Iwao Koyama

Alkaline phosphatases reduce toxicity of lipopolysaccharides in vivo and in vitro through dephosphorylation.

Intestinal alkaline phosphatase (AP), as a host defense factor, was first investigated in vivo using rats orally exposed to lipopolysaccharide (LPS). After the oral administration of LPS to rats, serum LPS content was increased within 2 hr and then decreased to 6 hr. In contrast, when L-phenylalanine (L-Phe), an inhibitor of intestinal-type AP isozymes, was simultaneously administered with LPS, serum LPS content significantly increased from 1 hr and the area under the concentration-time curve of serum LPS was augmented approximately 2-fold, suggesting that APs in the gastrointestinal tract reduced serum LPS content. In addition, LPS toxicity diminished by a treatment in vitro with intestinal APs, were recovered by the treatment in the co-presence of L-Phe. In the experiment using human aortic endothelial cells (HAECs), we observed that the cell viability decreased in a dose-dependent manner of LPS-exposure, and the LPS dose, exhibiting 50% viability of the cells, was 0.05 microg/ml. When the cells were exposed to LPS pretreated with 50 nIU/ml of intestinal AP at pH 10.0 and 8.0, the 50% viability was at 2.0 microg/ml of LPS. These results strongly suggest that the APs reduced the toxicity of LPS, as a host defense factor against LPS.

Sugar-chain heterogeneity of human alkaline phosphatases: differences between normal and tumour-associated isozymes

The sugar-chain heterogeneity of alkaline phosphatases (ALPs) from various human organs was investigated by using the serial lectin affinity technique. This technique revealed a possible structure of the sugar chain(s) of ALP isozymes and clarified a difference in affinity on the lectin column not only among three genetically different isozymes (liver/bone/kidney, intestinal and placental types) but also among liver, bone, and kidney ALPs. Lectin-binding profiles of ALPs in these human organs closely resembled those in the corresponding organs of the rat, as reported previously, suggesting that heterogeneities in sugar chains of ALPs have a specificity for the respective organs rather than being species-specific. Lectin-binding profiles of tumour-produced placental and liver ALPs were significantly different from those of ALPs in the respective normal organs. However, the two altered ALPs exhibited similar lectin-binding affinities. Isoelectric focusing analysis showed essentially no difference in protein charge between the normal and tumor-produced ALPs. Moreover, tumour-produced ALPs had the same N-terminal amino acid sequence and peptide mapping as normal ALPs. From these results, it is possible to suggest that organ-specific sugar chains in ALP isozymes are changed into those peculiar to tumours in association with malignant transformation.

Different lectin affinities in rat alkaline phosphatase isozymes: Multiple forms of the isozyme isolated by heterogeneities of sugar moieties

Differences among rat alkaline phosphatases from various organs were established by using the serial lectin affinity technique. Elution profiles of isozymes with various lectin columns were significantly different from each other, and it was possible to distinguish between isozymes by this technique. It has been shown by many workers that a high-mannose-type and/or hybrid-type sugar chain is contained in the fraction bound strongly to concanavalin A-Sepharose. The duodenal alkaline phosphatase had a low content of this fraction, although the content of this fraction obtained from duodenal explants was increased markedly when explants were cultured with swainsonine, which is an inhibitor of alpha-mannosidase II, and this leads to the accumulation of high-mannose-type and hybrid-type sugar chains in the pathway of sugar chain processing. From the present results, it is suggested that differences in the elution profiles of isozymes may be due to the structural differences of sugar chains in alkaline phosphatases.

A possible mechanism of induction and translocation into blood stream of rat alkaline phosphatase activity by bile duct ligation.

We investigated the effects of bile duct ligation on alkaline phosphatase (ALP) activities in liver, calvarium, duodenum, and ileum in rats and its possible mechanism of action. ALP isozyme activities in the ligated rats were significantly elevated in the liver and duodenum, while those in the ileum and calvarium were markedly decreased. The ALP isozyme activity elevated by the ligation was obviously suppressed by prior administration of indomethacin, an inhibitor of prostaglandin synthesis. Moreover, phorbol ester also elevated the ALP activity as well as the phosphatase level in the ligated rat. However, other drugs, such as an inhibitor of protein kinase C and calmodulin, showed different effects: calmodulin stimulated an 11.0-, 1.3-, or 1.5-fold increase in ALP activity in the ileum, duodenum, or calvarium, respectively; whereas the hepatic enzyme activity was not affected. The induction by calmodulin was markedly different from that by the ligation. Moreover, imipramine, an inhibitor of protein kinase C, had little effect. These results suggest that prostaglandin is a possible ALP inducer in ligated rats, probably working by elevating the cAMP level. On the other hand, the ligation induced simultaneously de novo synthesis of the membranous and soluble ALP isozymes; and the release rate of the soluble enzyme was greater than that of the membranous isozymes, indicating that the soluble enzyme might be a main source of the induced serum ALP. Lectin affinity chromatography indicated that the soluble enzyme or induced serum enzyme may contain more fucose than that of the membranous one, suggesting that the sugar moiety in the ALP molecule may relate to the clearance of ALP from or its release into the circulation.

Blood appearance of rat alkaline phosphatase originating from the duodenum in vitro.

The major source of rat serum alkaline phosphatase (ALP) is well known to be from the intestinal enzyme, but it is still unclear whether it is from the duodenal or the ileal enzyme. The organic origin was investigated by means of two-dimensional electrophoresis. Major isoelectric points and molecular masses for activities of duodenal enzyme treated with both phosphatidylinositol-specific phospholipase C and neuraminidase were identified apparently with those of the major serum enzyme. In organ culture, the normal duodenal enzyme was released in the highest amounts to the culture medium. These results indicate that the major source of serum ALP in adult rats is basically from the duodenal enzyme. On the other hand, lectin affinity chromatography for ALPs showed that the ALP in the medium from culture duodenum and liver had the same complex-type sugar chain as with the ALP in the duodenal tissue. Although the duodenal ALP induced by glucosamine in vitro had the hybrid-type chain, sugar chains of the induced ALP in the culture medium were of the complex type, indicating that medial ALPs possessing the same sugar chain as the native duodenal enzyme, complex type, are mainly released from their tissues in normal conditions.

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.

Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes

To understand the differences between the rat intestinal alkaline phosphatase isozymes rIAP‐I and rIAP‐II, we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP). Our models of rIAP‐I and rIAP‐II displayed a typical α/β topology, but the crown domain of rIAP‐I contained an additional β‐sheet, while the embracing arm region of rIAP‐II lacked the α‐helix, when each model was compared to hPLAP. The representations of surface potential in the rIAPs were predominantly positive at the base of the active site. The coordinated metal at the active site was predicted to be a zinc triad in rIAP‐I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP‐II. Using metal‐depleted extracts from rat duodenum or jejunum and hPLAP, we performed enzyme assays under restricted metal conditions. With the duodenal and jejunal extract, but not with hPLAP, enzyme activity was restored by the addition of zinc, whereas in nonchelated extracts, the addition of zinc inhibited duodenal IAP and hPLAP, but not jejunal IAP. Western blotting revealed that nearly all of the rIAP in the jejunum extracts was rIAP‐I, whereas in duodenum the percentage of rIAP‐I (55%) correlated with the degree of AP activation (60% relative to that seen with jejunal extracts). These data are consistent with the presence of a triad of zinc atoms at the active site of rIAP‐I, but not rIAP‐II or hPLAP. Although no differences in amino acid alignment in the vicinity of metal‐binding site 3 were predicted between the rIAPs and hPLAP, the His153 residue of both rIAPs was closer to the metal position than that in hPLAP. Between the rIAPs, a difference was observed at amino acid position 317 that is indirectly related to the coordination of the metal at metal‐binding site 3 and water molecules. These findings suggest that the side‐chain position of His153, and the alignment of Q317, might be the major determinants for activation of the zinc triad in rIAP‐I.

Intestinal type alkaline phosphatase hyperphosphatasemia associated with liver cirrhosis.

Hyperphosphatasemia due to increased intestinal type serum alkaline phosphatase was noted in a 48-year-old male who had asymptomatic liver cirrhosis. The alkaline phosphatase activity in the serum was 828 U/l (our reference range in adults: 57-194 U/l), 94% of which was of the intestinal type as measured by an immunoprecipitation method. The intestinal component of alkaline phosphatase was separated into two major and some minor components using electrophoresis and isoelectrofocusing. One of the major components had similar mobility to that of a standard intestinal enzyme purified from adult intestine. The components were heat-labile and neuraminidase-resistant. Serial lectin affinity chromatography, however, indicated that sugar chain compositions of the alkaline phosphatase were different from those of the standard tissue intestinal enzyme. These results and further enzymological studies suggest that the patients serum alkaline phosphatase basically consisted of several intestine-like isoforms.

Organ specific properties for human urinary alkaline phosphatases

We have re-evaluated the isolation and characteristics of human urinary alkaline phosphatases (ALPs). From the results of physicochemical properties and immunological identification, the urinary ALPs from healthy subjects and patients with hepatoma were found to be similar in nature to liver and/or bone-like ALP. In patients with chronic or acute nephritis, the ALPs contained a major band of kidney-like ALP with a minor band of bone and intestinal ALPs. However, the ALPs in pregnant women had not only liver and bone ALPs but also placental-like ALP. It is interesting that only bone-like ALP was detected in psychiatric patients administered chlorpromazine. In the conditions we investigated, the molecular sizes of the urinary ALPs were similar as those of original ALPs, except for the enzyme from renal failure. Moreover, the total activity of urinary ALP was closely related to the level of serum ALP, being in a ratio of 1/40. In general, urinary ALP may be derived from serum ALP by minor modification, suggesting that the identification of excreted ALP in urine is a good marker for disturbed organs in respective diseases.

Expression of α-amylase isozymes in rat tissues

Gene expressions of α-amylase isozymes in rat tissues were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR), followed by EcoRI digestion. This procedure is based on evidence that an RT-PCR product from mouse pancreas RNA is sensitive to EcoRI, but not the product from the salivary gland or liver RNAs. The method was applied to the analysis of α-amylase expression in rat liver after partial hepatectomy, in which a potent expression of pancreas type isozyme was observed. However, no expression of the pancreatic isozyme in the regenerating liver was found. We also analyzed the expression of α-amylase gene in several additional rat tissues. In intestine, stomach, testis and skeletal muscle, the corresponding PCR products were amplified, but few were detected in heart or spleen. Intestine and stomach expressed a pancreatic isozyme of α-amylase. Analyses of the α-amylase activity and protein indicated the presence of the enzyme in those tissues. Immunohistochemical analysis also indicated that the amylase proteins were specifically present in epithelial cells of rat intestinal mucosa. This is a convenient method for identification of α-amylase isozyme mRNA in rodent tissues.