Biomedical subjects

M H Montrose

Publications and source records attributed to M H Montrose.

At least 19 recordsLinked to original sources

PubMed1992

Assay of apical membrane enzymes based on fluorogenic substrates.

A series of enzymatic rate assays are described. The assays are based on coumarin derivatives that are fluorogenic substrates for the enzymes dipeptidase IV, aminopeptidase N, alkaline phosphatase, and gamma-glutamyltransferase. These simple assays are rapid and offer improved sensitivity over established colorimetric methods. The substrates have apparent affinities for the enzymes of 5-250 microM. L-Glutamic acid gamma-(7-amido-4-methylcoumarin) is characterized as a substrate of gamma-glutamyltransferase on the basis of inhibition of enzymatic cleavage when the glycylglycine acceptor molecule is omitted and inhibition of the enzymatic reaction by addition of glycine. Assay conditions for the four enzymes are established such that less than 0.6% of the substrate is consumed, fluorescence is proportional to enzymatic product, and results may be directly compared to established colorimetric assays. Intestinal epithelial cells are used both to establish appropriate assay conditions and to demonstrate the utility of the assays.

Alkaline Phosphatase↗

PubMed1992

Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin.

Regulation of Na+/H+ exchange by fetal bovine serum was studied in Caco-2 cells, an established cell line derived from a human colon carcinoma. Cells were grown as polarized monolayers on collagen-coated filters and intracellular pH measured fluorometrically with 2',7'-bis(2-carboxymethyl)-5,6-carboxyfluorescein. Na+/H+ exchange was reduced 64% when cells were deprived of serum for 4 h. In contrast to other cell types, readdition of serum for 10 min did not activate Na+/H+ exchange; however, readdition of serum for 4 h restored Na+/H+ exchange to control values. This long-term effect of serum on Na+/H+ exchange activity could not be explained by changes in intracellular buffering capacity or intracellular [Na+]. 4-h serum deprivation reduced the K(t) of the exchanger for external Na+ from 21 to 6 mM, and reduced the V(max) by 57%, but did not alter the IC50 for amiloride in the presence of 140 mM Na+. Inhibition of protein synthesis with cycloheximide (5 microM) did not alter the effect of serum removal or readdition on Na+/H+ exchange. Low temperature (13 degrees C) completely prevented the inhibition of Na+/H+ exchange caused by the removal of serum. In addition, once Na+/H+ exchange was inhibited by serum removal at 37 degrees C, maintaining cells at 13 degrees C also blocked the recovery of Na+/H+ exchange caused by serum readdition. Conversely, cytochalasin D (0.1-20 microM) blocked the reduction of Na+/H+ exchange which occurred due to 4-h serum deprivation, but did not block the restoration of Na+/H+ exchange when the cells were re-exposed to serum for a further 4 h. Colchicine (20 microM) did not alter the effect of serum removal or readdition. These data suggest that serum regulates Na+/H+ exchange activity by a posttranslational mechanism which is dependent on F-actin.

Actins↗

PubMed1992

CFTR protein expression in primary and cultured epithelia.

The gene responsible for the lethal disorder cystic fibrosis encodes a 1480-amino acid glycoprotein, CFTR. Using polyclonal antibodies directed against separate phosphorylation sites in the pre-nucleotide-binding fold (exon 9) and the R domain (exon 13), we have identified a 165-kDa protein in Xenopus laevis oocytes injected with recombinant CFTR cRNA transcribed from the full-length CFTR plasmid pBQ4.7. A protein of the same mobility was also detected with Western blotting techniques in whole cell extracts of cells that express CFTR mRNA (T84, FHTE, HT-29), including biopsied human nasal and bronchial tissue. Immunodetectable 165-kDa protein was concentrated in the apical membrane fraction of ileal villus tissue. We also report that the 165-kDa protein levels can be modulated pharmacologically, and these levels are appropriately correlated with second-messenger-regulated Cl- efflux. Thus, native or recombinant CFTR can be recognized by these anti-CFTR peptide polyclonal antibodies.

Animals↗

PubMed1992

H+ extrusion by an apical vacuolar-type H(+)-ATPase in rat renal proximal tubules.

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1991

Cellular differentiation regulates expression of Cl- transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells.

The gene defective in cystic fibrosis has recently been shown to code for a membrane protein designated the "cystic fibrosis transmembrane conductance regulator" (CFTR) protein. While it has been shown that detectable levels of the mRNA for the normal CFTR protein are present in epithelial cells from different tissues, factors which regulate CFTR expression have not been identified. A clonal cell line originating from a human colon adenocarcinoma (HT29-18) differentiates to multiple epithelial cell types when deprived of glucose in the culture medium. In these studies, mRNA isolated from these cells was examined by hybridization to a 1.45-kilobase cDNA probe which encodes transmembrane portions of the CFTR protein between exons 13 and 19. Cellular differentiation of HT29-18 causes a 9-18-fold increase in CFTR mRNA abundance versus the mRNA for the structural proteins actin and tubulin. Cellular differentiation also causes a 5-fold increase in second messenger-regulated Cl- transport which is sensitive to a Cl- channel blocker (diphenylamine 2-carboxylate). Subclones of HT29-18 which are committed to differentiate to either a mucin-secreting (HT29-18-N2) or an "enterocyte-like" (HT29-18-C1) phenotype have also been examined. In both subclones, elevated levels of CFTR mRNA are observed when compared with undifferentiated HT29-18 cells. However, during cellular differentiation, the regulation of CFTR mRNA abundance and membrane enzyme expression by the subclones is different from HT29-18. The results show that elevated CFTR mRNA occurs in multiple differentiated intestinal epithelial cell types, despite a phenotype-specific regulation of membrane protein expression. This suggests that CFTR expression plays a role in the differentiated functions of multiple epithelial phenotypes and that both cellular differentiation and cellular phenotypes are factors which regulate CFTR expression.

Biological Transport↗

PubMed1991

Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.

Amino Acid Sequence↗

PubMed1991

Polarized expression of Na+/H+ exchange activities in clonal LLC-PK1 cells (Clone4 and PKE20) I. Basic characterization.

We have analysed the mechanisms of Na(+)-dependent pHi recovery from an acid load in LLC-PK1/Clone4 and LLC-PK1/PKE20 cells by using the intracellular pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester. By analysis using single-cell microspectrofluorometry, we obtained evidence for polarized expression of Na+/H+ exchange activities with different properties in apical and basolateral cell surfaces, respectively. In Clone4 cells, Na+/H+ exchange activity is only visible on the basolateral cell surface; in PKE20 cells, Na+/H+ exchange activities with equal capacities are present on both cell surfaces. In Clone4 cells, the apparent Km value for Na+ is around 10 mM; in PKE20 cells it is around 20 mM and indistinguishable for the two cell poles. Ethylisopropylamiloride (EIPA) inhibition for all three activities measured in monolayer configuration is reduced by increasing Na+ concentration. Measured in the same cells, EIPA inhibition of transport of PKE20 cells is weaker for apical Na+/H+ exchange as compared to basolateral activity. In Clone4 and PKE20 cells kept in suspension, Na+/H+ exchange activities with similar properties for the two cell lines are observed. However, Na+/H+ exchange activities in cells in suspension are different from either activity measured in monolayer configuration: affinity for Na+ is higher (PKE20 cells) and inhibition by amiloride is weak and not influenced by increasing Na+ concentrations (PKE20 and Clone4 cells). It is concluded that PKE20 cells contain different Na+/H+ exchange activities on the two cell surfaces; this cell line should be a useful model to study regulatory aspect of different Na+/H+ exchange functions ("epithelial'/"housekeeping').(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1991

Studies on the kinetics of Na+/H+ exchange in OK cells: introduction of a new device for the analysis of polarized transport in cultured epithelia.

The present study describes a new perfusion technique--based on the use of a routine spectrofluorometer--which enables fluorometric evaluation of polarity, regulation and kinetics of Na+/H+ exchange at the level of an intact monolayer. Na+/H+ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na+/H+ exchange activity was measured by monitoring changes in intracellular pH (pHi) after an acid load, using the pH-sensitive dye 2'7'-bis (carboxyethyl) 5-6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pHi recovery mechanism, which is Na+ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pHi = 7.68; pHo = 7.4) where Na+/H+ exchange is not detectable, transport rate increased as pHi decreased. A positive cooperativity characterized the interaction of internal H+ with the exchanger, and suggests multiple H+ binding sites. In contrast, extracellular [Na+] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na+ was 19 mM at an intracellular pH of 7.1 and 60 mM at an intracellular pH of 6.6. Inhibition of Na+/H+ exchange activity by EIPA was competitive with respect to extracellular [Na+] and the Ki was 3.4 microM. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pHi and processes associated with their polarized expression and regulation.

Amiloride↗

PubMed1991

Kinetics and regulation of a polarized Na(+)-H+ exchanger from Caco-2 cells, a human intestinal cell line.

The kinetics and regulation of Na(+)-H+ exchange were studied using BCECF to measure pHi in Caco-2 cells grown on membrane filters. Na(+)-H+ exchange was defined as a Na(+)-dependent H+ efflux in response to an acid load imposed by an NH4Cl prepulse in the absence of added CO2. Na(+)-H+ exchange was present exclusively on the basolateral membrane, had a Kt (Na+) of 21 +/- 2 mM, and an ID50 for amiloride dependent on medium [Na+] with an apparent Ki for amiloride of 3 microM. Na(+)-H+ exchange rates had a greater than first-order dependence on intracellular [H+], suggesting the presence of an internal proton modifier site. Results also suggest that Na(+)-H+ exchange is kinetically inactivated at resting pHi (7.35 +/- 0.02), since neither removal of Na+ nor addition of amiloride affected resting pHi, although monensin alkalinized cells to pHi 7.6. To evaluate regulation of Na(+)-H+ exchange, cells were exposed to either forskolin, 1,9-dideoxyforskolin (a noncyclase-activating forskolin derivative), 8-BrcAMP, E. coli STa toxin, ionomycin, phorbol dibutyrate, or cellular shrinkage in hypertonic medium. Only forskolin and 1,9-dideoxyforskolin caused a significant change (inhibition) in Na(+)-H+ exchange rate. Experiments performed with the Ussing chamber-voltage clamp technique verified that forskolin, 8-BrcAMP, E. coli STa toxin, ionomycin, and phorbol dibutyrate increased transepithelial Isc, verifying that all the regulatory pathways tested were functional and responsive to agonists. Results suggested that the Isc was due to Cl- secretion, since no net transcellular Na+ or Cl- flux was detected in basal conditions, and the Isc response to forskolin was abolished by omission of serosal Cl-. Because forskolin, but not 1,9-dideoxyforskolin, increased both cellular cAMP and Isc, the inhibition of Na(+)-H+ exchange by forskolin derivatives was mediated by a mechanism not involving activation of adenylyl cyclase. In conclusion, Caco-2 cells use a basolateral Na(+)-H+ exchanger to regulate pHi, but this exchanger is not affected by cell shrinkage or second messenger pathways that regulate Na(+)-H+ exchangers in other cell systems.

Amiloride↗

PubMed1990

Parathyroid hormone regulation of Na+/H+ exchange in opossum kidney cells: polarity and mechanisms.

In previous work we have shown that parathyroid hormone (PTH) inhibits Na+/H+ exchange in cellular suspensions of OK (opossum kidney) cells (an established renal epithelial cell line) in a dose-dependent manner. PTH effects could be mimicked by pharmacological activation of both protein kinase A and protein kinase C (Helmle-Kolb et al. 1990). In the present paper we extend these observations and analyze the PTH-dependent control of Na+/H+ exchange in OK cells kept in epithelial configuration (monolayer). Na+/H+ exchange activity is examined by microfluorometry using the intracellularly trapped pH-sensitive dye 2'7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. Cells recovered from an acid load (NH4Cl prepulse) after addition of apical Na+. Ethylisopropylamiloride inhibits Na(+)-dependent pHi recovery at micromolar concentrations. PTH leads to an inhibition of apical Na+/H+ exchange activity; inhibition is observed even at a concentration of 5 pM PTH. PTH given at maximally effective concentrations (24 nM) reduces the total Na+/H+ exchange capacity by 60%-70%. Apical as well as basolateral hormone additions elicit an inhibitory response at low (5 pM) or high (24 nM) concentrations. Forskolin (activation of protein kinase A) and phorbol esters (activation of protein kinase C) lead to an inhibition of Na+/H+ exchange activity (60%-70% inhibition). These observations suggest that Na+/H+ exchange activity is preferentially located in the apical membranes of OK cells kept in monolayer configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1990

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters.

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na+/phosphate co-transport and Na+/H+ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na+/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na+/H+ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca2+ concentration do not induce inhibition of Na+/H+ exchange; PTH-dependent inhibition of Na+/H+ exchange is not prevented by ionomycin or by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Ca2+ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40-50% inhibition of Na+/H+ exchange. An analysis of intracellular pH sensitivity of Na+/H+ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced Vmax of Na+/H+ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na+/H+ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).

Animals↗

PubMed1990

Regulation of intracellular pH by cultured opossum kidney cells.

Opossum kidney (OK) cells (an epithelial cell line) were examined by flame photometry of cellular Na+ and K+ and by microfluorometric measurements of the intracellular pH (pHi) of single cells loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The work concentrates on defining resting pHi values under different experimental conditions and examines factors that contribute to the maintenance of resting pHi. To use nigericin to calibrate the intracellular response of BCECF, cellular K+ levels were measured by a null point analysis, and the stability and magnitude of cellular Na+ and K+ levels were determined vs. time. Resting pHi in medium without added CO2 was high when measured by null point analysis of the population (pHi 7.6) and from measurements of single cells that have recovered from an acid load caused by NH4 prepulse (pHi 7.76 +/- 0.03, n = 20 cells). In single-cell measurements, addition of CO2-HCO3- to the medium results in cellular acidification of the steady-state pHi by 0.35 +/- 0.04 pH units. In medium equilibrated with room air, the resting pHi is shown to be a dynamic steady state composed of net flux due to apical Na(+)-dependent transport (Na(+)H+ exchange) plus acidifying processes. It is concluded that although 5-[N-ethyl-N-isopropyl]amiloride (EIPA) inhibits the forward reaction of Na(+)-H+ exchange, EIPA is either ineffective as an inhibitor of the reverse reaction of Na(+)-H+ exchange or Na(+)-H+ exchange does not reverse measurably in the OK cells.

Amiloride↗

PubMed1990

Polarity and kinetics of Na(+)-H+ exchange in cultured opossum kidney cells.

Opossum kidney (OK) cells (an established cell line) were loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF; a fluorescent dye with a pH-sensitive spectrum), and intracellular pH (pHi) was examined by microfluorometry. Single cells, within a confluent monolayer and grown on a permeant support, were examined for the mechanism of recovery from an acid load as imposed by exposure to ammonium chloride (NH4 prepulse). The Na(+)-dependent recovery of pHi from an acid load (Na(+)-H+ exchange) is examined in terms of the Na+ activation kinetics of the recovery and the polarity of the response. In 80% of the cells examined (33/41), both apical and basolateral Na+ cause recovery from an acid load. The response of cells to apical Na+ is well fit by Michaelis-Menten kinetics [Kt(Na) = 35 mM], but the response to basolateral Na+ is not. The response to basolateral Na+ addition is modeled in terms of variable transepithelial leak of Na+ and variable amounts of basolateral Na(+)-H+ exchange. Despite an average response to basolateral (145 mM) Na+ that is 34% of the response to apical Na+, modeling suggests that basolateral Na(+)-H+ exchange must be less than 10% of the cellular total to fit the basolateral Na+ activation kinetics. The model, and experiments using ordered addition of Na+ from the apical vs. basolateral medium, also suggest that transepithelial leak (of basolateral Na+ to the apical compartment) is required to explain the pHi recovery observed due to addition of basolateral Na+. Direct estimation of (basolateral to apical) transepithelial leak demonstrates that the response due to basolateral Na+ addition is explained by transepithelial leak and a Na(+)-H+ exchange that is expressed solely in the apical membrane.

Amiloride↗

PubMed1989

Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells.

Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2',7'-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH4 prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na+ removal of EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na+/H+ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na+/H+ exchange. Despite this heterogeneity, the Na+/H+ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4-7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na+/H+ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.

Animals↗

PubMed1989

Regulatory volume decrease by cultured renal cells.

Volume regulatory responses of OK cells (a continuous epithelioid cell line from opossum kidney) are examined by electronic cell sizing and measurements of intracellular pH in cell suspensions. In response to a 40% reduction in osmolality, the cells swell and then subsequently shrink toward their starting volume. This regulatory volume decrease (RVD) is reduced by replacement of Cl- in the medium with acetate. Replacement of Cl- with NO3- accelerates the RVD. The RVD response is inhibited by 1 mM quinine or 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in the medium. The inhibitory effect of 100 microM DIDS (but not 1 mM quinine) is altered by replacement of Cl- by NO3- in the medium. Hypotonic challenge does not induce a DIDS-sensitive net flux of acid-base equivalents. Addition of (9 microM) valinomycin also inhibits the RVD response. It is suggested that the RVD response of OK cells involves activation of separate K+ and Cl- channels.

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

PubMed1988

Regulation of Na+-H+ exchange in cultured opossum kidney cells by parathyroid hormone, atrial natriuretic peptide and cyclic nucleotides.

The activity of Na+-H+, exchange was studied in a cultured cell line derived from opossum kidney (OK cells). The activity of the exchanger was measured either as the amiloride (2 mM) inhibitable 22Na flux in acid-loaded cells, or as the Na+-dependent and amiloride-sensitive recovery of intracellular pH (pHi) from an acid load. Initial rates of tracer flux were analyzed in confluent monolayers while changes in pHi were evaluated in suspensions of trypsinized cells which had been loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Both 8-bromo-cAMP and 8-bromo-cGMP inhibit the activity of the exchanger in a dose-dependent manner. Maximal inhibition due to 8-bromo-cAMP was about 50% and was attained with 0.75 mM of the cyclic nucleotide. Parathyroid hormone (10(-9)-10(-7) M) and atrial natriuretic peptide (10(-7) M) also inhibit the activity of the exchanger. By measuring the rate of Na+-dependent pHi recovery from different starting pHi values, evidence was obtained for a cyclic nucleotide-dependent decrease in the response of Na+-H+ exchange to intracellular acidification. We conclude that cAMP and cGMP are intracellular messengers in the hormone-dependent regulation of Na+-H+ exchange activity in renal epithelial cells.

Amiloride↗

PubMed1988

Bile acids increase cellular free calcium in cultured kidney cells (LLC-PK).

Suspensions of LLC-PK1 cells were used to determine the effect of bile acids on the cellular homeostasis of inorganic ions. It is determined that bile acids alter cellular free calcium (Cai) levels in LLC-PK1 cells. A series of bile acids were compared and found to produce increases in Cai in the order: lithocholate sulfate (LCS) greater than deoxycholate greater than chenodeoxycholate greater than lithocholate glucuronide greater than cholate. LCS (300 microM) produces changes in Cai (measured using Fura-2) qualitatively similar to those produced by 1 microM ionomycin, except that only ionomycin is able to release calcium from intracellular stores. The effect on Cai is roughly proportional to LCS concentration between 50 and 300 microM. The presence of 40 mM Na in the extracellular medium reduces the LCS-induced rise in Cai to 20% of that observed in the absence of Na. This effect is specific for Na versus 150 mM extracellular K, Li, or TMA. The effect is not dependent on the Na gradient across the membrane. At concentrations of LCS which induce changes in Cai, no significant effect of LCS is observed on either cellular Na or K levels, or intracellular pH.

Animals↗

PubMed1988

Separate control of regulatory volume increase and Na+-H+ exchange by cultured renal cells.

Suspensions of OK cells (a continuous epithelioid cell line from opossum kidney) are examined by electronic cell sizing, measurements of intracellular pH, and measurements of cellular Na+ and K+. The response of the cells to hypertonic solutions is evaluated in most detail. When shrunken by exposure to hyperosmotic medium (430 mosmol/kg), the cells do not demonstrate a regulatory volume increase (RVI) independent of the solute that is used to increase osmolality [NaCl, N-methyl-D-glucamine-HCl (NMGCl), or sucrose]. In contrast, when cells are preexposed to 190 mosmol/kg medium and then shrunken by exposure to 310 mosmol/kg medium, a volume increase is observed after the addition of 120 mosmol/kg NaCl or NMGCl, but not sucrose. This RVI is sensitive to 1 mM furosemide and removal of Na+ or K+ from the medium, but it is not inhibited by 1 mM amiloride. In the presence of a propionate-induced cellular acidification, a Na+-H+ exchanger in the cells is shown to have a large capacity for net solute uptake and to be inhibited by 1 mM amiloride. Net solute uptake by the Na+-H+ exchanger is sensitive to addition of parathyroid hormone or 8-bromoadenosine 3',5'-cyclic monophosphate but is not stimulated in response to cell shrinkage.

8-Bromo Cyclic Adenosine Monophosphate↗