Biomedical subjects

N L Simmons

Publications and source records attributed to N L Simmons.

At least 19 recordsLinked to original sources

PubMed2006

Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells.

We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn(2+) on whole-cell Ca(2+)-activated Cl(-) currents (I (CLCA)) in mouse inner medullary collecting duct cells (mIMCD-3). I (CLCA) was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10-400 microM) to the bathing solution resulted in a dose-dependent increase in I (CLCA) with little change in Cl(-) selectivity or biophysical characteristics, whereas gadolinium chloride (30 microM) and lanthanum chloride (100 microM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn(2+) (400 microM) stimulated an increase in intracellular Ca(2+) to an elevated plateau. The Zn(2+)-stimulated [Ca(2+)](i) increase was inhibited by thapsigargin (200 nM), the IP(3) receptor antagonist 2-aminoethoxydiphenyl borate (10 microM) and removal of bath Ca(2+). Pre-exposure to Zn(2+) (400 microM) markedly attenuated the ATP (100 microM)-stimulated [Ca(2+)](i) increase. These data are consistent with the hypothesis that extracellular Zn(2+) stimulates an increase in [Ca(2+)](i) by a release of calcium from thapsigargin/IP(3) sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca(2+)-sensing receptor, in IMCD cells is discussed.

Animals↗

PubMed2006

Expression and localization of monocarboxylate transporters and sodium/proton exchangers in bovine rumen epithelium.

Monocarboxylate-H+ cotransporters, such as monocarboxylate transporter (MCT) SLC16A, have been suggested to mediate transruminal fluxes of short-chain fatty acids, ketone bodies, and lactate. Using an RT-PCR approach, we demonstrate expression of MCT1 (SLC16A1) and MCT2 (SLC16A7) mRNA in isolated bovine rumen epithelium. cDNA sequence from these PCR products combined with overlapping expressed sequence tag data allowed compilation of the complete open reading frames for MCT1 and MCT2. Immunohistochemical localization of MCT1 shows plasma membrane staining in cells of the stratum basale, with intense staining of the basal aspects of the cells. Immunostaining decreased in the cell layers toward the rumen lumen, with weak staining in the stratum spinsoum. Immunostaining in the stratum granulosum and stratum corneum was essentially negative. Since monocarboxylate transport will load the cytosol with acid, expression and location of Na+/H+ exchanger (NHE) family members within the rumen epithelium were determined. RT-PCR demonstrates expression of multiple NHE family members, including NHE1, NHE2, NHE3, and NHE8. In contrast to MCT1, immunostaining showed that NHE1 was predominantly localized to the stratum granulosum, with a progressive decrease toward the stratum basale. NHE2 immunostaining was observed mainly at an intracellular location in the stratum basale, stratum spinosum, and stratum granulosum. Given the anatomic localization of MCT1, NHE1, and NHE2, the mechanism of transruminal short-chain fatty acid, ketone body, and lactate transfer is discussed in relation to a functional model of the rumen epithelium comprising an apical permeability barrier at the stratum granulosum, with a cell syncitium linking the stratum granulosum to the blood-facing stratum basale.

Acids↗

PubMed2006

Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells.

Mutations in CLCN5, which encodes the voltage-dependent Cl(-)/H(+)antiporter, CLC-5, cause Dent's disease. This disorder is characterized by low molecular-weight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent's disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis.

Animals↗

PubMed2005

UT-B is expressed in bovine rumen: potential role in ruminal urea transport.

The UT-A (SLC14a2) and UT-B (SLC14a1) genes encode a family of specialized urea transporter proteins that regulate urea movement across plasma membranes. In this report, we describe the structure of the bovine UT-B (bUT-B) gene and characterize UT-B expression in bovine rumen. Northern analysis using a full-length bUT-B probe detected a 3.7-kb UT-B signal in rumen. RT-PCR of bovine mRNA revealed the presence of two UT-B splice variants, bUT-B1 and bUT-B2, with bUT-B2 the predominant variant in rumen. Immunoblotting studies of bovine rumen tissue, using an antibody targeted to the NH2-terminus of mouse UT-B, confirmed the presence of 43- to 54-kDa UT-B proteins. Immunolocalization studies showed that UT-B was mainly located on cell plasma membranes in epithelial layers of the bovine rumen. Ussing chamber measurements of ruminal transepithelial transport of (14)C-labeled urea indicated that urea flux was characteristically inhibited by phloretin. We conclude that bUT-B is expressed in the bovine rumen and may function to transport urea into the rumen as part of the ruminant urea nitrogen salvaging process.

Amino Acid Sequence↗

PubMed2004

Functional organization of the bovine rumen epithelium.

The functional organization of the bovine rumen epithelium has been examined by electron and light microscopy combined with immunocytochemistry to define a transport model for this epithelium. Expression of connexin 43, an integral component of gap junctions, the tight-junction molecules claudin-1 and zonula occludens 1 (ZO-1), and the catalytic alpha-subunit of Na(+)-K(+)-ATPase was demonstrated by SDS-PAGE and Western blotting. From the lumen surface, four cell layers can be distinguished: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. Both claudin-1 and ZO-1 immunostaining showed plasma membrane staining, which was present at the stratum granulosum with decreasing intensity through the stratum spinosum to the stratum basale. The stratum corneum was negative for claudin-1 immunostaining. Transmission electron microscopy confirmed that occluding tight junctions were present at the stratum granulosum. Plasma membrane connexin 43 immunostaining was most intense at the stratum granulosum and decreased in intensity through stratum spinosum and stratum basale. There was intense immunostaining of the stratum basale for Na(+)-K(+)-ATPase, with weak staining of the stratum spinosum. Both the stratum granulosum and the stratum corneum were essentially negative. Stratum basale cells also displayed a high mitochondrial density relative to more apical cell layers. We conclude that epithelial barrier function may be attributed to the stratum granulosum and that cell-cell gap junctions allow diffusion to interconnect the barrier cell layer with the stratum basale where Na(+)-K(+)-ATPase is concentrated.

Animals↗

PubMed2004

Calcium phosphate and calcium oxalate crystal handling is dependent upon CLC-5 expression in mouse collecting duct cells.

Defects in an intracellular chloride channel CLC-5 cause Dent's disease, an inherited kidney stone disorder. Using a collecting duct model, mIMCD-3 cells, we show expression of dimeric mCLC-5. Transient transfection of antisense CLC-5 reduces CLC-5 protein expression. Binding of both calcium phosphate (hydroxyapatite) and calcium oxalate monohydrate (COM) crystals overlaid onto mIMCD-3 cultures was affected by altered CLC-5 expression. Calcium phosphate crystal agglomerations (>10 microm) were minimal in control (9%) and sense (13%) CLC-5-transfected cells, compared to 66% of antisense CLC-5-transfected cells (P<0.001). Small calcium phosphate crystals (<10 microm) were found associated with 45% of sense CLC-5-treated cells, of which the majority (11/14 cells) appeared to be internalised within the cell. Calcium oxalate agglomerations (>10 microm) were also largely absent for controls or sense mCLC-5 transfectants (11% and 9% of cells, respectively) with COM crystal agglomerates predominating in antisense CLC-5 transfectants (66%, P<0.0001). We conclude that collecting duct cells with reduced CLC-5 expression lead to a tendency to form calcium crystal agglomeration, which may help explain the nephrocalcinosis and nephrolithiasis seen in Dent's disease.

Animals↗

PubMed2003

Kinetics and regulation of a Ca2+-activated Cl- conductance in mouse renal inner medullary collecting duct cells.

Using the whole cell patch-clamp technique, a Ca2+-activated Cl- conductance (CaCC) was transiently activated by extracellular ATP (100 microM) in primary cultures of mouse inner medullary collecting duct (IMCD) cells and in the mouse IMCD-K2 cell line. ATP also transiently increased intracellular Ca2+ concentration ([Ca2+]i) from 100 nM to peak values of approximately 750 nM in mIMCD-K2 cells, with a time course similar to the ATP-induced activation and decay of the CaCC. Removal of extracellular Ca2+ had no major effect on the peak Cl- conductance or the increase in [Ca2+]i induced by ATP, suggesting that Ca2+ released from intracellular stores directly activates the CaCC. In mIMCD-K2 cells, a rectifying time- and voltage-dependent current was observed when [Ca2+]i was fixed via the patch pipette to between 100 and 500 nM. Maximal activation occurred at approximately 1 microM [Ca2+]i, with currents losing any kinetics and displaying a linear current-voltage relationship. From Ca2+-dose-response curves, an EC50 value of approximately 650 nM at -80 mV was obtained, suggesting that under physiological conditions the CaCC would be near fully activated by mucosal nucleotides. Noise analysis of whole cell currents in mIMCD-K2 cells suggests a single-channel conductance of 6-8 pS and a density of approximately 5,000 channels/cell. In conclusion, the CaCC in mouse IMCD cells is a low-conductance, nucleotide-sensitive Cl- channel, whose activity is tightly coupled to changes in [Ca2+]i over the normal physiological range.

Animals↗

PubMed2001

The voltage-dependent Cl(-) channel ClC-5 and plasma membrane Cl(-) conductances of mouse renal collecting duct cells (mIMCD-3).

1. We have tested the hypothesis that the voltage-dependent Cl(-) channel, ClC-5 functions as a plasma membrane Cl(-) conductance in renal inner medullary collecting duct cells. 2. Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl(-) conductance that was unaffected by external DIDS. 3. Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl(-) currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca(2+)-activated Cl(-) conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4. A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5. Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6. Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl(-)-selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl(-) conductances. Transient transfection with sense mClC-5 failed to induce the Cl(-) conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca(2+)-activated Cl(-) conductance 24 h post-transfection. 8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9. These data discount a major role for ClC-5 as a plasma membrane Cl(-) conductance in mIMCD-3 cells but suggest a role in endosomal function.

Algorithms↗

PubMed2001

Regulation of an outwardly rectifying chloride conductance in renal epithelial cells by external and internal calcium.

We have used perforated patch clamp and Fura-2 microfluorescence measurements to study Ca(2+)-activated Cl- currents in cultured mouse renal inner medullary collecting duct cells (mIMCD-3). The conductance was spontaneously active under resting conditions and whole cell currents were time and voltage-independent with an outwardly rectifying current-voltage relationship. The channel blockers DIDS, niflumic acid, glybenclamide and NPPB reversibly decreased the basal currents, whereas the sulfhydryl agent, DTT produced an irreversible inhibition. Increasing or decreasing extracellular calcium produced parallel changes in the size of the basal currents. Variations in external Ca2+ were associated with corresponding changes in free cytosolic Ca2+ concentration. Increasing cytosolic Ca2+ by extracellular ATP or ionomycin, further enhanced Cl- conductance, with whole cell currents displaying identical biophysical properties to the basal currents. However, the agonist-stimulated currents were now increased by DTT exposure, but still inhibited by the other channel blockers. Using RT-PCR, three distinct mRNA transcripts belonging to the CLCA family of Ca(2+)-activated Cl- channel proteins were identified, two of which represent novel sequences. Whether different channels underlie the basal and agonist-stimulated currents in mIMCD-3 cells is unclear. Our findings establish a novel link between alterations in external and internal Ca2+ and the activity of Ca(2+)-activated Cl- transport in these cells.

Adenosine Triphosphate↗

PubMed2001

Expression and role of sodium, potassium, chloride cotransport (NKCC1) in mouse inner medullary collecting duct (mIMCD-K2) epithelial cells.

Loop-diuretic-sensitive 86Rb+(K+) transmembrane fluxes were determined in cells of a mouse inner medullary collecting duct cell line (mIMCD-K2). The furosemide-sensitive (0.1 mM) influx was a substantial fraction of the total influx (0.39+/-0.04 or 0.42+/-0.03, n=5 in the presence or absence of ouabain, respectively). Furosemide also reduced 86Rb+(K+) efflux by a similar fraction (0.46). RT-PCR analysis revealed expression of mRNA for the Na+-K+-2Cl- cortransporter-1 (NKCC1), but not NKCC2. Loop-diuretic-sensitive 86Rb+(K+) influx was confined to the basolateral membrane, confirming its localisation there. The physiological properties of NKCC1 expressed in mIMCD-K2 cells, including the dependence upon medium Na+, K+ and Cl- and the relative sensitivity to loop diuretics as assessed by the concentration required for half-maximal inhibition (IC50) (bumetanide 3.3+/-1.4x10-7 M>piretanide 2.5+/-0.15x10-6 M>furosemide 2.3+/-1.2x10-5 M) were typical for NKCC1. Possible functions of NKCC1 were tested; furosemide did not inhibit the majority of forskolin-stimulated secretory short-circuit current (Isc) (83.5+/-5.3% of the maintained response at 5 min). Secondly, total 86Rb+(K+) influx was stimulated markedly when external osmolarity was increased to 600 mosmol/l by mannitol due to an increase via NKCC1 from 55+/-11 to 191+/-2 nmol/106 cells per 15 min, (both n=4, P<0.01). In contrast, 10-5 M forskolin did not stimulate total 86Rb+(K+) influx. Finally, the ability of both K+ and NH4+ to compete for ouabain-insensitive 86Rb+(K+) influx via NKCC1 was confirmed with similar concentrations for half-maximal influx reduction (K0.5). Apical exposure to NH4+ elicited rapid cytosolic alkalinisation in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded epithelial layers, consistent with selective permeability of the apical membrane to NH3. Conversely, NH4+ (5 mM) at the basal cell surface resulted in progressive acidification, the initial rate being reduced by 43% by furosemide. We conclude that NKCC1 participates in selective uptake of NH4+ at the basal surface, and that IMCD may function in direct NH4+ deposition to urine.

Ammonium Chloride↗

PubMed2001

Human intestinal cell monolayers are preferentially sensitive to disruption of barrier function from basolateral exposure to cholic acid: correlation with membrane transport and transepithelial secretion.

Unconjugated bile acids such as cholic acid cause diarrhoea, mucosal irritation and toxicity. We sought to define the mechanism of cholate permeation across intestinal mucosal cells to understand how cellular exposure and accumulation are deleterious to mucosal function. Human intestinal Caco-2 and T84 cell monolayers were prepared by high-density seeding and cultured for >14 days on permeable culture supports. Cholate transport and cellular accumulation were determined using [3H]cholic acid. Epithelial barrier function was assessed by measuring transepithelial electrical resistance (Rt) and [14C]mannitol fluxes. Exposure of Caco-2 epithelia to serosal cholate caused a dose- and time-dependent disruption of barrier function. Apical exposure was without disruptive effect. Similar responses were observed for T84 epithelia. Cholate was preferentially accumulated across the basolateral surfaces in both Caco-2 and T84 cells, but was subject to active transepithelial secretion in Caco-2 monolayers only. Net secretion was substantially reduced by ATP depletion, showed saturation kinetics, and was subject to competitive inhibition by other bile acids. Cholate secretion was also sensitive to inhibition by the leukotriene antagonist MK-571 but not by digoxin, suggesting that MRP2, not MDR1, was responsible. RT-PCR and Western blotting confirmed MRP2 expression in Caco-2 epithelia but indicated its apparent absence from T84 cells.

Absorption↗

PubMed2000

The swelling-activated anion conductance in the mouse renal inner medullary collecting duct cell line mIMCD-K2.

Swelling-activated Cl(-) currents (I(Cl,swell)) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I(-) > Br(-) > Cl(-) > Asp(-). NPPB (100 microm) inhibited the current in a voltage independent manner, as did exposure to 10 microm tamoxifen and 500 microm niflumic acid (NFA). In contrast, DIDS (100 microm) blocked the current with a characteristic voltage dependency. These characteristics of I(Cl, swell) in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I(Cl,swell) by hypotonicity. However, PDBu inhibition of I(Cl,swell) was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca(2+) activated Cl(-) conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I(Cl,swell). Control of I(Cl,swell) by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl(-) secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I(Cl,swell) is therefore capable of regulating transepithelial Cl(-) secretion and suggests that I(Cl,swell) is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl(-) secretion, but tamoxifen (100 microm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I(sc)). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line.

Animals↗

PubMed2000

Ca2+ and cAMP-activated Cl- conductances mediate Cl- secretion in a mouse renal inner medullary collecting duct cell line.

1. The nature of Cl- conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD-K2 cell line) was investigated. 2. Extracellular ATP (100 microM) stimulated a transient increase in both whole-cell Cl- conductance and intracellular free Ca2+. In contrast, ionomycin (10-100 nM) caused a sustained increase in whole-cell Cl- conductance. Pre-loading cells with the Ca2+ buffer BAPTA abolished the ATP-dependent responses and delayed the onset of the increase observed with ionomycin. 3. The Ca2+-activated whole-cell Cl- current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady-state current-voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at -80 mV. In BAPTA-loaded cells, ionomycin-elicited whole-cell currents exhibited pronounced outward rectification with time-dependent activation/inactivation. 4. Ca2+-activated and forskolin-activated Cl- conductances co-exist since ATP activation of whole-cell current occurred during a maximal stimulation by forskolin in single cell recordings. 5. In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (Isc) dependent upon basal medium Na+ and Cl-/HCO3- but independent of the presence of apical bathing medium Na+ and Cl-/HCO3-. This was identical to forskolin stimulation and consistent with transepithelial anion secretion. 6. PCR amplification of reverse-transcribed mRNA using gene-specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca2+-activated Cl- channel (mCLCA1) mRNA in mIMCD-K2 cells. 7. Ca2+ and forskolin-activated Cl- conductances participate in anion secretion by IMCD.

Adenosine Triphosphate↗

PubMed2000

Gamma-Aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.

1. Transintestinal absorption of gamma-aminobutyric acid (GABA) via a pH-dependent mechanism is demonstrated in the model human intestinal epithelial cell line Caco-2. 2. Experiments with BCECF [2',7',-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]-loaded Caco-2 cells demonstrate that GABA transport across the apical membrane is coupled to proton flow into the cell. 3. Short-circuit current (ISC) measurements using Caco-2 cell monolayers under voltage-clamped conditions demonstrate that pH-dependent GABA transport is a rheogenic process even in the absence of extracellular Na+, consistent with H+/GABA symport. 4. A range of GABA analogues were tested for their abilities to: (a) inhibit pH-dependent [3H]GABA uptake across the apical membrane; (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers; (c) increase inward ISC across voltage-clamped Caco-2 cell monolayers. 5. Nipecotic acid, isonipecotic acid, D,L-beta-aminobutyric acid, and 3-amino-1-propanesulphonic acid each caused a marked acidification of intracellular pH and an increase in ISC when superfused at the apical surface of Caco-2 cell monolayers. In contrast L-alpha-amino-n-butyric acid failed to induce proton flow or ISC. The ability of these compounds to induce proton or current flow across the apical surface of this intestinal epithelium was closely related to the relative inhibitory effects on [3H]GABA uptake. 6. These observations demonstrate H+/GABA symport and suggest that this transport mechanism may be accessible as a route for oral absorption of therapeutically-useful GABA analogues.

Biological Transport↗

PubMed2000

Bradykinin regulation of salt transport across mouse inner medullary collecting duct epithelium involves activation of a Ca(2+)-dependent Cl(-) conductance.

The mechanism by which bradykinin regulates renal epithelial salt transport has been investigated using a mouse inner medullary renal collecting duct cell-line mIMCD-K2. Using fura-2 loaded mIMCD-K2 cells bradykinin (100 nM) has been shown to induce a transient increase in intracellular Ca(2+) via activation of bradykinin B2 receptors localized to both the apical and basolateral epithelial cell surfaces. In mIMCD-K2 epithelial cell-layers clamped in Ussing chambers, 100 nM bradykinin via apical and basolateral bradykinin B2 receptors stimulated a transient increase in inward short-circuit current (I:(sc)) of similar duration to the increase in intracellular Ca(2+). Replacements of the bathing solution Na(+) by the impermeant cation, N-methyl-D-glucamine and of Cl(-) and HCO(3)(-) by the impermeant anion gluconate at either the apical (no reduction) or basal bathing solutions (abolition of the response) are consistent with the bradykinin-stimulated increase in inward I:(sc) resulting from basal to apical Cl(-) (anion) secretion. Using the slow whole cell configuration of the patch-clamp technique, bradykinin was shown to activate a transient Cl(-) selective whole cell current which showed time-dependent activation at positive membrane potentials and time-dependent inactivation at negative membrane potentials. These currents were distinct from those activated by forskolin (CFTR), but identical to those activated by exogenous ATP and are therefore consistent with bradykinin activation of a Ca(2+)-dependent Cl(-) conductance. The molecular identity of the Ca(2+)-dependent Cl(-) conductance has been investigated by an RT - PCR approach. Expression of an mRNA transcript with 96% identity to mCLCA1/2 was confirmed, however an additional but distinct mRNA transcript with only 81% of the identity to mCLCA1/2 was identified.

Animals↗

PubMed1999

H(+)/solute-induced intracellular acidification leads to selective activation of apical Na(+)/H(+) exchange in human intestinal epithelial cells.

The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces - transepithelial ion gradients - are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH(i)) regulation in response to H(+)-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na(+)/H(+) exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na(+)/H(+) exchange was determined by a combination of pH(i), (22)Na(+) influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H(+)/solute-induced acidification (using glycylsarcosine or beta-alanine) led to Na(+)-dependent, EIPA-inhibitable pH(i) recovery or EIPA-inhibitable (22)Na(+) influx at the apical membrane only. Selective activation of apical (but not basolateral) Na(+)/H(+) exchange by H(+)/solute cotransport demonstrates that coordinated activity of H(+)/solute symport with apical Na(+)/H(+) exchange optimizes the efficient absorption of nutrients and Na(+), while maintaining pH(i) and the ion gradients involved in driving transport.

Adenocarcinoma↗

PubMed1998

Substrate upregulation of the human small intestinal peptide transporter, hPepT1.

1. Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl-L-glutamine (Gly-Gln) of the proton-coupled peptide transporter, hPepT1, in the Caco-2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl-[14C]sarcosine was increased 1.64 (+/- 0.34)-fold after incubation of Caco-2 cells for 3 days in a peptide-rich medium (4 mM Gly-Gln replacing 4 mM Gln). 2. A full-length Caco-2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile-662 to Val. Transcript sizes, on Northern blots of Caco-2 poly(A)+ RNA probed with a 630 bp 5' hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide-induced increase in substrate transport was accompanied by a parallel increase of 1.92 (+/- 0.30)-fold (n = 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half-life from 8.9 +/- 1.1 to 12.5 +/- 1.6 h (n = 3), but the increase in half-life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide-mediated increase in hPepT1 transcription. 3. Anti-hPepT1-specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco-2 cells. Gly-Gln supplementation of media resulted in a 1.72 (+/- 0.26)-fold (n = 5) increase in staining intensity of Caco-2 cells. 4. We conclude that Caco-2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly-Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.

Adenocarcinoma↗

PubMed1998

Co-culture of two MDCK strains with distinct junctional protein expression: a model for intercellular junction rearrangement and cell sorting.

Distinct epithelial MDCK cell strains displaying extremes in transepithelial electrical resistance (paracellular permeability) have been established in co-culture and the subsequent cellular behaviour and formation of junctional complexes investigated. After high-density seeding, MDCK strain I and II cells in co-culture are initially randomly distributed but subsequently sort themselves out in a time-dependent manner to form separate homotypic aggregates. The final pattern of cell arrangement of homotypic aggregates depends on the relative seeding proportion of each cell type. Immunostaining of established marker proteins for junctional complexes has revealed that MDCK I and II cells differ in the degree of expression of the zonula-adherens-associated protein, E-cadherin, their cytoskeletal architecture and the junctional distribution of a desmosomal protein, and by showing subtle differences in tight junction staining for the zona-occludens-associated proteins, ZO-1 and occludin. The distinct pattern of junctional protein expression is maintained when the two MDCK strains are co-cultured; however, morphologically atypical intercellular junctions between heterotypic cells at the boundary of homotypic cell aggregates have been observed. It has been suggested that cell sorting, a phenomenon yet to be completely understood, is involved in important morphogenetic processes. We propose that co-culture of strains of the well-characterised MDCK cell line may be a novel but well-defined cell system for studying epithelial cell rearrangement and sorting in intact epithelial sheets.

Animals↗