate Na(+)-coupled transporters of organic solutes.

Amino Acid Sequence↗

PubMed1994

Human uterine cervical epithelial cells grown on permeable support--a new model for the study of differentiation.

The purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5-12 cell layers compared to 1-3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support. Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [35S]methionine- and [35S]cysteine-, [3H]fucose- and [14C]glucosamine-labeled molecules, and positive short-circuit current (Isc) under voltage-clamp conditions. Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7-22.10(-6) cm.sec-1, which was 5-100 fold smaller compared to blank inserts, with a cut-off MW of 40-70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5 mS.cm-2, indicating a leaky but confluent epithelia. Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenotypic characteristics of squamous metaplastic cervical epithelium and endocervical epithelium respectively.

Carcinoma, Squamous Cell↗

PubMed1994

ATP decreases acutely and reversibly transport through the paracellular pathway in human cervical cells.

We studied the effect of ATP on transepithelial transport through the paracellular pathway in human cervical cells. Transepithelial conductance and transepithelial permeability (determined from the measurements of unidirectional flux of inert molecules) were measured in Caski cells grown on permeable support. Transepithelial conductance was 55.9 +/- 17.7 mS/cm2 and permeability was 12.5 +/- 2.7 x 10(-6) cm/s for a 0.51-kDa probe. Addition of ATP to the medium decreased acutely and reversibly the conductance and the permeability to probes between 0.18 and 10 kDa by 23-31% in a dose-related fashion; the 50% effective concentration was 1 microM, with a maximal effect at 5-10 microM extracellular ATP. The ATP effect was observed regardless of the pressure gradient across the epithelium. These results indicate that extracellular ATP in micromolar concentrations decreases acutely and reversibly the permeability through the paracellular pathway in cervical cells, possibly by affecting the permeability of the tight junctions and the resistance of the intercellular space. On the basis of these data, we speculate that ATP may play a role in the regulation of solutes and fluid transport across the cervical epithelium in vivo.

Adenosine Triphosphate↗

PubMed1993

Modulation of phospholipase A2 activity and sodium transport by angiotensin-(1-7).

Angiotensin II (Ang II) receptors are coupled to a variety of signal transduction mechanisms. In the kidney, Ang II at nanomolar concentration binds to proximal tubular cells and stimulates phospholipase A2 (PLA2), which in turn catalyzes the hydrolysis of phosphatidylcholine into lysophosphatidylcholine (LPC) and fatty acid. This signal transduction pathway has been shown to be an important modulator of sodium transport. The kidney cortex possesses the enzyme necessary to convert angiotensin I (Ang I) directly to Ang-(1-7) bypassing Ang II as an intermediate. The present investigation was undertaken to determine whether Ang-(1-7) influences epithelial cell function by comparing this heptapeptide with Ang II as a modulator of PLA2 activity and sodium transport. Proximal tubular cells were labeled in tissue culture with 3H-choline and PLA2 activity was measured by quantitation of LPC. We found that Ang II (10(-9) M to 10(-6) M) significantly increased PLA2 activity (154 +/- 36% to 209 +/- 94%). Similar results were obtained with Ang-(1-7) (240 +/- 130% to 353 +/- 40%). The bioactivity of the peptides was assayed by its ability to regulate transcellular 22Na flux. Ang II (10(-9) M) inhibited 22Na flux by 12 +/- 2% while Ang-(1-7) (10(-9) M) inhibited 22Na flux by 20 +/- 5%. These results suggest that one potential role of Ang-(1-7) in the regulation of kidney epithelial electrolyte transport may involve activation of PLA2.

8,11,14-Eicosatrienoic Acid↗

PubMed1992

Development and characterization of rabbit proximal tubular epithelial cell lines.

We have isolated rabbit kidney proximal tubular epithelial cell lines. The selection was based on their ability to form confluent monolayers on porous supports and to maintain receptor-mediated signal transduction and ion transport, characteristic of the proximal tubule. The isolation method consisted of several steps: (1) superficial cortical proximal tubule segments were microdissected and cultured on a matrix-coated porous support until cells formed a confluent monolayer; (2) primary cultures showing hormone-regulated ion transport typical for the proximal tubule were selected and co-cultured with irradiated fibroblasts; and (3) the epithelial cells surviving after several passages were expanded and passaged on porous substrates. Most of the cell lines developed in this manner were obtained by co-culture with irradiated fibroblasts producing a recombinant retrovirus encoding SV40 large T antigen and G418 resistance. However, SV40 T antigen expression was not essential for immortalization, since neither T antigen nor G418 resistance was detected in the isolated cell lines and co-culture with non-producing 3T3 cells gave similar results. One cell line (vEPT) has been characterized in some detail with respect to morphological, biochemical, and ion transport properties. This line forms confluent monolayers with apical microvilli, tight junctions, and convolutions of the basolateral plasma membrane. Once confluent, monolayers maintain conductances of 25 to 32 mS/cm2 for several weeks in culture and possess phlorizin-sensitive short-circuit current (Isc) in glucose containing media, indicative of apical Na(+)-glucose co-transport. vEPT cells also retain receptor and signaling mechanisms for angiotensin II (Ang II). Apical and basal Ang II and 5,6-epoxy-eicosatrienoic acid (5,6-EET) modulate the Isc in a manner similar to primary cultures. The cell lines share with primary cultures expression of the cytokeratins K8, K10/K11, and K19 ("nomenclature" [21]). They also retain several receptor and signal transduction mechanisms. For example, Ang II, arachidonate, bradykinin, 5,6-EET, parathyroid hormone (residues 1 through 34), and purine nucleotides increase cytosolic Ca2+, PTH elevates cAMP levels, and Ang II enhances proximal tubule-specific arachidonic acid metabolism.

Animals↗

PubMed1991

An epoxygenase metabolite of arachidonic acid 5,6 epoxy-eicosatrienoic acid mediates angiotensin-induced natriuresis in proximal tubular epithelium.

Several laboratories have documented that angiotensin II (AII) induces natriuresis in proximal tubular epithelium by a mechanism that has as yet not been disclosed. The present studies were designed to test the hypothesis that cytochrome P450-dependent metabolites of arachidonic acid mediate this effect. In cultured rabbit proximal tubule a ketoconazole-sensitive product comigrating with 5,6-epoxy-eicosatrienoic acid (5,6-EET) on reverse phase and normal phase HPLC was stimulated two-fold by AII. We employed cultures of early S1 segments on a modified Ussing chamber as a bioassay to evaluate the effects of AII and 5,6-EET on unidirectional 22Na (apical to basolateral) flux (JNa). AII inhibited JNa, an effect that was abolished by ketoconazole. Furthermore, 5,6-EET decreased JNa in a manner analogous to AII, and the effect was potentiated by inhibition of endogenous 5,6-EET production. Employing the Ca2(+)-sensitive fluorescent probe, Fura 2, we observed a dose-dependent increase in [Ca2+]i with nM to microM 5,6-EET, effects that were abolished by depletion of extracellular Ca2+ and voltage-sensitive Ca-channel blockers. These observations support the hypothesis that 5,6-EET represents the second messenger that mediates AII-induced natriuresis via stimulation of Ca2+ influx through voltage-sensitive channels.

8,11,14-Eicosatrienoic Acid↗

PubMedDate not supplied

Angiotensin II actions in the rabbit proximal tubule. Angiotensin II mediated signaling mechanisms and electrolyte transport in the rabbit proximal tubule.

Angiotensin II (AngII) is a potent regulator of electrolyte transport with biphasic effects on salt and HCO3-resorption in proximal tubule epithelia (PCT). In cultured PCT cells, pM to nM AngII activates a GTP-binding protein to inhibit cAMP formation and thus releases inhibition of apical Na/H exchange. Phospholipase A2 is activated by nM to microM AngII releasing arachidonate which is metabolized by a novel P450 epoxygenase to form 5,6-epoxy-eicosatrienoic acid (5,6-EET). 5,6-EET and nM apical AngII cause dihydropyridine-sensitive Ca2+ influx from the extracellular space, inhibition of apical-to-basolateral Na flux, and decrease in epithelial monolayer short circuit current. 5,6-EET also inhibits Na/K-ATPase by 50%. This P450 epoxygenase is physiologically important in the AngII-signaling system because the P450 inhibitor ketoconazole blocks AngII effects while potentiating exogenous 5,6-EET effects. Finally, these AngII-mediated signaling systems are polarized in the PCT with pM basolateral AngII inhibiting adenylate cyclase and nM apical AngII activating PLA2 and subsequent generation of 5,6-EET.

Angiotensin II↗

PubMedDate not supplied

Characterization of paracellular permeability in cultured human cervical epithelium: regulation by extracellular adenosine triphosphate.

OBJECTIVE: The purpose of the present study was to compare the permeability and regulation of paracellular transport in human cervical cells with those in epithelial cells of other organs. METHODS: Cervical cells (ECE16-1, Caski, and HT3) were grown on filters, and transepithelial electrical conductance (GT) and the permeability to pyranine (PPyr) were determined. RESULTS: Cervical cultures were characterized by high GT (83-125 mS.cm-2) and high PPyr (6.2-18 x 10(-6).sec-1). The GT was not significantly affected by cell density but was increased by 20% by lowering extracellular calcium to 0.45 mmol/L or less. The high values of GT and PPyr and the regulation by extracellular calcium indicate that all three cervical cell lines have "leaky" tight junctional complexes. Addition of extracellular adenosine triphosphate (ATP) at 50 mumol/L to the cervical cultures evoked a biphasic change in GT that was unique to the cervical cells: an initial increase, followed by a sustained decrease by 30% from baseline GT. The decrease of GT was associated with a decrease in PPyr by 17%, indicating that ATP had an effect on the tight junctional/paracellular permeability. The ATP effect was reversible either by washing or by chemical hydrolysis with ATPase. The non-cervical cell lines all responded to extracellular ATP with a transient increase in GT, but not with the pronounced decrease. CONCLUSION: The permeability of the paracellular pathway can be regulated in cervical epithelia by mechanisms that may be different from those in epithelial cells from other organs.

Adenosine Triphosphate↗

PubMedDate not supplied

[P300 and auditory information processing during natural sleep].

INTRODUCTION: The P300 is elicited, in the waking state and during an 'attention condition', in response to deviant stimuli of an oddball paradigm. This component of event related potentials (ERP) may be a useful research tool in the assessing of cortical sensory processing during normal sleep since a subject does not need to be awake or totally conscious in order to generate a measurable response. OBJECTIVE: This study used an classical oddball paradigm as a means to assess the auditory information processing during sleep. PATIENTS AND METHODS: The auditory ERP were registered in twelve healthy volunteers during the waking state and sleep stages II, III-IV and REM. The amplitude, latency and scalp distribution parameters of the positivity observed were contrasted with the results obtained in the waking state. RESULTS: A 'P300-like' with a significantly smaller peak amplitude and an increment of latency was elicited during stage II and the REM stage of sleep. As in the waking state, the positivity during this sleep stages was maximal at central-parietal regions. CONCLUSIONS: The response obtained seems to correspond both for the morphology of the potential as for the centro-parietal predominance with a waking P3b component. These results suggest that certain processes of attention and memory-related operations involved in the auditory processing of simple signals remain operative during these sleep stages.

Adult↗