Intestinal Alkaline Phosphatase mRNA Research Articles

IIAEK Targets Intestinal Alkaline Phosphatase (IAP) to Improve Cholesterol Metabolism with a Specific Activation of IAP and Downregulation of ABCA1.

IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel cholesterol-lowering pentapeptide derived from bovine milk β-lactoglobulin. However, the molecular mechanisms underlying the IIAEK-mediated suppression of intestinal cholesterol absorption are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in a human intestinal model using Caco-2 cells. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1). Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Through photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered the common IIAEK-binding amino acid sequence, GFYLFVEGGR. IIAEK significantly increased IAP mRNA and protein levels while decreasing ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway involving the specific activation of IAP and downregulation of intestinal ABCA1.

The small intestinal apical hydrolase activities are decreased in the piglet with bowel inflammation induced by dextran sodium sulfate1

Inflammatory bowel disease (IBD) is characterized by cramping, abdominal pain, bloating, constipation, and diarrhea. We tested the hypothesis that compromised activities of the major small intestinal apical hydrolases contribute to the symptoms of IBD. Changes in hydrolytic kinetics, target protein abundances, and mRNA expression of intestinal alkaline phosphatase (IAP), lactase, maltase, sucrase-isomaltase (SI), maltase-glucoamylase (MGA), and aminopeptidase N (APN) in piglets with colonic inflammation chemically induced by dextran sodium sulfate (DSS) were investigated. Yorkshire piglets at 5 d of age, with an average initial BW of about 3 kg, were fitted with intragastric catheters and were divided into control (CON; n = 6) and treatment groups (DSS; n = 5). Both groups were infused with an equal volume of either saline or 1.25 g of DSS · kg BW(-1) · d(-1) in saline, respectively, for 10 d. Enzyme kinetic experiments for IAP, lactase, maltase, SI, MGA, and APN were measured at 37°C with isolated proximal jejunal apical membrane. Target hydrolase protein abundances in the apical membrane were analyzed by Western blotting and their mRNA abundances in the jejunum were measured by quantitative real-time reverse transcription (RT-) PCR with β-actin as the housekeeping gene. Expressed as percentage of the CON, DSS treatment decreased (P < 0.05) the maximal specific activities of IAP (53%), lactase (78%), maltase (56%), SI (72%), MGA (29%), and APN (22%) as well as the target hydrolase protein abundances of IAP (39%), lactase (35%), SI (36%), and APN (54%), respectively. Decreases (P < 0.05) in the mRNA abundances (% of the CON) for lactase (25%), SI (52%), MGA (75%), and APN (39%) were observed in the DSS group. However, DSS treatment increased (P < 0.05) the jejunal IAP mRNA abundance by 3.5 fold. We conclude that decreases in the small intestinal apical activities of these examined hydrolases likely contribute to overgrowth of pathogenic bacterial populations in the distal small intestine and the colon, leading to the pathogenesis of IBD.

Vitamin K1 (Phylloquinone) or Vitamin K2 (Menaquinone-4) Induces Intestinal Alkaline Phosphatase Gene Expression

Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters into inorganic acid and alcohol at a high optimum pH (pH 8-10). Previously, we identified a significant increase of intestinal ALP (IAP) activity in the rat intestine on long-term dietary vitamin K supplementation. However, it was unclear whether the induction of ALP gene expression was caused by vitamin K intake. In the present study, we examined the effects of vitamin K on IAP gene expression. A total of 21 male ICR strain mice (7 wk old) were divided into three groups: control, PK, and MK groups. Mice were orally administered a 0.1-mL solution of physiological saline in the control group, phylloquinone (3 mg/kg mouse) in the PK group, and menaquinone-4 (3 mg/kg mouse) in the MK group. Four hours after administration, we determined the ALP activity of the intestinal mucosa in three areas (duodenum, jejunum, and ileum). In the MK groups, the levels of ALP activity in the jejunum increased significantly compared with the control. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers revealed that IAP mRNA expression was significantly enhanced in the jejunum in both PK and MK groups. Interestingly, vitamin K administration also increased the expression of pregnane X receptor mRNA. This is the first report concerning IAP mRNA expression induced by oral administration of vitamin K. The results support the possible involvement of vitamin K in the regulation of IAP mRNA expression as a novel pharmacological effect of vitamin K.

Early Weaning Reduces Small Intestinal Alkaline Phosphatase Expression in Pigs1–3

Expression of the small intestinal alkaline phosphatase (IAP) is enterocyte differentiation dependent and plays essential roles in the detoxification of pathogenic bacterial lipopolysaccharide endotoxin, maintenance of luminal pH, organic phosphate digestion, and fat absorption. This study was conducted to examine the effect of early weaning on adaptive changes in IAP digestive capacity (Vcap) and IAP gene expression compared with suckling counterparts in pigs at ages 10–22 d. Weaning decreased (P < 0.05) IAP enzyme affinity by 26% and IAP maximal enzyme activity by 22%, primarily in the jejunal region, with the jejunum expressing 84–86% of the whole gut mucosal IAP Vcap [mol/(kg body weight·d)]. The majority (98%) of the jejunal mucosal IAP maximal activity was associated with the apical membrane and the remaining (2%) existed as the intracellular soluble IAP. Weaning reduced the abundance of the 60-kDa IAP protein associated with the proximal jejunal apical membrane by 64% (P < 0.05). Furthermore, weaning reduced (P < 0.05) the relative abundance of the proximal jejunal IAP mRNA by 58% and this was in association with decreases (P < 0.05) in the abundances of cytoplasmic (27%) and nuclear (29%) origins of IAP caudal-associated homeobox transcription factor 1. In conclusion, early weaning decreased small intestinal IAP Vcap, IAP catalytic affinity, and IAP gene expression, and this may in part contribute to the susceptibility of early-weaned piglets to increased occurrence of enteric diseases and growth-check.

Role of lysophosphatidylcholine in brush-border intestinal alkaline phosphatase release and restoration

Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased approximately 10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4-6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.

Intestinal alkaline phosphatase gene expression is activated by ZBP-89

Intestinal alkaline phosphatase (IAP) is an enterocyte differentiation marker that functions to limit fat absorption. Zinc finger binding protein-89 (ZBP-89) is a Kruppel-type transcription factor that appears to promote a differentiated phenotype in the intestinal epithelium. The purpose of this study was to investigate the regulation of IAP gene expression by ZBP-89. RT-PCR, quantitative real-time RT-PCR, Western blot analyses, and reporter assays were used to determine the regulation of IAP by ZBP-89 in HT-29 and Caco-2 colon cancer cells. ZBP-89 knockdown was achieved by specific short interfering (si)RNA. EMSA and chromatin immunoprecipitation (ChIP) were performed to examine the binding of ZBP-89 to the IAP promoter. The results of RT-PCR, quantitative real-time PCR, and Western blot analyses showed that ZBP-89 was expressed at low levels in Caco-2 and HT-29 cells, whereas IAP was minimally expressed and absent in these cells, respectively. Transfection with ZBP-89 expression plamid increased IAP mRNA and protein levels in both cell lines, whereas knockdown of endogenous ZBP-89 by siRNA reduced basal levels of IAP gene expression in Caco-2 cells. IAP-luciferase reporter assays, EMSA, and ChIP established that ZBP-89 activated the IAP gene through a response element (ZBP-89 response element: 5'-CCTCCTCCC-3') located between -1018 and -1010 bp upstream of the AUG start codon. We conclude that ZBP-89 is a direct transcriptional activator of the enterocyte differentiation marker IAP. These findings are consistent with the role that this transcription factor is thought to play as a tumor suppressor and suggests its possible function in the physiology of fat absorption.

Differential regulation of intestinal alkaline phosphatase gene expression by Cdx1 and Cdx2

We have examined the role that the caudal-related homeobox transcription factors Cdx1 and Cdx2 play in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). Human colon cancer Caco-2 cells were transiently transfected with Cdx1 and/or Cdx2, and semiquantitative RT-PCR was used to study the effects on IAP mRNA expression. Transfections with a variety of IAP-luciferase reporter constructs were used to identify a Cdx response element located within the human IAP gene promoter. Protein-DNA interactions were examined by EMSA. Results showed that Cdx1 markedly induced IAP mRNA expression, whereas Cdx2 did not, and, in fact, inhibited the Cdx1 effects. Functional analysis revealed that Cdx1 transactivates (fourfold, P < 0.05) the IAP promoter through a novel Cdx response element (GTTTAGA) located between -2369 and -2375 upstream of the translational start site. EMSA showed that both Cdx1 and Cdx2 could bind to the cis element, but in cotransfection experiments, Cdx2 inhibited the Cdx1 effects by approximately 50%. Thus we have identified a previously unrecognized interaction between two important gut transcription factors, Cdx1 and Cdx2, in the context of IAP gene regulation. Cdx1 activates the IAP gene via a novel cis element, whereas Cdx2 inhibits the Cdx1 effects.

Tissue-Engineered Small Intestine Improves Recovery After Massive Small Bowel Resection

Rescue with tissue-engineered small intestine (TESI) after massive small bowel resection (MSBR). Short bowel syndrome is a morbid product of massive small bowel resection. We report the first replacement of a vital organ by tissue engineering with TESI after MSBR. Ten male Lewis rats underwent TESI implantation with green fluorescent protein (GFP)-marked cells (TESI+, n = 5) or sham laparotomy (TESI-, n = 5) followed by MSBR. Side-to-side anastomosis of TESI to proximal small intestine was performed or omitted. TESIO animals underwent implantation of engineered intestine with no further surgery. Weights were measured QOD until day 40. Transit times were measured. DNA assay was performed with computer morphometry. Northern blots of RNA were probed for intestinal alkaline phosphatase (IAP) and villin. Hematoxylin and eosin, S100, and smooth muscle actin immunohistochemistry were performed. Blood was collected at sacrifice. All 10 rats initially lost then regained weight. The initial rate of weight loss was higher in TESI+ versus TESI-, but the nadir was reached a week earlier with more rapid weight gain subsequently to 98% preoperative weight on day 40 in animals with engineered intestine versus 76% (P < 0.03). Serum B12 was higher at 439 pg/mL versus 195.4 pg/mL. IAP mRNA appeared greater in TESI+ than TESIO, with constant villin levels. Histology revealed appropriate architecture including nerve. GFP labeling persisted. Anastomosis of TESI significantly improved postoperative weight and B12 absorption after MSBR. IAP, a marker of differentiation in intestinal epithelium, is present in TESI, and GFP labeling was accomplished.

Gene regulation by endogenous thyroid hormone receptors in intestinal epithelial cells

Abstract Introduction: Studies in knockout mice indicate that the levels of intestinal alkaline phosphatase (IAP) are a key determinant of the efficiency of dietary fat absorption and the subsequent development of obesity. IAP is a known target gene for thyroid hormone (T3), but no previous studies have examined the ability of endogenous T3 receptors to regulate the IAP gene. Methods: These studies employed Northern and Western blotting, IAP-luciferase reporter analysis, electrophoretic mobility shift assay, and in vitro site-directed mutagenesis. Results: The TRalpha1 and TRbeta1 proteins were expressed in Caco−2 cells; however, TRalpha1 was the predominant isoform. Northern blots showed that T3 induced IAP mRNA expression (3-fold, p Conclusions: In gut epithelial cells, the IAP gene is positively regulated by endogenous TRs via an atypical TRE, and these effects of thyroid hormone may partly explain the weight loss associated with hyperthyroidism.

Expression of intestinal brush-border membrane hydrolases and ferritin after segmental ischemia-reperfusion in rats.

Jejunal expression of three brush-border membrane (BBM) enzymes, intestinal alkaline phosphatase (IAP), lactose-phlorizin hydrolase (LPH), and sucrase-isomaltase (SI), and a cytosolic protein, ferritin (Ft), was investigated after transient segmental ischemia-reperfusion (I/R). I/R reduced mucosal IAP, LPH, and SI mRNAs to 36%, 11%, and 38% of normal jejunal levels after 3 h of reperfusion and to 22%, 8%, and 51% of normal jejunal levels after 6 h of reperfusion, respectively. Intriguingly, in the internal control jejunum IAP and LPH mRNAs also decreased significantly. LPH and SI mRNA rapidly recovered to levels significantly higher than those of normal jejunum at 12 h, whereas IAP mRNA levels did not recover until 48 h. Enzyme activity paralleled changes in mRNA levels in the ischemic reperfused jejunum. Electrophoretic mobility shift assays showed that I/R significantly increased SI footprinting 1 (SIF1) binding activity. The mobility of one of the DNA-protein complexes was further retarded in the presence of anti-Cdx-2 antibody, suggesting that either Cdx-2 or a related protein was interacting with the SIF1 sequences. Similar to BBM enzymes, cytosolic Ft mRNA and protein were significantly decreased at 3 and 6 h after I/R. By 12 h, Ft mRNA, but not Ft protein, had increased to higher than normal levels. We conclude that a rapid recovery of BBM mRNAs and enzymes occurs in regenerating mucosa after upper villus damage. The increase of SIF1 binding protein activity after I/R may enhance SI, and perhaps LPH, gene transcription. The expression of Ft is regulated at both pretranslational and translational levels.

Increased C/EBP in fetal rat small intestine precedes initiation of differentiation marker mRNA synthesis.

Morphogenesis, initiation of differentiation marker gene expression, and their correlation with CCAT/enhancer binding protein (C/EBP) expression were analyzed in the developing fetal rat small intestine. Expressions of mRNAs for lactase-phlorizin hydrolase (LPH), intestinal alkaline phosphatase (IALP), carbamoyl-phosphate synthetase (CPS), and three isoforms of C/EBP were simultaneously determined by Northern blot analysis from 15 to 19 days of gestation. At 17 days of gestation, prior to villus formation as demonstrated by light and electron microscopy, only CPS and C/EBPalpha, -beta, and -delta expression could clearly be detected. Both LPH and IALP mRNA were definitely detectable in proximal and middle intestine on day 18, as soon as the stratified epithelium of the early intestine had been transformed into a single layer of columnar epithelium lining villi. This distribution was confirmed by in situ hybridization for LPH mRNA. During the period of transformation when the columnar epithelium and villi were forming, no LPH or IALP mRNA was detectable in the immature distal one-third of the fetal intestine. Preceding villus morphogenesis, immunostaining demonstrated nuclear localization of C/EBPalpha protein in intestinal epithelial cells, with continued expression in all enterocytes through 19 days of gestation. Enhanced expression of C/EBPalpha mRNA and protein began 24 h prior to the initiation of the differentiation markers, suggesting that it may play a role in regulation of fetal intestinal differentiation.

Prenatal ethanol exposure alters the expression of intestinal hydrolase mRNAs in newborn rats.

To gain insight into the postnatal growth delay induced by ethanol in utero, we characterized functional impairments of the small intestine of neonatal rats prenatally exposed to ethanol using a well-described model of gestational alcoholism (25% ethanol w/v in the drinking water). Expression of the intestinal enzymes-lactase-phlorizin hydrolase (LPH) and intestinal alkaline phosphatase (IAP)-that are critical for enteral nutrition of neonates was studied. Characteristic patterns of LPH and IAP expression along the proximal-distal (horizontal) and crypt-villus (vertical) axes of the small intestine, as well as the intracellular localization of LPH and IAP mRNAs and immunoreactive proteins within absorptive enterocytes, were not altered by prenatal exposure to ethanol. However, a 10- to 15-fold increase in the number of LPH and IAP mRNA molecules per absorptive enterocyte was found throughout the intestine of ethanol-exposed neonates, compared with controls, whereas lactase and alkaline phosphatase activities per enterocyte remained unchanged. These findings suggest that ethanol in utero alters the mRNA abundance of epithelial enzymes in newborn rat small intestine. Changes in mRNA abundance could be an important aspect of enterocyte adaptation to high ethanol concentrations in gastrointestinal amniotic fluid of ethanol-exposed fetuses.

Pattern of rat intestinal brush-border enzyme gene expression changes with epithelial growth state.

Enterocyte growth and differentiation occur simultaneously within the epithelium, but little is known regarding any relationship between these two processes. Four rat models of small intestinal epithelial hypo- and hyperplasia (neonatal ontogeny, fasting/refeeding, hypo-/hyperthyroidism, and bombesin treatment) were used to study the regulation of enterocyte gene expression in relation to epithelial growth state. Mucosal scrapings, as well as crypt and villus cell populations, were subjected to Northern blot analyses using radiolabeled cDNA probes corresponding to lactase, intestinal alkaline phosphatase, villin, ornithine decarboxylase (ODC), and the actin control. In all four models, the hypoplastic (atrophic) condition is characterized by high levels of lactase and low levels of the 3.0-kb intestinal alkaline phosphatase mRNA, whereas under hyperplastic conditions this pattern is reversed. The changes in intestinal alkaline phosphatase and lactase are qualitatively similar along the longitudinal axis of the intestine and are proportional to the degree of hyperplasia, as verified by ODC mRNA levels. Furthermore, the crypt-villus axis of differentiation is maintained regardless of epithelial growth state. In conclusion, the pattern of brush-border enzyme gene expression changes as a function of epithelial growth state, indicating a previously unrecognized degree of plasticity to the state of enterocyte differentiation.

Development and hormonal modulation of postnatal expression of intestinal alkaline phosphatase mRNA species and their encoded isoenzymes

In the rat, intestinal alkaline phosphatase (IAP) activity in the duodenum, but not jejunum, increases on day 22-24 after birth and exhibits higher activity hydrolysing phenyl phosphate (PhP) than beta-glycerophosphate (beta GP) [Moog and Yeh (1973) Comp. Biochem. Physiol. 44B, 657-666]. The mechanism underlying these developmental changes remains unknown. To define possible mechanisms, we have measured IAP activity and mRNA levels, and analysed IAP mRNA species and isoenzymes on postnatal days 12, 18, 24 and 32. Duodenal IAP activity and mRNA content were identical on postnatal days 12 and 18, but were 7-fold and 3-fold higher on day 24, respectively than on day 18. The increased IAP activity exhibited a high PhP/beta GP ratio and was accompanied by initial appearance of the 3.0 kb mRNA and 90 kDa isoenzyme. On day 32, duodenal IAP activity did not increase over the levels on day 24, whereas mRNA levels doubled. The lack of enzyme increase might be related in part to increased apical release, as luminal IAP activity increased from 2% of total mucosal IAP on days 12 and 18 to 7% and 14% on days 24 and 32 respectively. In the jejunum, IAP activity decreased postnatally, but mRNA content was unaltered; only the 2.7 kb mRNA and 65 kDa IAP isoenzyme were present. Administration of cortisone or cortisone+thyroxine induced simultaneous appearance of the duodenal 3.0 kb mRNA and 90 kDa isoenzyme with an increased PhP/beta GP ratio. Thus postnatal increase in duodenal IAP activity is related to the expression of a 90 kDa PhP-preferring isoenzyme encoded by the 3.0 kb mRNA. The low-PhP/beta GP-ratio 65 kDa isoenzyme is expressed in the duodenum and in the jejunum and is encoded by the 2.7 kb mRNA.

Temporal pattern of rat small intestinal gene expression with refeeding

Studies were carried out to elucidate the molecular mechanisms underlying small intestinal epithelial growth. Adult rats were fasted for 4 days and then refed a chow diet for up to 48 h. Histological examination confirmed the sequential occurrence of mucosal atrophy and hyperplasia. Northern blot analyses of RNA derived from small intestinal mucosal scrapings revealed a striking pattern of alterations in the expression of two different categories of genes. There were very early increases in the expression of c-fos and c-jun, reflecting the mitogenic response to refeeding that occurs within the crypt compartment. Studies using the protein synthesis inhibitor cycloheximide suggest that c-fos and c-jun are part of the "immediate-early" response of the small intestine. At later time points after the refeeding stimulus, differential changes occurred in the expression of the brush-border enzymes, lactase, and intestinal alkaline phosphatase (IAP). Refeeding caused a decrease in lactase gene expression and an increase in the expression of the 3.0-kb IAP mRNA species, reflecting a return of the villus phenotype to the normal fed state. Thus we have demonstrated a complex and temporally related pattern of gene expression within the small intestinal epithelium upon refeeding. The results provide insight into the relationship between the processes of intestinal growth and differentiation.

Identification of the HeLa tumor-associated antigen, p75/150, as intestinal alkaline phosphatase and evidence for its transcriptional regulation.

Prior studies identified a cell-surface antigen, p75/150, that exclusively associated with the tumorigenic phenotype of the HeLa parent and the tumorigenic phenotype of the HeLa parent and the tumorigenic segregants of suppressed, nontumorigenic HeLa x human fibroblast cell hybrids. Candidate p75/150 cDNA clones were isolated from a D98/AH.2 (HeLa) cDNA library using oligonucleotide probes derived from p75/150 partial peptide sequence data. A data base search revealed close similarity of p75/150 with intestinal alkaline phosphatase (IAP) [Berger, J., Garantini, E., Hua, J. C. & Udenfriend, S. (1987) Proc. Natl. Acad. Sci. USA 84, 695-698]. We demonstrate that p75/150 is identical to HeLa IAP by the following criteria: (i) 47/49 amino acid identity of p75 peptide sequence with IAP, (ii) restriction maps for the p75/150 candidate cDNA clone and IAP are identical, (iii) partial DNA sequence analysis of p75/150 candidate cDNA clones revealed complete nucleotide identity with IAP, except for a single nucleotide substitution in the 5' untranslated region, (iv) transfection of a p75/150 cDNA expression vector into the nontumorigenic hybrid, CGL1, yielded p75/150 antibody-positive transfectants that also expressed partially heat-resistant alkaline phosphatase activity. Northern blot analysis demonstrated that high levels of HeLa IAP mRNA were expressed in D98/AH.2 and the tumorigenic segregant CGL4; however, no mRNA was detected in CGL1. Nuclear run-on analyses indicate that HeLa IAP mRNA expression in the HeLa x fibroblast hybrids is regulated at the level of transcription initiation. Furthermore, evidence is discussed supporting the involvement of a chromosome 11 tumor suppressor locus in the regulation of HeLa IAP gene expression.

The mechanism of elevated alkaline phosphatase activity after bile duct ligation in the rat.

Alkaline phosphatase activity in the liver and intestine increases after bile duct ligation, reportedly by increased enzyme synthesis. To ascertain the mechanism of this increased synthesis in the absence of a cDNA clone encoding the enzyme, we have estimated the concentration of liver and intestinal alkaline phosphatase mRNA by translational analysis. Monospecific antiserum to rat placental alkaline phosphatase was raised. The resulting antiserum precipitated two peptides of 53 and 56 kd after translation of liver poly(A) + RNA. The precipitation of both peptides was blocked by the single 64 kd placental alkaline phosphatase. Processing of the cell-free products by microsomal membranes produced peptides of 62 and 64 kd. Antiserum to rat intestinal alkaline phosphatase also identified two peptides as products of intestinal RNA translation. After bile duct ligation, we confirmed a transient 2-fold increase in alkaline phosphatase activity in the intestine and a more constant 7-fold increase in the liver. However, the alkaline phosphatase mRNA concentration remained unchanged in both organs. We conclude that increased alkaline phosphatase synthesis after bile duct ligation results from an enhanced rate of translation of mRNA.