Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate.

Butyrate and the other short-chain fatty acids (SCFAs) are the most abundant anions in the colonic lumen. Also, butyrate is the preferred energy source for colonocytes and has been shown to regulate colonic electrolyte and fluid absorption. Previous studies from our group have demonstrated that the HCO(3)(-)/SCFA(-) anion exchange process is one of the major mechanisms of butyrate transport across the purified human colonic apical membrane vesicles and the apical membrane of human colonic adenocarcinoma cell line Caco-2 and have suggested that it is mainly mediated via monocarboxylate transporter-1 (MCT-1) isoform. However, little is known regarding the regulation of SCFA transport by various hormones and signal transduction pathways. Therefore, the present studies were undertaken to examine whether hydrocortisone and phorbol 12-myristate 13-acetate (PMA) are involved in a possible regulation of the butyrate/anion exchange process in Caco-2 cells. The butyrate/anion exchange process was assessed by measuring a pH-driven [(14)C]butyrate uptake in Caco-2 cells. Our results demonstrated that 24-h incubation with PMA (1 microM) significantly increased [(14)C]butyrate uptake compared with incubation with 4alphaPMA (inactive form). In contrast, incubation with hydrocortisone had no significant effect on butyrate uptake in Caco-2 cells compared with vehicle (ethanol) alone. Induction of butyrate uptake by PMA appeared to be via an increase in the maximum velocity (V(max)) of the transport process with no significant changes in the K(m) of the transporter for butyrate. Parallel to the increase in the V(max) of [(14)C]butyrate uptake, the MCT-1 protein level was also increased in response to PMA incubation. Our studies demonstrated that the butyrate/anion exchange was increased in response to PMA treatment along with the induction in the level of MCT-1 expression in Caco-2 cells.

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.

Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1.

The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na(+) independent. Butyrate uptake was unaffected by DIDS; however, alpha-cyano-4-hydroxycinnamate and the butyrate analogs propionate and L-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.

Expression of a recombinant apolipoprotein(a) in HepG2 cells. Evidence for intracellular assembly of lipoprotein(a).

Apolipoprotein(a) (apo(a)), a large glycoprotein with extensive homology to plasminogen, forms a complex with apolipoprotein B100 (apoB100), which circulates in human plasma in the form of lipoprotein(a) (Lp(a)). Evidence indicates that the association of apo(a) with apoB100 occurs in the extracellular environment. We have reevaluated the possibility that apo(a)-B100 association can also occur as an intracellular event through studies with HepG2 cells stably transfected with an apo(a) minigene. Several lines of evidence support this possibility. First, continued Lp(a) production was demonstrated following incubation of transfected HepG2 cells with anti-apo(a) antisera, conditions that effectively block the fluid-phase association of apo(a) and apoB100 in vitro. Second, an apo(a)-B100 complex was detectable in Western blot analyses of transfected HepG2 lysates following immunoprecipitation with anti-apo(a) antisera. These studies incorporated precautions to eliminate cell-surface attachment of preformed apo(a)-B100 complexes to the low density lipoprotein receptor and were conducted in the presence of the lysine analog epsilon-aminocaproic acid, which precludes apo(a)-B100 association occurring during the isolation and analyses. Third, the presence of an intracellular apo(a)-B100 complex was demonstrated in lipoproteins isolated from microsomal contents. Of particular significance was the observation that this complex contained the precursor form of apo(a), which is not secreted, in addition to the mature, recombinant form. Finally, direct evidence was provided for the synthesis of a precursor form of apo(a) in a nascent intracellular complex with apoB100 following treatment of transfected HepG2 cells with brefeldin A plus N-acetyl-leucyl-leucyl-norleucinal. Taken together, these data suggest that apo(a)-B100 association can occur as an intracellular event in a human hepatoma-derived cell line, raising important implications for the regulation of Lp(a) secretion from human liver.

Molecular cloning of a human small intestinal apolipoprotein B mRNA editing protein.

Mammalian small intestinal apolipoprotein B (apo B) mRNA undergoes posttranscriptional cytidine deamination with the production of an in frame stop codon and the translation of apo B48. We have isolated a cDNA from human jejunum which mediates in vitro editing of a synthetic apo B RNA template upon complementation with chicken intestinal S100 extracts. The cDNA specifies a 236 residue protein which is 69% identical to the apo B mRNA editing protein (REPR) cloned from rat small intestine [Teng, B., Burant, C. F. and Davidson, N. O. (1993) Science 260, 1816-1819] and which, by analogy, is referred to as HEPR. HEPR does not contain the carboxyl-terminus leucine zipper motif identified in REPR but contains consensus phosphorylation sites as well as the conserved histidine and both cysteine residues identified as a Zn2+ binding motif in other cytidine deaminases. The distribution of HEPR mRNA was predominantly confined to the adult small intestine with lower levels detectable by reverse-transcription polymerase chain reaction amplification in the stomach, colon and testis. These differences in the structure and distribution of the human as compared to the rat apo B mRNA editing protein suggest an important evolutionary adaptation in the mechanisms restricting apo B48 production to the small intestine.

Complementation of apolipoprotein B mRNA editing by human liver accompanied by secretion of apolipoprotein B48.

Mammalian small intestine secretes a truncated apolipoprotein B (apoB48) species as a result of tissue-specific post-transcriptional RNA editing. The human liver, by contrast, contains only unedited apoB mRNA and secretes only apoB100. We have recently isolated a cDNA clone from rat small intestine which encodes an apoB mRNA editing protein, REPR (Teng, B., Burant, C.F., and Davidson, N.O. (1993) Science 260, 1816-1819). The current study demonstrates that homogenates of Xenopus oocytes expressing REPR confer editing ability upon S100 extracts prepared from human liver when tested on a synthetic apoB RNA template in vitro. Transfection of REPR into HepG2 cells resulted in editing of endogenous apoB mRNA and the appearance of an apoB48-like protein in the media. Extracts prepared from these transfected cells edit mammalian apoB RNA templates when incubated alone and with enhanced efficiency in the presence of chicken intestinal S100 extracts. The results suggest that human liver expresses factor(s) which are critical to apoB mRNA editing and which allow functional complementation of REPR in vivo.

Beta-oxidation of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid by MOLT-4 lymphocytes.

MOLT-4 lymphocytes metabolize 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE via beta-oxidation with retention of the hydroxyl group at the omega 9 carbon atom. The isolation of 6-hydroxy-4,8-tetradecadienoic acid documents that these cells have the capacity to catabolize the conjugated diene system. 12(S)-HETE was also metabolized to 3,12-dihydroxy-8,10,14-eicosatrienoic acid and 1,9-dihydroxy-5,7,11-heptadecatriene as well as to 17- and 19-carbon aldehydes. When MOLT-4 cells were incubated with the beta-oxidation product, 10-hydroxy-6,8,12-octadecatrienoic acid, it was in part further catabolized but in addition it served as an anabolic precursor as defined by the accumulation 3,12-dihydroxy-8,10,14-eicosatrienoic acid as well as 1,11-dihydroxy-7,9,13-nonadecatriene. Neither 10-hydroxy-6,8,12-octadecatrienoic acid nor 13-hydroxy-5,8,11-octadecatrienic acid was as potent in inhibiting phytohemagglutin-induced lymphocyte mitogenesis as were their parent compounds--i.e., 12(S)- and 15(S)-HETE. These findings argue against the hypothesis that beta-oxidation products of 12(S)- and 15(S)-HETE are the potential modulators of lymphocyte function. However, neither the pathway for synthesis, nor the role of odd chain aldehydes and diols as potential lipid mediators was determined in this study.

Metabolism of 13-hydroxy-9,11-octadecadienoic acid by MOLT-4 lymphocytes.

MOLT-4 lymphocytes metabolize 13-hydroxy-9,11-octadecadienoic acid, via the beta-oxidation pathway with retention of the omega 6 hydroxyl group and the conjugated diene system. The products which accumulate include 11-hydroxy-7,9-hexadecadienoic acid and 9-hydroxy-5,7-tetradecadienoic acid. In addition, it was possible to isolate two beta-hydroxy acids which were shown to be 3,13-dihydroxy-9,11-octadecadienoic acid and 3,11-dihydroxy-7,9-hexadecadienoic acid. The odd chain aldehyde, 12-hydroxy-8,10-heptadecadien-1-al, also was detected. However, neither the pathway nor the immediate precursor for the synthesis of this compound was established.

Metabolism of 15-hydroxy-5,8,11,13-eicosatetraenoic acid by MOLT-4 cells and blood T-lymphocytes.

MOLT-4 lymphocytes metabolize 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) via beta-oxidation with retention of the hydroxyl group at the omega 6-carbon atom. 15-HETE oxidation is accompanied by the time-dependent accumulation of both beta-hydroxy acids and metabolites produced by repetitive cycles of the beta-oxidation spiral. Detection of 7-hydroxy-5-dodecenoic acid shows that these cells continue to beta-oxidize the substrate when the conjugated diene is allylic to a hydroxyl group. When 15-HETE was the substrate, it was also possible to detect 12-hydroxy-5,8,10-heptadecatrien-1-al and 3,15-dihydroxy-8,11,13-eicosatrienoic acid. The former product may be produced by alpha-oxidation of 13-hydroxy-6,9,11-octadecatrienoic acid followed by its decarboxylation. Detection of a 20-carbon metabolite, lacking a double bond at position 5, suggests that an intermediate of beta-oxidation was used as a substrate for chain elongation. When 13-hydroxy-6,9,11-octadecatrienoic acid was used as a substrate, it was indeed possible to detect 3,15-dihydroxy-8,11,13-eicosatrienoic acid as well as 15-hydroxy-8,11,13-eicosatrienoic acid. In addition, 13-hydroxy-6,9,11-octadecatrienoic acid was a precursor for the biosynthesis of both 14-hydroxy-7,10,12-nonadecatrien-1-al and 1,14-dihydroxy-7,10,12-nonadecatriene. These studies with MOLT-4 cells as well as with T-lymphocytes isolated from blood show that products of the 15-lipoxygenase pathway are metabolized with the accumulation of a variety of compounds. Since 15-HETE has been implicated as a modulator of T-cell function, these findings raise the possibility that the newly described metabolites may be involved in regulating lymphocyte function.

Formation of 8-hydroxyhexadecatrienoic acid by vascular smooth muscle cells.

Smooth muscle cells derived from the human umbilical vein produce four radioactive metabolites when they are incubated in culture with [3H]-12-hydroxyeicosatetraenoic acid. This conversion does not require the addition of an agonist for eicosanoid formation. The main product, which accounts for 60% of the radioactivity converted to these metabolites, has been identified as 8-hydroxyhexadecatrienoic acid.

Docosahexaenoic acid metabolism and effect on prostacyclin production in endothelial cells.

Bovine aortic endothelial cultures readily take up docosahexaenoic acid (DHA). Most of the DHA was incorporated into phospholipids, primarily in ethanolamine and choline phosphoglycerides, and plasmalogens accounted for 34% of the DHA contained in the ethanolamine fraction after a 24-h incubation. The retention of DHA in endothelial phospholipids was not greater than other polyunsaturated fatty acids and unlike arachidonic and eicosapentaenoic acids, DHA did not continue to accumulate in the ethanolamine phosphoglycerides after the initial incorporation. About 15% of the [14C(U)]DHA uptake was retroconverted to docosapentaenoic and eicosapentaenoic acids in 24 h. Some of the newly incorporated [14C(U)]DHA was released when the cells were incubated subsequently in a medium containing serum and albumin. The released radioactivity was in the form of free fatty acid and phospholipids and after 24 h, 11% was retroconverted to docosapentaenoic and eicosapentaenoic acids. Total DHA uptake was decreased only 10% by the presence of a 100 microM mixture of physiologic fatty acids, but as little as 10 microM docosatetraenoic acid reduced DHA incorporation into phospholipids by 25%. DHA was not converted to prostaglandins or lipoxygenase products by the endothelial cultures. When DHA was available, however, less arachidonic acid was incorporated into endothelial phospholipids, and less was converted to prostacyclin (PGI2). Enrichment of the endothelial cells with DHA also reduced their capacity to subsequently produce PGI2. These findings indicate that endothelial cells can play a role in DHA metabolism and like eicosapentaenoic acid, DHA can inhibit endothelial PGI2 production when it is available in elevated amounts.

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells.

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI2) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 microM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI2 formation. The inhibition was not specific for PGI2; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-14C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI2 formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI2.

Eicosapentaenoic acid utilization by bovine aortic endothelial cells: effects on prostacyclin production.

We have investigated whether the presence of other fatty acids in physiologic amounts will influence the effects of eicosapentaenoic acid on cellular lipid metabolism and prostaglandin production. Eicosapentaenoic acid uptake by cultured bovine aortic endothelial cells was time and concentration dependent. At concentrations between 1 and 25 microM, most of the eicosapentaenoic acid was incorporated into phospholipids and of this, 60-90% was present in choline phosphoglycerides. Eicosapentaenoic acid inhibited arachidonic acid uptake and conversion to prostacyclin (prostaglandin I2) but was not itself converted to eicosanoids. Only small effects on the uptake of 10 microM eicosapentaenoic acid occurred when palmitic, stearic or oleic acids were added to the medium in concentrations up to 75 microM. In contrast, eicosapentaenoic acid uptake was reduced considerably by the presence of linoleic, n-6 eicosatrienoic, arachidonic or docosahexaenoic acids. Although a 100 microM mixture of palmitic, stearic, oleic and linoleic acid (25:10:50:15) had little effect on the uptake of 10 or 20 microM eicosapentaenoic acid, less of this acid was channeled into endothelial phospholipids. However, the fatty acid mixture did not prevent the inhibitory effect of eicosapentaenoic acid on prostaglandin I2 formation in response to either arachidonic acid or ionophore A23187. An 8 h exposure to eicosapentaenoic acid was required for the inhibition to become appreciable and, after 16 h, prostaglandin I2 production was reduced by as much as 60%. These findings indicate that the capacity of aortic endothelial cells to produce prostaglandin I2 is decreased by continuous exposure to eicosapentaenoic acid. Even if the eicosapentaenoic acid is present as a small percentage of a physiologic fatty acid mixture, it is still readily incorporated into endothelial phospholipids and retains its inhibitory effect against endothelial prostaglandin I2 formation. Therefore, these actions may be representative of the in vivo effects of eicosapentaenoic acid on the endothelium.