Rat Small Intestinal Membrane Research Articles

The effect of dinoprost on transport of water and imipramine through rat small intestinal membranes.

The relation between transmucosal fluid movement and its effect on absorption and exsorption of imipramine was studied with the in-situ single-pass perfusion technique in rats. Dinoprost (prostaglandin F2 alpha, PGF2 alpha) caused a dose-related inhibition of both absorption and secretion of water across the intestinal membrane. When PGF2 alpha was infused at a rate of 5 mumols kg-1 h-1, the absorption rate of water decreased from 51.7 to 21.5 mL h-1 and the secretion rate decreased from 48.9 to 26.8 mL h-1. Net water flux changed from net water absorption (0.9 mL h-1) to net water secretion (5.33 mL h-1) by infusion of PGF2 alpha. However, absorption and exsorption of imipramine were little affected by infusion of PGF2 alpha. The absorption rates of imipramine were 3.03 and 2.36 mg h-1 in the absence and presence of PGF2 alpha, respectively. Furthermore, the average amounts of imipramine exsorbed into the intestinal lumen in 2 h were 7.82 and 8.10% in the absence and presence of PGF2 alpha, respectively. Infusion of PGF2 alpha also enhanced motility of the small intestine compared with the control. From these results, it appears that PGF2 alpha has no effect on the absorption and exsorption of imipramine across the intestinal membrane although it is reasonable to use PGF2 alpha in the case of patients with overdoses of drugs which decrease gastrointestinal motility.

Site-specific contribution of proton-coupled folate transporter/haem carrier protein 1 in the intestinal absorption of methotrexate in rats

Abstract Objectives Methotrexate is reportedly a substrate for proton-coupled folate transporter/haem carrier protein 1 (PCFT/HCP1) and reduced folate carrier 1 (RFC1). In this study, we examined the contribution of PCFT/HCP1 and RFC1 in the intestinal absorption of methotrexate in rats. Methods Western blot analysis was carried out to evaluate the protein levels of PCFT/HCP1 and multidrug resistance-associated protein 2 in brush-border membrane of rat small intestine. Mucosal uptake of methotrexate was studied in the rat everted small intestine and an in-situ intestinal perfusion study of methotrexate was also carried out in rats. Key findings In transport studies using everted intestine, the mucosal methotrexate influx rate in proximal intestine at pH 5.5 was significantly greater than that at pH 7.4. Coadministration of folate or its analogues, such as folinate and 5-methyltetrahydrofolate, substrates for both PCFT/HCP1 and RFC1, significantly suppressed the methotrexate influx at pH 5.5, whereas thiamine pyrophosphate, an inhibitor for RFC1 alone, exerted no significant effect. Western blot analysis showed higher PCFT/HCP1 expression in proximal than distal small intestine. In distal small intestine, methotrexate influx rate was low and was not pH dependent. Also, folate and its analogues exerted no significant effect on methotrexate absorption. Conclusions Based on the present and our previous results, the site-specific contributions of various transporters including PCFT/HCP1 in methotrexate intestinal absorption were discussed. The variation in luminal pH and the involvement of multiple transporters in methotrexate absorption may cause variation in oral bioavailability among patients.

Site-specific contribution of proton-coupled folate transporter/haem carrier protein 1 in the intestinal absorption of methotrexate in rats

Methotrexate is reportedly a substrate for proton-coupled folate transporter/haem carrier protein 1 (PCFT/HCP1) and reduced folate carrier 1 (RFC1). In this study, we examined the contribution of PCFT/HCP1 and RFC1 in the intestinal absorption of methotrexate in rats. Western blot analysis was carried out to evaluate the protein levels of PCFT/HCP1 and multidrug resistance-associated protein 2 in brush-border membrane of rat small intestine. Mucosal uptake of methotrexate was studied in the rat everted small intestine and an in-situ intestinal perfusion study of methotrexate was also carried out in rats. In transport studies using everted intestine, the mucosal methotrexate influx rate in proximal intestine at pH 5.5 was significantly greater than that at pH 7.4. Coadministration of folate or its analogues, such as folinate and 5-methyltetrahydrofolate, substrates for both PCFT/HCP1 and RFC1, significantly suppressed the methotrexate influx at pH 5.5, whereas thiamine pyrophosphate, an inhibitor for RFC1 alone, exerted no significant effect. Western blot analysis showed higher PCFT/HCP1 expression in proximal than distal small intestine. In distal small intestine, methotrexate influx rate was low and was not pH dependent. Also, folate and its analogues exerted no significant effect on methotrexate absorption. Based on the present and our previous results, the site-specific contributions of various transporters including PCFT/HCP1 in methotrexate intestinal absorption were discussed. The variation in luminal pH and the involvement of multiple transporters in methotrexate absorption may cause variation in oral bioavailability among patients.

Region-Dependent Role of the Mucous/Glycocalyx Layers in Insulin Permeation Across Rat Small Intestinal Membrane

The regional difference in the contribution of the mucous/glycocalyx layers in rat small intestine, as a diffusional or enzymatic barrier, to the absorption of insulin was investigated by in vitro studies. The mucous/glycocalyx layers from the duodenum, the jejunum, and the ileum in rat were successfully removed without damaging membrane integrity, by exposing them to a hyaluronidase solution in situ. In an in vitro transport experiment, the apparent permeability coefficient (P(app)) of insulin for the hyaluronidase-pretreated group was significantly increased compared to the PBS-pretreated (control) group in all small intestinal regions, and the P(app) of insulin in both PBS- and hyaluronidase-pretreated groups increased in the following order: duodenum < jejunum < ileum. On the other hand, irrespective of small intestinal regions, the P(app) of FD-4 and of antipyrine, respectively the passive para- and transcellular permeation marker, exhibited no significant differences between PBS- and hyaluronidase-pretreated group. In addition, a significant amount of insulin was degraded in the mucous/glycocalyx layers compartment removed by hyaluronidase pretreatment, and the degradation activity in the mucous/glycocalyx layers showed regional differences in the following order: duodenum > jejunum > ileum. These findings suggest that, irrespective of small intestinal regions, the mucous/glycocalyx layers contributed to insulin permeation predominantly as an enzymatic barrier, and not as a diffusional barrier. Furthermore, the variation of the enzymatic activities in the mucous/glycocalyx layers and in the brush-border membrane would be one factor that accounts for the regional differences in the transport of insulin.

Quercetin-3-Glucoside Is Transported by the Glucose Carrier SGLT1 across the Brush Border Membrane of Rat Small Intestine

Quercetin-3-Glucoside Is Transported by the Glucose Carrier SGLT1 across the Brush Border Membrane of Rat Small Intestine

Quercetin-3-Glucoside Is Transported by the Glucose Carrier SGLT1 across the Brush Border Membrane of Rat Small Intestine

In the present study we investigated a possible involvement of the intestinal sodium-dependent glucose transporter (SGLT)1 in the absorption of quercetin-3-glucoside (Q3G). Pieces of rat jejunum or proximal colon were mounted in Ussing-type chambers and incubated under short-circuited conditions. Test flavonols were added to the mucosal or serosal bathing solution (initial concentration, 100 micromol/L) and disappearance from the donor compartment was monitored for 2 h. With jejunal tissue, only 13.6 +/- 3.5% of the initial dose of Q3G was found in the mucosal compartment 2 h after mucosal addition. Simultaneous addition of D-glucose (10 mmol/L) significantly reduced the disappearance of Q3G (remaining concentration, 33.4 +/- 6.9%) as did a Na(+)-free buffer solution containing phloridzin (final mucosal concentration of Q3G, 54.2 +/- 7.7%). In these experiments, disappearance of Q3G was paralleled by the appearance of quercetin in the mucosal solutions. In contrast, D-fructose (10 mmol/L) did not influence the disappearance of Q3G (Na(+)-free conditions). With proximal colon, 78.2 +/- 11.5% of the initial concentration of Q3G was still present in the mucosal solution after 2 h. When added to the serosal side, the concentration of Q3G decreased only slightly (jejunum, 96.1 +/- 2.1%; proximal colon, 90.7 +/- 1.2%). The concentration of rutin did not change after mucosal or serosal addition. Neither transport of intact glycosides nor of free quercetin from the donor into the acceptor compartment was observed under our experimental conditions. Taken together, the results clearly indicate a role of SGLT1 in mucosal uptake of the Q3G.

Na +-dependent d-mannose transport at the apical membrane of rat small intestine and kidney cortex

The presence of a Na +/ d-mannose cotransport activity in brush-border membrane vesicles (BBMV), isolated from either rat small intestine or rat kidney cortex, is examined. In the presence of an electrochemical Na + gradient, but not in its absence, d-mannose was transiently accumulated by the BBMV. d-Mannose uptake into the BBMV was energized by both the electrical membrane potential and the Na + chemical gradient. d-Mannose transport vs. external d-mannose concentration can be described by an equation that represents a superposition of a saturable component and another component that cannot be saturated up to 50 μM d-mannose. d-Mannose uptake was inhibited by d-mannose≫ d-glucose>phlorizin, whereas for α-methyl glucopyranoside the order was d-glucose=phlorizin≫ d-mannose. The initial rate of d-mannose uptake increased as the extravesicular Na + concentration increased, with a Hill coefficient of 1, suggesting that the Na +: d-mannose cotransport stoichiometry is 1:1. It is concluded that both rat intestinal and renal apical membrane have a concentrative, saturable, electrogenic and Na +-dependent d-mannose transport mechanism, which is different from SGLT1.

Tachykinin NK2 receptors: characterization in the rat small intestine by the use of a peptide agonist and a nonpeptide antagonist as radioligands.

The tachykinin NK2 receptors present in membranes of rat small intestine were characterized by means of tachykinin receptor agonists and antagonists, by using the natural agonist, NKA, or the nonpeptide antagonist SR 48968, as radioligands. The affinity of the antagonists was independent of the radioligand used, whereas the NK2 receptor selective agonists showed a different binding profile according to the radioligand. In particular, when using [125I]NKA, NKA and NKA(4-10) bound to a high affinity site, whilst [beta-Ala8]NKA(4-10) and GR 64349 bound to a high and a low affinity site; the high affinity site was still detected in the presence of 100 microM GppNHp which produced a strong inhibition of the specific binding of [125I]NKA. On the other hand, when using [3H]SR 48968, NKA bound to a high affinity site, [beta-Ala8]NKA(4-10) bound to a low affinity site and NKA(4-10) and GR 64349 bound to a high and a low affinity site; in this case, the high affinity site was no longer detected in the presence of 1 microM GppNHp, which did not reduce the specific binding of [3H]SR 48968 to NK2 receptors. We interpreted these data as an indication that in the rat small intestine membranes [125I]NKA labels multiple conformations of G protein-coupled NK2 receptor which are distinguished by the use of selective receptor agonists, but not by peptide or nonpeptide antagonists.

Intestinal alkaline phosphatase can transphosphorylate thiamin to thiamin monophosphate during intestinal transport in the rat.

Intestinal alkaline phosphatase (IAP) purified from calf intestine and IAP present in the brush border membrane of rat small intestine effectively transphosphorylated thiamin (T) to thiamin monophosphate (TMP) using Na2-beta-glycerophosphate or Na2-creatine phosphate as phosphate donors at pH 8.5. TMP production in the brush border membrane was very small and corresponded to 0.001-0.01 percent of the total inorganic phosphate simultaneously released by the enzyme activity. This reaction, however, could account for TMP formation independently from that much more important due to the hydrolysis of thiamin pyrophosphate during T intestinal absorption.

Transport of citrate across the brush border and basolateral membrane of rat small intestine

It was the aim of the present study to investigate the transport of tricarboxylates (citrate, tricarballylate) across the basolateral membrane (BLM) of the small intestine. Experiments were performed using BLM vesicles isolated from the jejunum of rats. For comparison, some experiments with brush border membrane (BBM) vesicles were also performed. Finally, transfer of citrate and tricarballylate across the intestinal wall was investigated using sacs of everted small intestine. Uptake of citrate by BBM vesicles occurs by a Na + gradient-driven transport mechanism specific for tri- and dicarboxylates. The partially protonated forms of citrate seem to be much better transported than the completely dissociated form, since lowering the extravesicular pH from 7.8 to 5.6 resulted in a marked stimulation of Na +-dependent citrate uptake. In contrast to citrate uptake across the BBM, uptake of citrate across the BLM was neither influenced by Na 2+ nor by pH changes. Neither structurally related tri- and dicarboxylates (tricarballylate, succinate) nor other organic and inorganic anions (e.g. lactate, P-aminohippurate, sulfate, chloride, bicarbonate) significantly influenced citrate uptake by BLM vesicles under cis-conditions. Uptake of citrate as a function of the extravesicular substrate concentration was linear over a concentration range from 0.1 to 10mmol/l. Thus, citrate uptake under these conditions seems to be Na +-independent and not to be mediated by a carrier. However, preloading the BLM V with citrate clearly mi/is-stimulated the uptake of citrate and tricarballylate, respectively. Furthermore, citrate significantly inhibited tricarballylate uptake into BLMV preloaded with citrate. These results indicate uptake of tricarboxylates across the BLM by an exchange mechanism. Using sacs of everted small intestine, no transfer of intact citrate against a concentration gradient occurred, but some evidence for metabolization of citrate within the intestinal wall was obtained. In contrast, the non-metabolizable tricarboxylate tricarballylate was significantly accumulated in the serosal compartment of everted intestinal sacs.

A retinyl ester hydrolase activity intrinsic to the brush border membrane of rat small intestine.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A. Hydrolysis of these esters in the lumen of the small intestine is required prior to absorption. Bile salt-stimulated retinyl esterase activity was present with purified rat intestinal brush border membrane, with the maximum rate of ester hydrolysis at approximately pH 8, the physiological luminal pH. Taurocholate, a trihydroxy bile salt, stimulated hydrolysis of short-chain fatty acyl retinyl esters more than hydrolysis of long-chain fatty acyl esters. Deoxycholate, a dihydroxy bile salt, primarily stimulated hydrolysis of long-chain esters. Calculated Kms of 0.74 microM for retinyl palmitate (16:0) hydrolysis and 9.6 microM for retinyl caproate (6:0) hydrolysis suggested the presence of two separate activities. Consistent with that, the activity responsible for retinyl caproate hydrolysis could be inactivated to a greater degree than retinyl palmitate hydrolysis by preincubation of the brush border membrane at 37 degrees C for extended times. Brush border membrane from animals who had undergone common duct ligation 48 h prior to tissue collection showed little ability to hydrolyze retinyl caproate but retained 70% of retinyl palmitate hydrolytic activity, compared to sham-operated controls. Thus, two distinguishable retinyl esterase activities were recovered with purified brush border membranes. One apparently originated from the pancreas, was stimulated by trihydroxy bile salts, and preferentially hydrolyzed short-chain retinyl esters, properties similar to cholesterol ester hydrolase, known to bind to the brush border. The other was intrinsic to the brush border, stimulated by both trihydroxy and dihydroxy bile salts, and preferentially hydrolyzed long-chain retinyl esters, providing the majority of activity of the brush border against dietary retinyl esters.

N-terminal amino acid sequence, immunohistochemical localization and tissue distribution of a plasma membrane protein (Prot17) of rat enterocytes

Prot17, a protein of the basolateral membrane of rat small intestine with a mol.wt. of 17 kDa, can be isolated using a previously described method (Schiechl 1988). It occurs in the membrane as an oligomer with a mol.wt. of 90 kDa. In the present study a polyclonal antibody specific for Prot17 was used to explore by immunohistochemical techniques the tissue distribution of Prot17 and its ultrastructural localization within the cells. Furthermore the amino acid sequence of the N-terminal part of this molecule up to position 17 could be analyzed. The results are summarized as follows: Prot17 is a membrane anchored protein. Its partial amino acid sequence suggests that it is neither identical nor related to other known proteins. Immunofluorescence studies revealed, that it occurs only in epithelial cells. It is mainly found in the absorptive and goblet cells of the intestine and the acinar cells of the pancreas. Smaller quantities are found also in the bile duct epithelium of the liver, in the proximal tubule cells of the kidney and in the cells of the respiratory epithelium. Ultrastructural localization of Prot17 was possible in the intestinal epithelium and pancreas acinar cells. In both cell types it was found in the basolateral and microvillous membrane. In pancreas, Prot17 was also detected in the membrane of the zymogen granules. In the absorptive cells of the intestine Prot17 was found in both the membrane and the contents of subluminal vesicles. Furthermore, in apical granules of secretory cells of the respiratory epithelium binding of Prot17 specific antibody was found in the granular content, the membrane being negative.

Diabetes mellitus and the sodium electrochemical gradient across the brush border membrane of rat intestinal enterocytes.

The effects of chronic diabetes mellitus of 4-5 weeks duration on the potential difference across the brush border membrane of rat small intestine (Vm) and on Na+-dependent uptake of D-glucose by jejunal brush border vesicles have been studied. Diabetes increased Vm in the jejunum from a mean value of -47.2 mV in control tissue to -57.4 mV in diabetic tissue (P less than 0.001) but was without effect on ileal Vm. Measurements of Vm during ion-substitution experiments revealed that the conductance of Na+ of the jejunal brush border was reduced by diabetes, whilst K+ and Cl- permeabilities were unaltered. Uptake studies using brush border vesicles and an Na+-electrochemical gradient showed that diabetes caused a 56% increase in the initial rate of uptake of D-glucose but was without effect on the peak to equilibrium ratio. Taken together, data from these two studies suggest that the lower Na+ permeability of the brush border in diabetes enhances the electrical and chemical driving force for active Na+-dependent uptake of glucose by reducing glucose-independent movement of Na+ across this membrane. Finally, the possible humoral factors involved in this response to diabetes are discussed.

Acute and chronic exposure to ethanol and the electrophysiology of the brush border membrane of rat small intestine.

In this study we have investigated the effects of (a) chronic ethanol intake on glucose and galactose absorption across the rat jejunum in vivo and on the potential difference across the isolated brush border membrane (Vm) and (b) acute exposure to ethanol (4% or 8%) and acetaldehyde (0.25%) on changes in Vm associated with Na(+)-dependent galactose absorption across the jejunum and ileum. Chronic ethanol intake was associated with hyperpolarization of Vm and an enhanced galactose but not glucose transport. Acute ethanol and acetaldehyde were without effect on Vm whether or not galactose was present. We conclude that while a greater electrochemical gradient across the brush border membrane is a likely explanation for the stimulation of galactose absorption induced by ethanol feeding, factors other than changes in Vm are responsible for the inhibitory effects of acute ethanol.

The binding of manganese to the brush-border membrane vesicles of rat small intestine

This study was carried out to elucidate the first step of manganese absorption in small intestine. The uptake of manganese by brush-border membrane vesicles isolated from rat small intestine was studied by rapid filtration technique. Manganese in the vesicles was determined by atomic absorption spectrophotometry using a graphite furnace. The uptake of manganese by brush-border membrane vesicles represented the binding of manganese to them. The binding rate of manganese in the presence of Na + was accelerated compared with the rate in the presence of K +. The binding of manganese had the saturable component (apparent Km: 12μM) and was inhibited by iron, cobalt and zinc. These data suggest that manganese has specific binding sites on the brush-border membranes of rat small intestine.

Identification of proline-specific carboxypeptidase localized to brush border membrane of rat small intestine and its possible role in protein digestion.

A proline-specific carboxypeptidase (carboxypeptidase P, EC 3.4.12.-) was identified and partially characterized in the brush border membrane fraction of rat intestinal enterocytes and shown to be distinct from pancreatic proteases. The carboxypeptidase activity of isolated brush border membranes, with Z-Gly-Pro-Leu as substrate, was 43 nmol/min/mg protein representing a 16-fold purification when compared with mucosal cell homogenates. Activity was maximal in the middle region of the small intestine, and villus cells had twice the activity of crypt cells. Carboxypeptidase activity was maximal at pH 7.0, was stimulated by divalent cations, and was inhibited by metal chelating agents, suggesting that it is a metalloenzyme. The enzyme had the highest activity with synthetic peptides containing proline penultimate to the carboxy terminus. In vivo patterns of hydrolysis and absorption of amino acids from Z-Pro-Trp were examined using an intestinal perfusion technique. These studies indicate that brush border membrane carboxypeptidase may play an important role in the digestion of proline-containing peptides and proteins.

Hydrolysis of alpha-D-glucopyranosyl-1,6-sorbitol and alpha-D-glucopyranosyl-1,6-mannitol by rat intestinal disaccharidases.

The hydrolyzing activities in rat small intestine for newly developed sugar substitutes, alpha-D-glucopyranosyl-1,6-sorbitol (GPS) and alpha-D-glucopyranosyl-1,6-mannitol (GPM), both of which are produced by hydrogenation of palatinose, were characterized. GPS and GPM were hydrolyzed in mucosal homogenate as well as in brush border membranes of rat small intestine at a slower rate than the rate of hydrolysis of palatinose (30%) and sucrose (6-7%). Gel filtration column chromatography of disaccharidases solubilized from brush border membranes revealed that GPS and GPM were hydrolyzed mainly by sucrase-isomaltase complex and its degradation product, i.e. isomaltase monomer. The isomaltase monomer, purified from small intestine of rats, possessed similar Km values for GPS (2.47 mM) and GPM (5.38 mM) as compared to those of purified sucrase-isomaltase complex. The Vmax values of isomaltase monomer for GPS and GPM were twice as high as those of sucrase-isomaltase, suggesting that GPS and GPM are hydrolyzed by the active site of isomaltase. On the other hand, a small amount (up to 17%) of GPS- and GPM-hydrolyzing activities was ascribed to glucoamylase, which possessed relatively high Km values for GPS (18.7 mM) and GPM (32.9 mM). To examine a physiological significance of GPS- and GPM-hydrolyzing activities, the transmural potential difference (delta PD) evoked by Na+-dependent active transport of glucose, produced by the hydrolysis of these disaccharide alcohols, was measured in everted segments of rat jejunum. The relative rates of absorption of glucose produced by the hydrolysis of GPS and GPM were 36% and 27% of that of palatinose, directly reflecting the hydrolyzing activities determined in jejunal homogenate. These results suggest that the process of hydrolysis is the rate limiting step in digestion-absorption process of palatinose, GPS and GPM in small intestine.

Effect of hyperglycemia on d-glucose transport across the brush-border and basolateral membrane of rat small intestine

Experimental hyperglycemia leads to an increase in the capacity of the rat small intestine to absorb glucose. This effect occurs within hours from the onset of hyperglycemia and is thought to involve an induction of glucose transport in the brush-border and/or basolateral membrane of the intestinal epithelium. We devised a protocol for the simultaneous preparation of brush-border vesicles and basolateral vesicles from rat small intestine to determine the locus for the inductioof glucose transporter in hyperglycemic rats. A 6 h period of intravenous infusion with a 30% glucose solution had no effect on the initial rate of glucose uptake across jejunal or ileal brush-border vesicles when measured in the absence of a Na + gradient, suggesting that enhanced glucose uptake is not dependent on an increase in the number of Na +-dependent secondary active glucose transporters in the brush-border. Hyperglycemia did not effect the rate of glucose uptake across ileal basolateral vesicles but did cause a 78% increase in the initial rate of carrier-mediated d-glucose uptake across jejunal basolateral vesicles. The induction of glucose transport in the jejunal basolateral membrane was characterized by a rapid rate of glucose equilibration across the vesicles ( t 1 2 = 46 s sorbitol infused controls, 18 s hyperglycemia) and a 75% increase in the V max for carrier-mediated glucose uptake with no significant change in K t. When the rats were pretreated with cycloheximide prior to intravenous infusion, the initial rate of d-glucose uptake dropped to 13% of that seen in jejunal basolateral vesicles prepared from untreated rats. These results suggest a rapid turnover rate for the Na +-independent glucose transporter in the basolateral membrane of the enterocyte. An increase in the number of functioning glucose transporters in the basolateral membrane may play an important role in the short-term induction of glucose absorption by the jejunum of the hyperglycemic animal.

Changes in D-glucose uptake by brush-border vesicles from small intestine of rats treated with mitomycin C

The effect of mitomycin C pre-administration on the d-glucose transport system in the intestinal brush-border membrane of rat small intestine was examined by a rapid filtration technique. Forty-eight hours following the intravenous administration of mitomycin C, there were extensive and severe mucosal derangements. At this time point, membrane vesicles were prepared from the mitomycin-C-pretreated and control rats. Binding studies indicated that d-glucose entered into the intravesicular space of vesicles even in the case of mitomycin-C-pretreated rats. Vesicles obtained from both the mitomycin-C-pretreated and the control rats showed sodium-dependent uptake of d-glucose, but the initial uptake at 15 sec was significantly greater in control rats than in mitomycin-C-pretreated rats. Comparison of kinetic parameters of d-glucose transport indicated that K m was not significantly different between control and mitomycin-C-pretreated rats. The pretreatment with mitomycin C decreased V max and increased the diffusional permeability to d-glucose considerably. These changes induced by mitomycin C seemed to derive not from a direct effect on mature enterocytes but from an indirect effect secondary to mitotic inhibition in the crypts.

Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. I. The release from rat organs.

From various rat organs, alkaline phosphodiesterase I was liberated by the action of phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis. Especially, a large amount of alkaline phosphodiesterase I was released from slices of small intestine, testis, lung, and kidney, but not from pancreas and liver. The release of the enzyme induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentrations of the phospholipase C and the weight of the slices of small intestine or testis. Furthermore, little enzyme was released from the homogenate of pancreas. These results suggest an important role of phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membranes of rat small intestine and pancreas. The alkaline phosphodiesterase I released from slices of rat small intestine and testis had a molecular weight of about 240,000, and was activated by Mg2+ and Ca2+ but inhibited by EDTA. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9, having the Km values of 0.36 mM (small intestine) and 0.25 mM (testis). The intestinal enzyme differed from the testis enzyme in pI values, thermostability, and Arrhenius plot having a single breakpoint.