Protein fractionation byproduct from canola meal for dairy cattle.

Fiber-protein is a byproduct arising from a process for fractionating high-quality protein from canola meal. The objective of this study was to evaluate the fiber-protein fraction by examining the chemical profiles, rumen degradation, and intestinal digestive characteristics and determining the nutritive value of the fiber-protein fraction as dietary components for dairy cattle in comparison with commercial canola meal and soybean meal. Available energy values were estimated based on National Research Council guidelines, whereas total true protein content potentially absorbable in the small intestine (DVE) were predicted using the predicted DVE/degraded protein balance (OEB) model. The results show that fiber-protein was a highly fibrous material [neutral detergent fiber (NDF): 556; acid detergent fiber (ADF): 463; acid detergent lignin: 241 g/kg of dry matter (DM)] compared with canola meal (NDF: 254; ADF: 212; acid detergent lignin: 90 g/kg of DM) due to the presence of a higher level of seed hulls in fiber-protein. Compared with canola meal, fiber-protein contained 90 g/kg of DM less crude protein (CP), 25% of which consisted of undegradable acid detergent-insoluble CP. Most of the ruminally undegradable nutrient components present in canola meal appeared to be concentrated into fiber-protein during the manufacturing process and, as a result, fiber-protein showed a consistently lower effective degradability of DM, organic matter, CP, NDF, and ADF compared with both canola meal and soybean meal. Available energy content in fiber-protein contained two-thirds of that of canola meal. The DVE was one-third that of soybean meal and one-fifth that of canola meal [DVE value: 58 vs. 180 (soybean) and 291 g/kg of DM (canola meal)]. The OEB value of fiber protein was positive and about half of that of soybean and canola meal [OEB value: 74 vs. 162 (soybean) and 137 g/kg of DM (canola meal)]. Fiber-protein can be considered as a secondary source of protein in ruminant feed.

Absorption of methionine and 2-hydroxy-4-methylthiobutoanic acid in conventional and germ-free chickens.

The apparent absorption of 3H-labeled L-Met and L-2-hydroxy-4-methylthiobutoanic acid (MHA-FA) was compared in germ-free and conventional broiler chickens to determine the effect of intestinal bacteria on the absorption of Met and MHA-FA. The two diets contained 0.236% of added Met or MHA-FA. Nineteen germ-free birds were maintained in two isolators and fed diets that had been sterilized by gamma irradiation (50 kilogreys). Nineteen conventional birds were reared in batteries and received nonirradiated feed. Diets were fed ad libitum for 3 wk. On d 21 of the experiment, the birds fasted overnight and were refed the experimental diets to which 1.11 x 10(7) Bq of 1-[methyl3H]MHA-FA or 1-[methyl3H]Met/kg of feed had been added. 51CrCl3 (1.11 x 10(7) Bq/kg of feed) was added as an indigestible marker. After 3 h the birds were euthanized, and their intestinal tracts were removed and partitioned into six sections. Residual Met and MHA-FA in digesta were calculated as the ratio of 3H:51Cr in each sample divided by the ratio of 3H:51Cr in the feed. The residual MHA-FA in the distal ileum of germ-free broilers was lower than in conventional birds (4.7 and 10.2% respectively; P < 0.05). In contrast the residual Met in the distal ileum of germ-free broilers was not different than in conventional birds (3.0 and 3.7% respectively; P > 0.05). This study demonstrates that intestinal bacteria significantly reduce the apparent absorption of MHA-FA from the intestinal tract of broiler chickens.

Stability of porcine and microbial lipases to conditions that approximate the proventriculus of young birds.

In vitro experiments were conducted to characterize the activity and the stability of lipase from animal (crude porcine, CPL; lyophilized porcine, LPL), fungal (Rhizopus arrhizus, RAL; Aspergillus niger, ANL), and bacterial (two Pseudomonas spp., PL1, PL2; and Chromobacterium viscosum, CVL) sources when exposed to conditions associated with the glandular stomach. Activity was measured at pH 3 to 8, 40 C and then monitored in response to temperature (40 C), time of exposure (0 and 30 min), pH (3 and 7), and pepsin level (5, 50, and 500 U/mL). All lipases except ANL and CVL had maximum activity at pH 7 to 8. The optimal pH for ANL and CVL were 5 and 6 to 8, respectively. Exposure of lipases to 40 C and pH 7 for 30 min reduced the activity of all lipases except ANL. In contrast, 40 C increased ANL activity 2.5-fold. Although activity of all lipases was reduced by exposure to pH 3, it was nearly eliminated for CPL and LPL. Pepsin concentration had only minor effects on lipase activity and then only at high concentration. The results demonstrate that bacterial lipases (PL1, PL2, and CVL) and ANL are more stable under conditions that approximate the glandular stomach and may explain why dietary porcine lipase has been ineffective in preventing fat malabsorption in previous in vivo studies.

Stability of porcine and microbial lipases to conditions that approximate the small intestine of young birds.

In vitro experiments were conducted to study the stability of lipase activities from bacterial, fungal, and animal sources under conditions that approximate the small intestine. In the first experiment, the effects of preincubation with trypsin (500, 1,000, and 2,000 U/mL), chymotrypsin (200, 400, and 800 U/mL), and trypsin plus chymotrypsin (TC; 2,000 U/mL trypsin + 800 U/inL chymotrypsin) for 30 min at 40 C, on lipase activities from sources of Pseudomonas spp. (PL1, PL2), Chromobacterium viscosum (CVL), and Aspergillus niger (ANL) were determined. None of the enzymes were inhibited by trypsin. The chymotrypsin decreased the activity of all of the lipases. The TC had no additional negative effect on the activities of PL1 and PL2; however, ANL and CVL activities were further decreased relative to the chymotrypsin only treatment. In the second study, the effects of Na taurodeoxycholate (0.1 to 16 mM) on the activities of PL1, PL2, CVL, ANL, and crude porcine lipase (CPL) at 23 and 40 C were evaluated. At 23 C, in order of potency, Na taurodeoxycholate inhibited the activities of ANL, CPL, and CVL. At this temperature, Na taurodeoxycholate did not inhibit PL1 and PL2. An increase in the temperature to 40 C increased the activity of all of the enzymes tested. At 40 C, Na taurodeoxycholate had similar effects on lipase activities; however, higher Na taurodeoxycholate levels were required to inhibit ANL activity, and only a partial inhibition of CPL occurred. At 23 C, porcine colipase restored the activity of CPL but had no effect on ANL and CVL in the presence of inhibitory levels of Na taurodeoxycholate. At 40 C, porcine colipase had no effect on Na taurodeoxycholate inhibition of lipase activity. The results of this study indicate that PL is more stable than CVL and ANL, and that colipase addition has no beneficial effects on microbial lipase activities under conditions that approximate the avian small intestine.

Phytase activity in the small intestinal brush border membrane of the chicken.

The kinetics, mineral dependency, and pH dependency of phytate hydrolysis by preparations of chicken small intestinal brush border membrane vesicles were determined. Substantial phytate hydrolysis occurred over the pH range from 5 to 6.5 with a maximum hydrolysis at pH of 6. Inclusion of 25 mM MgCl2 in the media doubled the rate of phytate hydrolysis. The brush border was shown to have no nonspecific acid phosphatase activity and excess phytate had no effect on alkaline phosphatase activity at pH 11. Under optimal conditions of pH 6 plus 25 mM MgCl2, a kinetic model of a single Michaelis-Menten type of enzymatic activity with a Km of 0.160 +/- 0.008 mM and a Vmax of 42.5 +/- 1.0 nmol/mg vesicle protein per min plus a small unsaturable component converged to the data (P < 0.05). The specific and total activities of intestinal brush border phytase were highest in the duodenum (P < 0.05) and decreased progressively down the length of the gut. Intestinal brush border vesicles prepared from broiler chicks and mature laying hens had comparable specific phytase activity. However, the total activity of brush border phytase was 35% higher in the small intestine of laying hens (P < 0.05). Intestinal brush border phytase could contribute to phytate-phosphorus digestibility and may be subject to regulation in response to the dietary phosphorus and vitamin D status of the chicken.

Pre-steady-state and steady-state function of the ileal brush border SO4(2-)-OH- exchanger.

Rapid-sampling analysis of the detailed time course of 35SO4(2-) uptake under pH-gradient (pHin = 7.5; pHout = 5.5) conditions converged to a model of an initial burstlike pre-steady-state with relaxation to linear steady-state uptake across pig ileal brush border vesicles. A model of low affinity transport (K(m) = 7.7 mM) plus an unsaturable component described the steady-state component of 35SO4(2-) uptake. Steady-state transport was maximal under pH-gradient conditions and sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Significant steady-state 35SO4(2-) transport sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was found under acidic (pHin = pHout = 5.5) and neutral (pHin = pHout = 7.5) pH-equilibrated conditions. Varying conditions from pH gradient to neutral pH equilibrated had no effect on the amplitude of the pre-steady-state burst or on the time constant for relaxation from pre-steady-state to steady-state conditions. However, maximal amplitude was obtained under acidic pH-equilibrated conditions. These results are incorporated into a model for the low affinity ileal brush border SO4(2-)-OH- exchanger whereby, under 0-trans (0 internal substrate concentration) conditions, translocation of inner-facing to outer-facing conformations defines the overall rate-limiting step of the transporter. Provision for slippage of the unloaded carrier could account for 35SO4(2-) transport under acidic pH-equilibrated conditions.

Methionine and 2-hydroxy-4-methylthiobutanoic acid are partially converted to nonabsorbed compounds during passage through the small intestine and heat exposure does not affect small intestinal absorption of methionine sources in broiler chicks.

Broiler chicks were fed diets supplemented with DL-methionine or DL-2-hydroxy-4-methyl-thiobutanoic acid. At 4 wk of age the chicks were subdivided into thermoneutral (22 degrees C) and heat-exposed (32 degrees C) groups and maintained under these conditions for 48 h. Highly purified 3H-L-methionine (3H-L-Met) and 3H-L-2-hydroxy-4-methyl-thiobutanoic acid (3H-L-HMB) were used to evaluate treatment effects on the small intestinal passage of sources of supplemental methionine and on the transport of methionine sources across purified small intestinal brush border vesicles. 3H-L-Met was efficiently absorbed in the upper regions of the small intestine; however, 2.5-3.5% of dietary 3H from birds fed 3H-L-Met remained unabsorbed in the distal small intestine. Dietary 3H (15%) initially associated with 3H-L-HMB was not absorbed during passage down the length of the gut. The HPLC analysis indicated that only 10% of the radiolabeled material remaining in the terminal ileum eluted at the time expected for HMB. Partial breakdown of HMB to nonabsorbed, nonmethionine products during passage down the small intestine may contribute to the difference in biopotency of the two sources of supplemental dietary methionine. Heat exposure did not affect in vivo small intestinal passage or in vitro transport of 3H-L-Met and 3H-L-HMB across small intestinal brush border membrane vesicles.

Methionine and 2-hydroxy-4-methylthiobutanoic acid are transported by distinct Na(+)-dependent and H(+)-dependent systems in the brush border membrane of the chick intestinal epithelium.

The pathways that facilitate the uptake of L-methionine (L-Met), D-methionine (D-Met) and L-2-hydroxy-4-methylthiobutanoic acid (L-HMB) were characterized in chick intestinal brush border membrane vesicles. A model of high affinity transport (Km approximating 0.1 mmol/L) converged to the data obtained for 35S-L-Met uptake under Na(+)-gradient and Na(+)-free conditions. The maximal velocity of 35S-L-Met transport was almost threefold greater in the presence of a Na(+)-gradient. Concentrations (100 mmol/L) of D-Met, the L-isomers of neutral amino acids and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid completely inhibited 35S-L-Met transport. A model of low affinity competitive inhibition (Ki approximately 17 mmol/L) described D-Met inhibition of 35S-L-Met transport. These data indicate that L- and D-Met are transported by a broad specificity system B type transporter. Maximal rates of 3H-L-HMB uptake were obtained under conditions of an internally directed H(+)-gradient (pHin = 7.5, pHout = 5.5). A model of intermediate affinity transport (Km approximately 1 mmol/L) described H(+)-dependent 3H-L-HMB uptake across the vesicles. The data from this and other studies indicate that a H(+)-dependent, nonstereo-specific system facilitates the uptake of the hydroxy analogues of linear amino acids, including HMB.

The influence of the mineral level in drinking water and the thermal environment on the performance and intestinal fluid flux of newly-weaned pigs.

The effects of drinking water containing high levels of dissolved minerals including sulphate (HMW) and a chilled environment on the performance of newly-weaned pigs were evaluated in three replicate 10-d trials. In each trial, 12, 28-d-old pigs were taken from the sow and allocated by weight and litter to treatment groups following a 2 x 2 factorial arrangement of HMW vs low-mineral drinking water (LMW) and normal (heat lamp) vs chilled (21 degrees C) pen temperature. No interactive effects of water mineral level and pen temperature on any of the measurements of health and productivity were found. Pigs given the HMW consumed more water on d 7 to 10 and 1 to 10 (P < .05) and more feed from d 4 to 6, 7 to 10, and 1 to 10 (P < .05), had greater weight gains from d 7 to 10 and 1 to 10 (P < .05), and had higher scour scores on d 4 and 7 (P < .05). Pigs maintained in a chilled environment had lower body weights on d 3, 6, and 10 (P < .05), lower feed conversion efficiency from d 7 to 10 (P < .05) and 1 to 10 (P < .05) and 7 to 10 (P < .01). Pen temperature had no effect on feed intake and scour scores. There was a correlation (P < .05) between feed intake and growth rates throughout the trial, between feed intake and water intake on d 4 to 6, 7 to 10, and 1 to 10, and between water intake and growth rate on d 7 to 10.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterotropic effects of dipolar amino acids on the activity of the anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles.

D-Aspartic acid was used as a specific substrate to evaluate the effects of dipolar amino acids on the high affinity anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles. At pH 6, increasing L-phenylalanine concentrations caused a saturable activation of 0.05 mM D-aspartic acid uptake (Ka = 2.4 mM), and a saturating concentration of effector increased the Vmax of transport (2.6-fold) without any significant effect on the Km. At pH 8, however, a complex activation/inhibition curve was obtained with increasing L-phenylalanine concentrations, and a saturating concentration of effector increased both the Vmax (1.5-fold) and Km (2.1-fold) for transport. Increasing concentrations of L-valine, L-isoleucine, L-methionine, and L-threonine also showed complex activation/inhibition curves of D-aspartic acid uptake at both pH 6 and 8. The maximum level of activation, the plateau reached at saturating concentrations, and the concentration of effector producing either maximum activation or inhibition were, however, different at these two pH values. By using an optimum concentration of 10 mM L-valine at pH 6, the absence of trans-activation and of further activation by a cis-gradient of effector could be demonstrated. These results suggest that two allosteric sites directly accessible from the external medium are responsible for the heterotropic activation of intestinal system X-AG by dipolar amino acids and that, under physiological conditions, such effects might compensate for the lack of specificity of the neutral brush-border system.

L-threonine transport in pig jejunal brush border membrane vesicles. Functional characterization of the unique system B in the intestinal epithelium.

Uptake and inhibitory kinetics of [3H]L-threonine were evaluated in preparations of pig jejunal brush border membrane vesicles. Uptake of [3H]L-threonine under O-trans, Na+ gradient, and O-trans, Na(+)-free conditions was best described by high affinity transport (Km < 0.01 mM) plus a nonsaturable component. The maximal velocity of transport was 3-fold greater under Na+ gradient conditions. 100 mM concentrations of all of the dipolar amino acids and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid caused complete inhibition of [3H]L-threonine transport under Na+ gradient and Na(+)-free conditions. Imino acids, anionic amino acids, cationic amino acids, and methylamino-isobutyric acid caused significant partial inhibition of L-threonine uptake. Inhibitor concentration profiles for proline and lysine were consistent with low affinity competitive inhibition. The Ki values of alanine and phenylalanine approximated 0.2 and 0.5 mM, respectively, under both Na+ gradient and Na(+)-free conditions. These data indicate that the transport system available for L-threonine in the intestinal brush border membrane (system B) is functionally distinct from other amino acid transport systems. Comparison of kinetics parameters in the presence and absence of a Na+ gradient suggests that both partially and fully loaded forms of the carrier can function to translocate substrate and that Na+ serves to accelerate L-threonine transport by a mechanism that does not involve enhanced substrate binding.

pH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush border membrane vesicles.

Initial rates of Na(+)-dependent L-glutamic and D-aspartic acid uptake were determined at various substrate concentrations using a fast sampling, rapid filtration apparatus, and the resulting data were analyzed by nonlinear computer fitting to various transport models. At pH 6.0, L-glutamic acid transport was best accounted for by the presence of both high (Km = 61 microM) and low (Km = 7.0 mM) affinity pathways, whereas D-aspartic acid transport was restricted to a single high affinity route (Km = 80 microM). Excess D-aspartic acid and L-phenylalanine served to isolate L-glutamic acid flux through the remaining low and high affinity systems, respectively. Inhibition studies of other amino acids and analogs allowed us to identify the high affinity pathway as the X-AG system and the low affinity one as the intestinal NBB system. The pH dependences of the high and low affinity pathways of L-glutamic acid transport also allowed us to establish some relationship between the NBB and the more classical ASC system. Finally, these studies also revealed a heterotropic activation of the intestinal X-AG transport system by all neutral amino acids but glycine through an apparent activation of Vmax.

Improved stability of rabbit and rat intestinal brush border membrane vesicles using phospholipase inhibitors.

The initial rates of Na(+)-dependent D-aspartate and D-glucose uptakes were shown to decline from the time of resuspension of brush border membrane vesicles isolated from rabbit and rat jejunum by standard divalent cation precipitation procedures. The former were however more stable than the latter and followed quite closely the decrease in the intravesicular volume, thus suggesting that the loss of transport activity may involve both nonspecific opening of the vesicles and either direct or indirect specific inactivation of the transporters. Uptake rates for both substrates did tend to stabilize at 6-24 h from resuspension, however this final 'next day' uptake activity was too low to be of practical use in kinetic studies. Freezing aliquots of rabbit jejunal vesicles in liquid N2 until the time of assay resulted in complete stabilization of D-glucose uptake. A modified homogenate buffer designed to inhibit a broad spectrum of phospholipase activities resulted in a partial stabilization of glucose transport by rabbit jejunal vesicles with, on average, an over 6-fold enrichment in the 'next day' stable specific activity of uptake as compared to unfrozen vesicles. The modified homogenate buffer also improved the stability and the 'next day' specific activities of D-glucose uptake in rat jejunal brush border vesicles and D-aspartic acid uptake in rabbit jejunal vesicles. It also completely stabilized the intravesicular volume in the latter preparation. An evaluation of the kinetic parameters of Na(+)-dependent D-glucose transport in rabbit vesicles prepared from either the standard homogenate media and frozen in liquid N2 or the modified media and allowed to stabilize overnight, revealed a single transport system with a Km of 0.31-0.32 mM as the best model to fit the data. As such the modifications to the homogenate media do not appear to effect the functional properties of D-glucose transport in the membrane. While being less efficient in stabilizing the vesicles than the rapid freezing protocol, it is shown that the modified homogenate should however be preferred when dealing with slowly permeant ions like choline since it provides in this case the only alternative to reliable measurement of uptake rates across a stable and equilibrated vesicle preparation.

Rapid regulation of D-glucose transport in basolateral membrane of rat jejunum.

D-Glucose transport and D-glucose inhibitable [3H]cytochalasin B binding to jejunal basolateral membrane vesicles were measured to investigate the possible association between changes in transport activity seen in hyperglycemia and density of transporter sites. Comparison was made between hyperglycemic animals, noninfused rats, and a group infused with sorbitol. Vascular infusion of D-glucose produced a rapid increase in D-glucose transport followed by a delayed and smaller increase in [3H]cytochalasin B binding. The Vmax for glucose uptake was increased after only 30 min of glucose infusion and continued to rise up to 6 h. Comparison with noninfused and sorbitol-infused controls showed that 2 h of glucose infusion produced a 3.5-fold increase in the Vmax for D-glucose uptake while D-glucose-inhibitable binding of [3H]cytochalasin B was unaffected. Six hours of hyperglycemia resulted in the further stimulation of glucose transport (4.1-fold) and a significant 1.8-fold increase in cytochalasin B binding over that for noninfused animals. Vesicles prepared from animals 4 h after an in vivo injection of cycloheximide showed an 80% reduction in glucose transport with no significant change in the cytochalasin B binding density. These results suggest that D-glucose transport in the basolateral membrane is regulated by a combination of a modulation of carriers already in the membrane and subsequent changes in carrier site density.

Calcium transport affinity, ion competition and cholera toxin effects on cytosolic Ca concentration.

The physiological relevance of an apparent ionophore activity of cholera toxin towards Ca2+ has been examined in several different systems designed to measure affinity, specificity, rates of ion transfer, and effects on intracellular ion concentrations. Half-maximal transfer rates across porcine jejunal brush-border vesicles were obtained at a concentration of 0.20 microM Ca2+. When examined in the presence of competing ions the transfer process was blocked by very low concentrations of La3+ or Cd2+, Sr2+, Ba2+ and Mg2+ were relatively inefficient competitors for Ca2+ transport mediated by cholera toxin. The relative affinities observed would be compatible with a selectivity for Ca2+ transfer at physiological ion concentrations, as well as an inhibition of this ionophore activity by recognized antagonists of cholera toxin such as lanthanum ions. Entry rates of Ca2+ into brush-border vesicles exposed to cholera toxin were large enough to accelerate the collapse of a Ca2+ gradient generated by endogenous Ca, Mg-ATPase activity. The treatment of isolated jejunal enterocytes with cholera toxin caused a significant elevation in cytosolic Ca2+ concentrations as measured by Quin-2 fluorescence. This effect was specifically prevented by prior exposure of the cholera toxin to excess ganglioside GM1. We conclude that cholera toxin has many of the properties required for promoting transmembranes Ca2+ movement in membrane vesicles and appears to be an effective Ca2+ ionophore in isolated mammalian cells.

The Na+-independent D-glucose transporter in the enterocyte basolateral membrane: orientation and cytochalasin B binding characteristics.

Phloridzin-insensitive, Na+-independent D-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed a Kt of 48 mM and a Vmax of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lower Kt of 23 mM and a Vmax of 0.20 nmol glucose/mg protein/sec. D-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. The Ki for cytochalasin B as an inhibitor of glucose transport was 0.11 microM and the KD for binding to the carrier was 0.08 microM. D-glucose-sensitive sensitive binding of cytochalasin B to the membrane preparation was maximized with L- and D-glucose concentrations of 1.25 M. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na+-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and the Kt for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.

A novel imino-acid carrier in the enterocyte basolateral membrane.

Basolateral membrane vesicles prepared from rat small intestinal epithelial cells were used to study the sodium-independent transport of L-proline. The uptake of L-proline was unaffected by the presence of sodium and showed saturation kinetics (Kt = 0.5 mM and Vmax = 23.3 pmol/mg protein per s). Competition experiments indicated that other amino acids had an affinity for the carrier system with L-leucine greater than L-alanine greater than sarcosine greater than glycine greater than L-lysine greater than OH-proline greater than taurine greater than beta-alanine greater than D-alanine greater than D-proline greater than L-serine greater than phenylalanine greater than valine greater than D-serine greater than phenylalanine greater than valine greater than D-serine greater than MeAIB greater than methionine greater than threonine. This pathway does not resemble those previously described either in the brush-border membrane of intestinal epithelial cells or the plasma membrane of other cell types. The lack of effect of methionine and threonine indicate that proline is not using the L-type system, while the very low affinity for MeAIB and the Na+ independence suggest that this is a novel system for imino acids. The relatively high capacity of this system and its low Kt, which is almost identical to the proline system in the brush-border membrane, strongly suggest that this is an important pathway in the final step for proline absorption by the small intestine.

Use of calcium depletion and chlorpromazine to study calcium dependence of secretory detergent action in the colon.

The role of Ca2+ in the in situ secretory response of rat colon and pig ileum was studied by chelation depletion of Ca2+ and by treatment with chlorpromazine. The effect of depleting lumenal Ca2+ by chelation and the effect of intraperitoneal administration of chlorpromazine were determined relative to colonic permeability and net fluid flux measured across the rat colon or pig ileum. Replacement of Ca2+ in the perfusate by 1.0 mM ethyleneglycol-(bis-beta-ethylaminoether) (EGTA) did not produce significant changes in the net absorptive fluid flux measured in the control state or in the net secretory fluid flux caused by secretory detergent agents. The concentration of EGTA used in the perfusate did not alter mucosal permeability. Nonsecretory bile acids or A23187 had no effect on net colonic fluid flux or on colonic permeability to mannitol in the rat. The known correlation between net fluid flux and increased colonic permeability to polar molecules has been confirmed for the secretory detergent compounds. Chlorpromazine pretreatment caused a partial reversal of net secretory fluid fluxes induced by deoxycholate and high concentrations (6.0 mM) of ricinolate and dioctyl sulfosuccinate without significantly altering mucosal permeability to mannitol. We conclude that depletion of lumenal Ca2+ is not an effective method for determining the possible Ca2+ dependence of these intestinal secretory events. The antisecretory actions of chlorpromazine may provide some indirect evidence for Ca2+ involvement in the secretory effects of the detergent class of laxative compounds. Permeability may be essential for secretion caused by these agents, but the driving force would appear to be provided by the active transfer of electrolytes from the blood to the lumen of the colon.

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 induction of 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 Vmax for carrier-mediated glucose uptake with no significant change in Kt. 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.

Cholera toxin facilitates calcium transport in jejunal brush border vesicles.

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1. This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.