Absorption of 5-methyltetrahydrofolate in rat jejunum with intact blood and lymphatic vessels

Transport results from in vitro studies may not be applicable to in vivo situations. In this study, we extended our previous in vitro observations regarding the intestinal transport of 5-methyltetrahydrofolate to in vivo studies in the unanesthetized rat and examined the effect of the unstirred water layer on the absorption process. We used a well defined intestinal perfusion technique. Absorption of 0.5 and 5 microM 5-methyltetrahydrofolate proceeded in a linear manner for 40 min of perfusion at 0.31 and 1.74 nmol/100 cm per min, respectively. Absorption of 0.5 microM 5-methyltetrahydrofolate increased with increasing perfusate flow-rate from 0.5 to 2 to 4 ml/min, indicating an unstirred water layer influence on the absorption rate. Absorption of the substrate was saturable with an apparent Kt of 5.7 microM and Vmax of 3.45 nmol/100 cm per min. Absorption was pH-dependent, and was inhibited by structural analogues. In contrast to the in vitro data, addition of glucose (20 mM) to the perfusate was unnecessary for in vivo absorption to proceed. Unconjugated cholic (5 mM) and deoxycholic (1 mM) acids and the organic anion rose bengal (0.1 mM) inhibited the absorption of 0.5 microM 5-methyltetrahydrofolate when added to the perfusate. Conclusions: the results of previous in vitro studies of 5-methyltetrahydrofolate intestinal transport are applicable to in vivo situations, except that luminal glucose was found to be unnecessary in the latter. The unstirred water layer modulated the absorption of 5-methyltetrahydrofolate, while unconjugated bile acids and rose bengal inhibited it.     

Intestinal anion exchange in teleost water balance

Simultaneous measurements of all major electrolytes including HCO3(-) and H+ as well as water demonstrated that fluids absorbed by the anterior intestine of the marine gulf toadfish under in vivo-like conditions on an overall net basis are hypertonic at 380 mOsm and acidic ([H+] = 27 mM). This unusual composition of fluids absorbed across the intestinal epithelium is due to the unusual intestinal fluid chemistry resulting from seawater ingestion and selective ion and water absorption along the gastro-intestinal tract. Measurement under near symmetrical conditions with high NaCl concentrations and low MgSO4 concentrations revealed absorption of iso-osmotic and much less acidic fluids by the intestinal epithelium, a situation resembling that of other water absorbing leaky vertebrate epithelia. Reduced luminal NaCl concentrations seen in vivo results in lower absolute water absorption rates but higher Cl-/HCO3(-) exchange rates which are associated with higher net H+ absorption rates. It appears that apical anion exchange is important for net Cl- uptake by the marine teleost intestine especially when luminal NaCl concentrations are low and/or when MgSO4 concentrations are high. Observations indicate that fluid absorption from solutions of low NaCl but high MgSO4 concentrations is energetically more demanding than absorption from NaCl rich solutions at the level of the intestinal epithelium. Furthermore, the high luminal MgSO4 concentration which is an unavoidable consequence of seawater ingestion projects a demand for renal and branchial compensation for intestinal MgSO4 uptake and absorption of hypertonic and acidic fluid by the intestine.     

Pancreatic trypsin cleaves intestinal alkaline sphingomyelinase from mucosa and enhances the sphingomyelinase activity

Sphingomyelin (SM) hydrolysis in the gut has implications in colonic tumorigenesis and cholesterol absorption. It is triggered by intestinal alkaline sphingomyelinase (Alk-SMase) that is present in the intestinal mucosa and content. The mechanism by which the enzyme is released into the lumen is not clear. We studied whether trypsin can dissociate Alk-SMase from the mucosa and affect its activity. During luminal perfusion of rat intestine, addition of trypsin to the buffer increased Alk-SMase activity in the perfusate output by about threefold. Treating COS-7 cells transfected with Alk-SMase cDNA with trypsin increased the SMase activity in the medium and reduced that in the cell lysate dose dependently. The appearance of Alk-SMase in the perfusate and culture medium was confirmed by Western blot analysis. The effect of trypsin was blocked by trypsin inhibitor, and neither chymotrypsin nor elastase had a similar effect. We also expressed the full length and COOH-terminal truncated Alk-SMase in COS-7 cells and found that the activity of the full-length enzyme is mainly in the cells, whereas that of the truncated form is mainly in the medium. Both forms were active, but only the activity of the full-length Alk-SMase was enhanced by trypsin. By linking a poly-His tag to the constructed cDNA, we found that the first tryptic site Arg440 upstream of the signal anchor was attacked by trypsin. In conclusion, trypsin cleaves the Alk-SMase at the COOH terminal, releases it from mucosa, and meanwhile enhances its activity. The findings indicate a physiological role of trypsin in SM digestion.     

Increased plasma pyridoxal-5′-phosphate when alkaline phosphatase activity is reduced in moderately zinc-deficient rats

It is generally believed that the zinc metalloenzyme alkaline phosphatase is required to hydrolyze phosphorylated forms of vitamin B-6 prior to their use. To test this hypothesis, rats were fed a liquid diet containing either adequate or moderately low zinc during gestation and lactation. Zinc deficiency was produced in dams evidenced by significant reductions in zinc concentration of plasma (49%), liver (25%), and femur (24%), and plasma alkaline phosphatase activity (48%). Plasma pyridoxal-5′-phosphate (PLP), which significantly increased (61%) in these same rats, was negatively correlated (r=−0.74,P<0.02) with plasma alkaline phosphatase activity. Maternal liver PLP concentration was unaffected by zinc status. The zinc and vitamin B-6 relationship seen in dams was less observable in offspring. Stimulation of erythrocyte alanine aminotransferase activity by exogenously added PLP in vitro tended to be higher in both moderately zinc-deficient mothers and their offspring, but the difference was not significant. Our results support the hypothesis that alkaline phosphatase activity is required for the hydrolysis of plasma PLP. Our results also suggest that zinc status as alkaline phosphatase activity should be defined in an individual if plasma PLP is to be used as an indicator of vitamin B-6 status.     

Quantitative histochemical study of alkaline phosphatase isoenzymes in the uteri of ovariectomized golden hamsters during estrogenization

The activity of two isoenzymes of alkaline phosphatase was studied in the uteri of ovariectomized golden hamsters. Animals belonging to different groups were daily injected with 10 micrograms of estrogen (octoestrol) once, for 4 and 16 days. The estrogen did not affect the overall activity lf alkaline phosphatase in the uterine luminal epithelium, but decreased the enzyme activity in stroma. Moreover, it was found that prolonged estrogen treatment increases significantly the proportion of alkaline phosphatase isoenzyme of the intestinal type in the uterine luminal epithelium.     

The mechanism by which interleukin-1 beta reduces net fluid absorption from the rat colon

IL-1beta is suspected to be involved in the diarrhea that always accompanies inflammatory bowel disease. This work was aimed at studying the in vivo effect of IL-1beta on the net absorption of fluid, Na(+) and Cl(-) from the rat colon, and at delineating its mechanism of action. Rats were injected i.p. with IL-1beta (1 mug/kg body weight) and the colon was perfused, four hours later, with Krebs-Ringer buffer. Net fluid absorption was calculated as the difference between the total volume of the buffer infused and collected per cm(2) of perfused intestine. Chloride in both buffers was determined by titration according to Mohr's method and net Cl- absorption was calculated in the same way. IL-1beta reduced the net absorption of water and chloride. The cytokine also reduced the percentage recovery of the Na(+)-K(+) ATPase activity in crude homogenates of membranes from surface and crypt colonic cells as revealed by the determination of inorganic phosphate released. In addition IL-1beta decreased the protein expression of the Na(+)-K(+) pump and increased that of the NaKCl(2) symporter. It is concluded that IL-1beta has a dual effect: it inhibits the Na(+)-K(+) pump and consequently NaCl absorption, and up-regulates the NaKCl(2) transporter and increases Cl(-) secretion. The ultimate effect of the two processes is a net decrease in Na(+)+ and Cl(-) absorption and an increase in water retention in the colon leading to the observed diarrhea in inflammatory bowel disease.     

Kinetics of the absorption of amino acids by the rat intestine in vivo

The kinetics of l-phenylalanine and l-lysine absorption by the rat small intestine in vivo have been studied by perfusing intestinal segments and monitoring simultaneously the uptake of the substrate into the intestinal tissue and its disappearance from the perfusate.The rate of phenylalanine disappearance is a linear function of the substrate concentration. Its uptake into the tissue is rapid and obeys saturation kinetics, but is not concentrative. Both tissue uptake and disappearance rate can be inhibited by leucine or methionine, but are not influenced by hydrophilic neutral or dibasic amino acids.Lysine disappearance from the perfusate and its uptake into the tissue both display saturation kinetics. Lysine transport is quantitatively smaller than that of phenylalanine. Both uptake and disappearance are inhibited by arginine and leucine, but are unaffected by other neutral amino acids or sugars.To analyse the kinetic results, integrated equations were developed to express the final concentration in the perfusate in terms of the original concentration. The disappearance rate was considered as a mixed process (saturable and non-saturable in parallel) in a one-compartment system, and the uptake by the tissue was treated as a two-compartment system in which the amino acid entered the cells by a mixed process but left them by a pure non-saturable mechanism.The results concerning disappearance from the lumen are compatible with the one-compartment model. Phenylalanine absorption can be described by a major non-saturable component and a minor saturable one, while lysine absorption occurs almost entirely by a saturable process. The two-compartment model does not adequately describe the tissue uptake results.     

Improved absorption of caseinophosphopeptide-bound iron: role of alkaline phosphatase

Hydrolysis of proteins could lessen their inhibiting effect on the poor absorption of cow's milk iron (Fe), which is responsible for the high incidence of Fe deficiency worldwide. When bound to Fe, caseinophosphopeptides (CPP) derived from milk proteins resist luminal digestion, enhance Fe solubility and could improve its bioavailability; brush border enzyme alkaline phosphatase activity could influence iron absorption by releasing free Fe; this study assessed its role in the absorption of CPP-bound Fe. Rat duodenal loops were perfused with Fe gluconate or Fe bound to the CPP of beta casein [beta-CN (1-25)], with or without the addition of an inhibitor of alkaline phosphatase, Na2WO4. The uptake of Fe-beta-CN (1-25) was greater than Fe gluconate. Na2WO4 enhanced the uptake of Fe-beta-CN (1-25) and not of Fe gluconate. So the release of free, insoluble Fe, by alkaline phosphatase seems to be prevented by providing Fe in the Fe-beta-CN (1-25) complex form. Its good disappearance rate makes beta-CN (1-25)-bound Fe a candidate for food fortification.     

Lack of relationship between activity of intestinal alkaline phosphatase and calcium or phosphate absorption

The effects of vitamin D3 and the aqueous extract of Solanum malacoxylon on intestinal alkaline phosphatase and tissue phosphate content were studied on rachitic chicks treated with large doses of ethane-1-hydroxy-1,1 diphosphonate (EHDP). The EHDP treatment blocks the increase of intestinal calcium or phosphate absorption induced by the vitamin D3, while it has no effects on the rise of intestinal alkaline phosphatase activity or the increment in tissue phosphate content. The lack of correlation between the increment of alkaline phosphatase and that of Ca or phosphate absorption in vitamin D3 plus EHDP treated chicks excludes a participation of the alkaline phosphatase in the mechanism of Ca or P intestinal absorption. The Ca or phosphorus absorption are elicited specifically by 1,25-(OH)2-D3, while alkaline phosphatase activity and phosphate tissue concentration respond to a broader spectrum of stimuli.     

Effects of a single dose of menadione on the intestinal calcium absorption and associated variables

The effect of a single large dose of menadione on intestinal calcium absorption and associated variables was investigated in chicks fed a normal diet. The data show that 2.5 micro mol of menadione/kg of b.w. causes inhibition of calcium transfer from lumen-to-blood within 30 min. This effect seems to be related to oxidative stress provoked by menadione as judged by glutathione depletion and an increment in the total carbonyl group content produced at the same time. Two enzymes presumably involved in calcium transcellular movement, such as alkaline phosphatase, located in the brush border membrane, and Ca(2+)- pump ATPase, which sits in the basolateral membrane, were also inhibited. The enzyme inhibition could be due to alterations caused by the appearance of free hydroxyl groups, which are triggered by glutathione depletion. Addition of glutathione monoester to the duodenal loop caused reversion of the menadione effect on both intestinal calcium absorption and alkaline phosphatase activity. In conclusion, menadione shifts the balance of oxidative and reductive processes in the enterocyte towards oxidation causing deleterious effects on intestinal Ca(2+) absorption and associated variables, which could be prevented by administration of oral glutathione monoester.     

Intragastric pH regulates conversion from net acid to net alkaline secretion by the rat stomach

Our previous report showed gastric mucosal surface pH was determined by alkali secretion at intragastric luminal pH 3 but by acid secretion at intragastric pH 5. Here, we question whether regulation of mucosal surface pH is due to the effect of luminal pH on net acid/base secretions of the whole stomach. Anesthetized rats with a gastric cannula were used, the stomach lumen was perfused with weakly buffered saline, and gastric secretion was detected in the gastric effluent with 1) a flow-through pH electrode and 2) a fluorescent pH-sensitive dye (Cl-NERF). During pH 5 luminal perfusion, both pH sensors reported the gastric effluent was acidic (pH 4.79). After perfusion was stopped transiently (stop-flow), net acid accumulation was observed in the effluent when perfusion was restarted (peak change to pH 4.1-4.3). During pH 3 luminal perfusion, both pH sensors reported gastric effluent was close to perfusate pH (3.0-3.1), but net alkali accumulation was detected at both pH sensors after stop-flow (peak pH 3.3). Buffering capacity of gastric effluents was used to calculate net acid/alkaline secretions. Omeprazole blocked acid secretion during pH 5 perfusion and amplified net alkali secretion during pH 3 perfusion. Pentagastrin elicited net acid secretion under both luminal pH conditions, an effect antagonized by somatostatin. We conclude that in the basal condition, the rat stomach was acid secretory at luminal pH 5 but alkaline secretory at luminal pH 3.     

Phosphatase activity in the larva of the euryhaline mosquito, Aëdes togoi Theobald,with special reference to sea-water adaptation

The alkaline and acid phosphatases in larvae of the euryhaline mosquito, Aëdes togoi Theobald, were measured and the distribution of alkaline phosphatase was examined histochemically. The optima pH for alkaline and acid phosphatases in the larvae were ≈9.0 and 3.2. respectively. The thorax region showed the highest activity of alkaline phosphatase. The enzyme activity of the thorax of seawater adapted larvae was about twice as high as that of freshwater larvae. When the larvae were transferred from fresh water to sea water, the alkaline phosphatase activity of the thorax increased greatly for 3 days, and thereafter decreased to the normal level of sea-water adapted larvae within seven days. In larvae transferred from sea water to fresh water, the activity of the thorax decreased gradually and after 7 days remained at the level of freshwater adapted larvae. No change in acid phosphatase activity was detected following transfer of the larvae from fresh water to sea water or vice versa. A strong alkaline phosphatase reaction was found only in the luminal border of the gastric caeca in the thorax region. The locality of this enzyme did not vary according to the salinity of environmental water.The activity change of alkaline phosphatase of the gastric caeca is discussed in relation to the absorption of the ingested medium from the gastric caeca.     

Na transport and enzyme activities in the intestine of the freshwater and sea-water adapted trout (Salmo gairdnerii R.)

Unidirectional fluxes of Na+ obtained in perfused preparation and mucosal enzyme equipment (alkaline phosphatase, ouabain-sensitive Na+, K+-ATPase) have been determined in the middle and posterior intestine of freshwater (FW) and sea-water (SW) adapted trout. In FW, influxes and outfluxes were higher in the middle than in the posterior intestine, although net fluxes were similar. SW adaptation induced an increase of influxes and net fluxes mainly in the posterior intestine. SW adaptation decreased the alkaline phosphatase activity only in the posterior intestine. Na+,K+-ATPase activity was always higher in the middle than in the posterior intestine in FW and SW and increased in both parts by SW adaptation. Thus, it seems that SW adaptation of rainbow trout modifies Na intestinal absorption principally in its posterior part and in relation with the Na+, K+-ATPase activity.     

myo-inositol action on gerbil intestine: alterations in alkaline phosphatase activity upon phosphatidylinositol depletion and repletion in vivo

The influence of phosphatidylinositol (PI) on intestinal alkaline phosphatase activity was studied in myo-inositol deficient gerbils. A reduction of membrane PI in intestinal mucosa to 30-40% of the control was produced by feeding female gerbils a myo-inositol-deficient diet containing coconut oil for 2 weeks. As expected, the animals developed typical intestinal lipodystrophy with abnormal fat accumulation. In the PI-depleted animal, intestinal alkaline phosphatase activity was reduced to 20-30% of the control group. The levels of both membranous and soluble enzymes in intestinal mucosa were affected, but there were no changes in liver, kidney and plasma levels. When the lipodystrophic gerbils were given dietary myo-inositol, the complete repletion of intestinal membrane PI to the control level occurred 36 h later, whereas membrane-bound alkaline phosphatase activity in intestine was not restored to the control level until 72 h later. Administration of cycloheximide or actinomycin D did not block this enzyme induction. Lymphatic output of triacylglycerol into the bloodstream was stimulated 10-fold at 18 h of myo-inositol repletion, but there was no parallel increase in the activity of alkaline phosphatase in plasma during this early phase of intestinal recovery. Thus, these data suggest a possible regulatory role of PI in the processing and/or turnover of alkaline phosphatase in vivo, but a negative role of alkaline phosphatase in lipid transport across gerbil intestine.     

Effects of perfusate buffer capacity on capillary CO2-HCO3(-)-H+ reactions: theory.

The importance of perfusate nonbicarbonate buffer capacity (beta nonHCO3) to intracapillary CO2-HCO3(-)-H+ reactions was assessed by theoretical analysis of CO2 exchange in saline-perfused pulmonary capillaries. Time courses for perfusate PCO2, [HCO3-], and [H+] were computed for capillaries containing different activities of luminal vascular carbonic anhydrase and different amounts of perfusate nonbicarbonate buffers. Mobilization of perfusate HCO3- toward CO2 during capillary transit is determined by the availability of HCO3- and H+. A supply of protons from the nonbicarbonate buffer pool is necessary to maintain a high rate of HCO3- dehydration. The analyses indicate that beta nonHCO3 has marked nonlinear effects on transcapillary CO2 exchange and intravascular pH equilibration. These nonlinear effects differ from those previously computed for CO2 reactions in an open system because the present model system consists of a sequential combination of open (within capillary proper) and closed (within postcapillary vasculature) systems. The role of luminal vascular carbonic anhydrase in capillary CO2 reactions is strongly dependent on beta nonHCO3. Perfusate nonbicarbonate buffer capacity must be considered when the results of experimental studies of transcapillary CO2 exchange and/or intravascular pH equilibration are interpreted.     

Galactosyltransferase, pyrophosphatase and phosphatase activities in luminal plasma of the cauda epididymidis and in the rete testis fluid of some mammals

Galactosyltransferase activity was measured in the luminal plasma of the cauda epididymidis of mice, rats, rabbits, rams and boars, and in the rete testis fluid of rams and boars. The activities of nucleotide pyrophosphatase and alkaline phosphatase, which compete with galactosyltransferase for substrate, were also determined. In these species, galactosyltransferase activity in the luminal plasma of the cauda epididymidis was similar when the inhibitory effect of pyrophosphatase and phosphatase was minimized by assay conditions. However, under assay conditions that did not minimize the effect of these enzymes, the galactosyltransferase activities of these species were very different and were inversely correlated with the activities of pyrophosphatase and phosphatase. The ratio of galactosyltransferase activity to pyrophosphatase and phosphatase activity was much higher in the rete testis fluid than in the luminal plasma of the cauda epididymidis in both rams and boars. In rams, galactosyltransferase in the luminal plasma of the cauda epididymidis was more heat resistant than that in serum. These results suggest that there is a species difference in the availability of galactosyltransferase activity in the luminal plasma of the cauda epididymidis and that in some species, galactosyltransferase in the luminal fluid is unlikely to have any function. The results are also discussed with respect to the possible function of galactosyltransferase, pyrophosphatase and phosphatase in epididymal luminal plasma and rete testis fluid.     

Stability of isoenzymes of alkaline phosphatase in various buffer systems.

The stability of isoenzymes of alkaline phosphatase from liver, bones and small intestine was compared after addition to inactivated serum in the buffer systems: glycine, 2-amino-2-methyl-1-propanol, diethanolamine and 2-amino-2-methyl-1,3-propandiol at 37 degrees C. The mentioned isoenzymes were inactivated to different extents in glycine and 2-amino-2-methyl-1-propanol buffers. In diethanolamine and 2-amino-2-methyl-1,3-propandiol buffers sufficient stability of isoenzymes is obtained so that only these buffers are suitable for activity determinations of alkaline phosphatase at 37 degrees C.     

Kinetics of phenylalanine absorption by the rat intestine in vivo after distal resection

The kinetics of L-phenylalanine absorption across rat small intestine in sham and 50% distal resected animals, in vivo, have been studied by perfusing jejunal loops and monitoring the disappearance of the substrate from the perfusate. After 5 months postresection the total phenylalanine absorption was increased. The relationship between total absorption of substrate and its concentration in the bulk phase shows a non-saturable component and a saturable one that can be inhibited by methionine, both in control and remnant jejunum. The slope of the line that represents the non-saturable component is greater in remnant jejunum, indicating that the apparent mass-transfer coefficient, K'D, was increased by distal resection. The kinetic analysis of the saturable component shows that Jmax was unaltered and the apparent semisaturation constant, K'M, was slightly decreased by distal small intestine resection. Correction of the kinetic constant for the unstirred water layer effects shows that the differences between 'real' KD values of the two experimental groups increase whereas 'real' KM values do not change significantly. This indicates that the observed increase in total intestinal absorption in resected animals appears to result from an increase in the intestinal passive permeability.     

Effects of epinephrine, clonidine, L-phenylephrine, and morphine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in pig jejunum

Perfusion of pig jejunum with Escherichia coli heat-stable enterotoxin (strain 1261) reversed net absorption of water and electrolytes to net secretion. Addition of the alpha-adrenergic agonists clonidine (5 X 10(-7) M) or L-phenylephrine (5 X 10(-6) M), or the opiate agonist morphine (3.6 X 10(-6) M) to the perfusate reduced the secretory response to enterotoxin and stimulated absorption in normal jejunum. Epinephrine (5 X 10(-5) M) did not stimulate absorption in controls but reduced chloride loss in the presence of enterotoxin. Mucosal sodium--potassium adenosine triphosphatase was unchanged but disaccharidase activity was decreased in the presence of enterotoxin. The results suggest that alpha-adrenergic agonists and opiate agonists may exert an antidiarrheal action by increasing net transport across intestinal epithelium.     

The effect of vitamin D3 metabolites on membrane proteins of chick duodenal brush borders.

Chick intestinal brush border proteins were examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Following injection of 25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃, a large molecular weight protein present in the vitamin D-deficient brush borders diminishes and a larger protein appears. This change occurs before calcium binding protein can be detected by Chelex assay and prior to the increase in total alkaline phosphatase but correlates closely with increased intestinal calcium absorption in response to the metabolites. The two brush border proteins have been solubilized with n-butanol and partially characterized. The vitamin D-deficient protein has a molecular weight of about 200,000 and has alkaline phosphatase activity but no detectable calcium binding activity. The protein which appears in response to metabolites has a molecular weight of 230,000, binds calcium, and also has alkaline phosphatase activity.