Human intestinal anion exchanger isoforms: expression, distribution, and membrane localization.

A family of anion exchangers (AEs) including AE1, AE2 and AE3 has been described. AE3 gene has been shown to encode two alternatively spliced isoforms termed as bAE3 (brain subtype) and cAE3 (cardiac subtype). The identity of the AE(s) involved in the human intestinal NaCl absorption is not fully understood. Current studies were undertaken to identify the AE isoforms expressed in the human intestine, to define their regional and vertical axis (crypt vs. surface cells) distribution, and to elucidate their membrane localization in the epithelial cells along the entire length of the human intestine. Our studies utilizing reverse transcription (RT)-PCR with total RNA extracted from pinch biopsies from various regions of the human intestine demonstrate that AE2 and bAE3 but not AE1 or cAE3 were expressed in all the regions of the human intestine. Utilizing in situ RT-PCR, we demonstrated that the message of AE2 was expressed throughout the vertical surface--crypt axis of the colon. Our Western blotting studies demonstrated that AE2 and bAE3 are localized to the basolateral but not the apical membranes of the intestinal epithelial cells from the human ileum and colon. In conclusion, our results demonstrated that in the human intestine, AE2 and bAE3, but not AE1 or cAE3, are expressed throughout the tract with the highest expression in the colon compared to the ileum and jejunum. Both the isoforms were found to be localized to the basolateral but not the apical membranes of the epithelial cells. We speculate that, in the human intestine, AE2 and bAE3 may be the 'housekeeping' isoforms, and the apical AE, the potential candidate for chloride absorption, remains to be identified.

Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes.

1. Troglitazone was the first thiazolidinedione approved for clinical use in the treatment of non-insulin-dependent diabetes mellitus. During clinical investigations of drug-drug interactions with therapeutics (terfenadine and cyclosporine) known to be metabolized by CYP3A4, pharmacokinetic interactions were noted upon troglitazone multiple-dose treatments. The nature of the interactions suggested induction of CYP3A enzymes. 2. Primary cultures of human hepatocytes were used to investigate the induction potential of troglitazone with respect to CYP3A4, CYP2B6 and CYP1A1/2. In human hepatocytes, troglitazone induced both immunoreactive CYP3A4 protein and testosterone 6beta-hydroxylase activity in a dose-dependent fashion (EC50 = 5-10 microM), accompanied by an increase in CYP3A4 mRNA. The capacity of troglitazone to induce CYP3A4 was between that of rifampin (EC50 = 0.8 microM) and dexamethasone (40-50 microM). Troglitazone increased CYP2B6 immunoreactive protein but did not significantly effect CYP1A1/2 activity, immunoreactive protein or mRNA. 3. Troglitazone produced significant increases in CYP3A message, protein and activity in primary rat hepatocytes, a slight increase in CYP2B1/2 activity and no change in CYP1A1/2 message or activity. 4. These results provide evidence that troglitazone can induce CYP3A and CYP2B enzymes while apparently not altering CYP1A. This provides a rationale for the clinically observed interactions of troglitazone with selected CYP3A4 substrates.

Ion transport during growth and differentiation.

The major function of the adult colon is to reabsorb fluid from the chyme. This ability to conserve salt and water is especially important in newborns, where reserves are small and diarrhea is frequent. Although much is known about regulation of Cl- transport in the adult colon, postnatal changes in electrolyte transport are not well characterized. We have established an in vitro model to study colonic epithelial cells (colonocytes) at different stages of development. Primary cultures were isolated from newborn, weanling, and adult rabbit colon and properties such as growth and Cl- transport characterized. The isolation procedure yielded a crypt-enriched population of cells, and the cell yield per gram mucosa increased with age. The colonocytes also showed an age-related decrease in attachment to extracellular matrix, with maximum attachment seen with Matrigel and collagen IV. The crypt enrichment was confirmed by demonstrating that the cell population was capable of transporting Cl-, which was stimulated by agents such as forskolin and phorbol esters at all ages. Agents that increased intracellular cGMP, however, did not increase Cl- transport at any age. It was interesting to observe that the secondary bile acid, taurodeoxycholate, stimulated Cl- transport only in the adult but not newborn or weanling distal colonocytes. We have demonstrated that rabbit distal colonocytes can be kept viable in culture and transport Cl- at all ages. However, the regulation of Cl- transport changes during ontogeny and depends on the signaling pathway.

Cl- transport in an immortalized human epithelial cell line (NCM460) derived from the normal transverse colon.

Cells of a newly described, immortalized, epithelial, human transverse colonic cell line, NCM460, reach approximately 90% confluence on plastic and develop transepithelial resistances of 120-250 Omega . cm2 on porous substrates. Its utility as a model for the transverse human colon was validated by comparing second messenger-mediated Cl- transport, using the fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NCM460 cells and colonocytes isolated from human transverse crypts. Basal Cl- influx was increased (P < 0.01) by PGE1 (1 microM), forskolin (1 microM), 8-bromoadenosine 3'5'-cyclic monophosphate (100 microM), heat-stable Escherichia coli enterotoxin (STa; 1 microM), 8-bromoguanosine 3'5'-cyclic monophosphate (100 microM), histamine (1 microM), and phorbol 12,13-dibutyrate (1 microM) in both cell types. The Cl- channel blocker diphenylamine 2-carboxylic acid (50 microM) and the Na+-K+-2Cl- cotransport inhibitor furosemide (1 microM), but not the K+ channel blocker Ba2+ (3 mM), inhibited these Cl- permeabilities. These cells possess transcripts for cystic fibrosis transmembrane conductance regulator, Na+-K+-2Cl- cotransporter, STa receptor, and intestine-specific cGMP-dependent protein kinase II. Thus cAMP-, cGMP-, and Ca2+-dependent secretagogues act on NCM460 and primary colonocytes to stimulate Cl- transport. This validates the utility of NCM460 as a model for transverse colonic crypts and is the first demonstration of a colonic cell line whose origin is known.

Chloride transport in primary cultures of rabbit colonocytes at different stages of development.

BACKGROUND & AIMS: Ontogeny of colonic Cl- transport and its regulation has been characterized inadequately. The aim of this report was to study developmental changes in Cl- transport in primary cultures of rabbit distal colonocytes. METHODS: Colonocytes from newborn (7-9 days old), weanling (25-28 days old), and adult (6 months old) rabbits were cultured for 24 hours on a collagen IV matrix, and Cl- transport was measured using the fluoroprobe 6-methoxyquinolyl acetoethyl ester. RESULTS: Cl- permeabilities were dependent on [Cl-]o with maximal rates (in millimoles per liter per second) at [Cl-]o = 75 mmol/L (newborns; 0.15 +/- 0.04; weanlings; 0.2 +/- 0.02; and adults, 0.32 +/- 0.06). Influx was inhibited significantly by the Cl- channel (50 mumol/L diphenylamine-2-carboxylate) and the Na(+)-K(+)- 2Cl- cotransport (10 mumol/L furosemide) inhibitors. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues, prostaglandin E1 (1 mumol/L), forskolin (1 mumol/L), and 8-bromo-cAMP (100 mumol/L), and the protein kinase C activator, phorbol 12-13 dibutyrate (1 mumol/L), increased Cl- influx significantly in all groups with adults showing greatest stimulation. However, taurodeoxycholate (0.025-1 mmol/L) had an effect only in the adult and the guanosine 3',5'-cyclic monophosphate (cGMP) activators STa and 8-bromo-cGMP had no effect. CONCLUSIONS: Rabbit distal colonocytes possess inhibitor-sensitive Cl- permeabilities even in neonates. However, the ontogeny of their regulation depends on the secretagogue-signaling pathway.

Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes.

To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit Cl- secretion, their effects on Cl- transport and intracellular Ca(2+) concentrations ([Ca2+]i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl- sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca(2+)-sensitive fluorescent probe, fura-2AM were used to assess Cl- transport and [Ca2+]i, respectively. Basal Cl- transport (0.274 +/- 0.09 mM/sec) was inhibited significantly by the Cl- channel blocker diphenylamine-2-carboxylate (DPC, 50 microM, 0.068 +/- 0.02 mM/sec; P < 0.001) and the Na+/K+/ 2Cl- cotransport inhibitor furosemide (1 microM, 0.137 +/- 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be l- > F- > or = Cl- > Br-. DPC inhibited l- influx by approximately 50%, F- influx by 80%, Cl-influx by 85%, and Br- influx by 90%. Furosemide significantly inhibited influx of Br- (84%) and Cl- (81%) but not of F- and l-. The effects of agents known to alter biological response by increasing [Ca2+]i in other epithelial systems were used to stimulate Cl- transport. Cl- influx in mM/second was stimulated by 1 microM histamine (0.58 +/- 0.05), 10 microM neurotensin (2.07 +/- 0.32), 1 microM serotonin (1.63 +/- 0.28), and 0.1 microM of the Ca2+ ionophore A23187 (2.05 +/- 0.40). The Cl- permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl- influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl- permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl- transport by activating the Na+/K+/2Cl- cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl- channels and the cotransporter, and their actions are cyclooxygenase-independent.

Altered growth and attachment of rabbit crypt colonocytes isolated from different developmental stages.

The rabbit colon was used to establish an in vitro model for examining development-related cellular changes in colonocyte function. Colonic epithelia from newborn, weanling, and adult animals were separated from the muscle and subjected to enzymatic digestion. A mixture of 0.05% Pronase, 0.015% collagenase IV, and 0.023% DTT was determined to be optimal for the isolation of newborn and weanling colonocytes. This solution yielded significantly more cells and of greater viability than a 0.1% Pronase, 0.03% collagenase IV, 0.07% DTT mixture that is optimal for adult colonocytes. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining of cytokeratins. The isolation procedure resulted in a crypt-enriched population and the cell yield/g of mucosa increased with age as did the crypt depth. Colonocyte viability of adults but not of newborns and weanlings, declined from 24 to 72 h. When grown on plastic, the newborn and weanling colonocytes show a approximately 2-fold increase in number, DNA and protein content over 48 h. In contrast, for all three parameters the adult colonocytes revealed only a approximately 10% increase. The colonocytes also showed an age-related decline in attachment to extracellular matrices. Colonocytes showed maximal attachment to Matrigel and collagen IV; newborn and weanling colonocytes show > 80% attachment, whereas adult colonocytes showed only a 45% attachment. The efficacy of attachment to Matrigel compared with that on plastic also differed with age, representing 9.3-, 5.5-, and 4.4-fold increase in adult, weanling, and newborn colonocytes, respectively. Newborn and weanling colonocytes grown on Matrigel for 48 h, showed a significant, 15% increase in cell number, DNA, and protein content compared with those grown on plastic. There was no difference in these parameters when adult colonocytes grown on Matrigel were compared with those grown on plastic. In summary, we have established an in vitro model for studying colonic epithelial cells at different stages of development.

Escherichia coli heat-stable enterotoxin-mediated colonic Cl- secretion is absent in cystic fibrosis.

BACKGROUND/AIMS: Calcium- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretions in the human colon are abnormal in cystic fibrosis, but the effect of guanosine 3',5'-cyclic monophosphate (cGMP) is unknown. This study examined the effects of the cGMP activator Escherichia coli heat-stable enterotoxin (STa) on rectal ion transport of controls and subjects with cystic fibrosis. METHODS: In vivo rectal potential difference (PD) was measured in response to 10(-7) mol/L STa in adult cystic fibrosis (n = 6) and control subjects (n = 7). Cl- transport was also evaluated in 24-hour primary cultures of human colonocytes using 6-methoxy-quinolyl-acetoethyl ester in response to STa (1 mumol/L) and 8-bromo-cGMP (100 mumol/L) with or without Cl- transport inhibitors. RESULTS: Whereas STa increased rectal potential difference in controls, there was no effect in cystic fibrosis subjects. STa stimulated the cGMP concentration in rectal biopsy specimens from both control and cystic fibrosis subjects approximately twofold. In vitro Cl- transport in non-cystic fibrosis colonocytes increased threefold and fivefold with STa and 8-bromo-cGMP, respectively. These transport increases were inhibited by furosemide and the Cl- channel blocker diphenylamine-2-carboxylate. CONCLUSIONS: Human colonocytes secrete Cl- in response to STa and cGMP in normal subjects, but this response is absent in cystic fibrosis.

Cyclic AMP- and phorbol ester-regulated Cl- permeabilities in primary cultures of human and rabbit colonocytes.

Chloride transport in 24-h primary cultures of human and rabbit distal colonic crypt cells (90 +/- 5% viable) were characterized using the Cl(-)-sensitive fluorescent probe 6-methoxyquinolyl acetoethyl ester. To calculate the Cl- influx in millimolar per second, the Stern-Volmer quenching constant was determined to be 24.3 M-1 for human and 24.6 M-1 for rabbit colonocytes. Cl- influx was dependent on extracellular Cl- concentration ([Cl-]0), with maximal influx at [Cl-]0 > or = 20 mM. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues forskolin (1 microM), prostaglandin E1 (1 microM), and 8-bromoadenosine 3',5'-cyclic monophosphate (100 microM) increased Cl- influx in human colonocytes from 0.35 +/- 0.08 to 2.14 +/- 0.65, 1.85 +/- 0.51, and 0.84 +/- 0.04 mM/s (n = 4), respectively, and in rabbit colonocytes from 0.22 +/- 0.03 to 1.04 +/- 0.11, 1.24 +/- 0.12, and 1.08 +/- 0.07 mM/s (n = 5), respectively. Depending on the secretagogue, this influx was inhibited 50-90% by the Cl- channel blocker diphenylamine-2-carboxylate (DPC; 50 microM) and > or = 65% by the Na-K-2Cl cotransport inhibitor furosemide (10 microM). Phorbol 12,13-dibutyrate, an activator of protein kinase C, increased Cl- permeability 3.8-fold in human and 2.4-fold in rabbit colonocytes. The phorbol 12,13-dibutyrate-stimulated Cl- permeabilities were sensitive to DPC and furosemide but not to indomethacin. These studies demonstrate DPC and furosemide-sensitive Cl- permeabilities in isolated cultured human and rabbit colonocytes, which can be activated by cAMP and protein kinase C stimulators.

Isolation, characterization, and attachment of rabbit distal colon epithelial cells.

The authors investigated various enzymatic digestion procedures for isolating epithelial cells from the distal colon of New Zealand White male rabbits. Rabbit mucosa was washed, diced, and digested for 90 minutes in one of five different solutions, including a new combination consisting of 0.03% collagenase IV and 0.1% pronase (solution V). Solution I (0.3% dispase) yielded 14.2 +/- 8.2 x 10(6) colonocytes/g mucosa, solution II (0.15% dispase and 0.03% collagenase) yielded 7.7 +/- 2.8 x 10(6) colonocytes/g mucosa, and solution III (0.03% collagenase IV) yielded 15.4 +/- 10(6) cells/g mucosa. Solutions I-III have previously been described for the isolation of colonocytes. Solution IV (0.1% pronase and 325 U/mL DNAase) was originally described for the isolation of nasal epithelial cells but yielded only 2.5 +/- 1.2 x 10(6) cells/g mucosa when applied to the isolation of colonocytes. The new combination of pronase and collagenase, solution V, yielded significantly more colonocytes, 34.5 +/- 3.0 x 10(6) cells/g mucosa, than previously described methods (P less than 0.01). Inclusion of 5 mmol/L ethylenediaminetetraacetic acid in any of the solutions enhanced neither viability nor yield. The digestion product of solution V could be enriched for crypts by serial low-speed centrifugations. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining for cytokeratins. Functional viability was tested by determining the presence of a Na+/H+ exchanger, using the pH fluorescent dye bis(carboxymethyl)-5(6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH. The authors document that sodium-dependent restoration of intracellular pH in colonocytes acid-loaded to a pH of 6.30 occurred at a rate of 0.19 +/- 0.02 pH U/min. Amiloride at concentrations of 1 mmol/L completely inhibited operation of the exchanger, as did sodium substitution with choline or tetramethylammonium. Lineweaver-Burke analysis at this intracellular pH showed a Michaelis constant of 10.71 mmol/L Na+ and a maximum velocity of 0.12 pH U/min. Exposing the colonocytes to 100 nmol/L phorbol 12,13-dibutyrate increased antiporter activity by 62.0%. Finally, the authors describe the synthesis of a new biomatrix composed of the basement membrane of 3T3 NIH fibroblasts that permits significantly improved colonocyte attachment than to glass, plastic, collagen types I or IV, or matrigel.