Application of wavelet denoising to improve compression efficiency while preserving integrity of digital micrographs.

Modern microscopy methods require efficient image compression techniques owing to collection of up to thousands of images per experiment. Current irreversible techniques such as JPEG and JPEG2000 are not optimized to preserve the integrity of the scientific data as required by 21 CFR part 11. Therefore, to construct an irreversible, yet integrity-preserving compression mechanism, we establish a model of noise as a function of signal in our imaging system. The noise is then removed with a wavelet shrinkage algorithm whose parameters are adapted to local image structure. We ascertain the integrity of the denoised images by measuring changes in spatial and intensity distributions of registered light in the biological images and estimating changes of the effective microscope MTF. We demonstrate that the proposed denoising procedure leads to a decrease in image file size when a reversible JPEG2000 coding is used and provides better fidelity than irreversible JPEG and JPEG2000 at the same compression ratio. We also demonstrate that denoising reduces image artefacts when used as a pre-filtering step prior to irreversible image coding.

Intracrine role of progesterone in follicle-stimulating hormone- and cyclic adenosine 3',5'-monophosphate-induced fibronectin production and deposition by chicken granulosa cells: influence of follicular development.

The role of progesterone in FSH- and 8-bromo-cAMP (8-Br-cAMP)-induced fibronectin production by chicken ovarian granulosa cells was examined. Granulosa cells isolated from the third largest (F3; developing; 15-20 mm in diameter) preovulatory follicle and a pool of immature small yellow follicles (SYF; 6-8 mm in diameter) were incubated in serum-free medium 199, and the total amount of fibronectin produced (deposited, secreted into the medium, and associated with cells) was measured by enzyme-linked immunosorbent assay. Unstimulated F3 cells deposited greater amounts of fibronectin than unstimulated SYF cells. FSH or 8-Br-cAMP significantly increased fibronectin deposition. Similarly, both agents increased the quantity of fibronectin secreted into the medium and that associated with cells. The magnitude of FSH- and 8-Br-cAMP-enhanced fibronectin deposition or secretion into medium by SYF cells was greater than that by F3 cells. Cyanoketone (an inhibitor of progesterone synthesis) significantly suppressed basal fibronectin production by F3 cells, but not that by SYF cells. Cyanoketone completely blocked FSH- or 8-Br-cAMP-induced fibronectin production by F3 cells, but caused only a modest inhibition (nonsignificant) of agonist-induced fibronectin production by SYF cells. Exogenous progesterone completely reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The nondegradable synthetic progestin R5020 also reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The antiprogestin, ZK 98.299, inhibited basal and FSH-stimulated fibronectin production. The data demonstrate that FSH- and cAMP-stimulated fibronectin production by chicken granulosa cells is dependent (at least in part) on de novo progesterone synthesis. Furthermore, they indicate that fibronectin production and deposition by these cells are stimulated by progesterone, perhaps in an intracrine/autocrine manner, and that the role of progesterone increases with advancing stages of follicular development.

Luteinizing hormone-induced intracellular calcium mobilization in granulosa cells: comparison with forskolin and 8-bromo-adenosine 3',5'-monophosphate.

Digitonin-permeabilized avian granulosa cells were used as a model to study the effects of LH, 8-bromo-cAMP (8-Br-cAMP), and forskolin on intracellular calcium mobilization. LH caused a biphasic calcium efflux. There was a small but significant (P less than 0.05) calcium release within 1-2 min (rapid phase) with an ED50 of 35 ng/ml, followed by a larger one after 5 min (slow phase) with an ED50 of 10 ng/ml. The intracellular calcium antagonist TMB-8 inhibited the action of LH in both phases. Forskolin had no effect on calcium efflux during the rapid phase. However, it stimulated intracellular calcium efflux in a dose-dependent manner in the slow phase with an ED50 of 30 microM. Low doses of 8-Br-cAMP caused a small but significant increase in calcium uptake corresponding to the rapid phase, although high concentrations of 8-Br-cAMP caused efflux in the slow phase (ED50, 0.7 mM). Both LH and 8-Br-cAMP mobilized calcium from a nonmitochondrial pool, whereas forskolin induced calcium efflux from a nonendoplasmic reticular site. Apparently receptor-mediated (LH) and nonreceptor-mediated (forskolin and 8-Br-cAMP) intracellular calcium mobilization are qualitatively different, suggesting that rapid intracellular calcium mobilization may represent one of the initial steps in the mechanism of action of LH.

Epidermal growth factor elevates intracellular pH in chicken granulosa cells.

Many bioregulators, such as epidermal growth factor (EGF), induce intracellular alkalinization by activating a membrane bound Na+/H+ antiporter. The present studies were designed to examine the influence of EGF on intracellular pH (pHi) in chicken granulosa cells. pHi in granulosa cells from the two largest preovulatory follicles of hens was determined spectrofluorometrically using the dye 2',7'-bis(carboxyethyl-5(6)-carboxyfluorescein. The resting pHi was 6.81 +/- 0.006 (n = 30) when the extracellular pH and sodium concentration (Na+o) were 7.3 and 144 mM, respectively. EGF (5-100 ng/ml) induced a concentration-dependent increase in pHi, which reached a maximum of 0.217 +/- 0.009 pH units at a concentration of 100 ng/ml EGF. Cytosolic alkalinization was observed within 10 min of the addition of EGF and lasted over the 60 min observation period. The increase in pHi was dependent upon the presence of Na+o, since the EGF effect was attenuated when Na+o was substituted with equimolar concentrations of nonpermeant choline chloride. The EGF-induced pHi change was also inhibited by amiloride, dimethyl amiloride, and ethylisopropyl amiloride, inhibitors of the Na+/H+ antiporter. The alkalinization effect of EGF was mimicked by transforming growth factor-alpha but not by insulin, insulin-like growth factor-I, or transforming growth factor-beta. These studies suggest for the first time that intracellular alkalinization resulting from activation of the Na+/H+ antiporter may be a part of the transmembrane signaling pathway in the action of EGF on chicken granulosa cells.

T- and L-calcium channels in steroid-producing chicken granulosa cells in primary culture.

Steroidogenesis and other functions in granulosa cells are calcium dependent. Using the patch-clamp technique to record single ion channel activity, we have identified for the first time two kinds of calcium channels through which the divalent ion may enter chicken granulosa cells. The cells were maintained in primary culture whose basal and hormone-stimulated progesterone production was evaluated at different times in culture and at different temperatures. A small channel, with a conductance of 4-5 picosiemens (pS), had short openings and inactivated rapidly. A large channel had a conductance of 20-30 pS, a high activation threshold, and slow inactivation kinetics. The dihydropyridine compound Bay K-8644, a L-calcium channel agonist, significantly increased the activity of the large channel, and nifedipine, a dihydropyridine calcium channel blocker, inhibited it completely. Both types of channels were seen in functionally competent signal-responsive granulosa cells cultured for up to 48 h. Whether these channels are involved in steroidogenesis, protein production, and/or secretion remains to be established.

Ionic currents in avian granulosa cells.

Transmembrane ionic currents have been recorded in single granulosa cells from the laying hen using the whole-cell patch-clamp technique. Under voltage-clamp conditions, depolarizing voltage steps evoked currents composed of a fast inactivating inward component and a delayed outward component. The former was activated at voltages more positive than -50 mV and was fully inactivated within 500 ms. It was blocked by D600 (methoxyverapamil) and by cobalt, suggesting that it is a calcium current. The latter displayed inward rectification and did not inactivate during long duration pulses. It was blocked by tetraethylammonium indicating that it is a potassium current. This is the first evidence of the existence of potassium and calcium transmembrane currents in granulosa cells.

Sodium-dependent intracellular pH regulation in granulosa cells of the domestic hen (Gallus domesticus).

The existence and importance of the Na+/H+ exchanger in intracellular pH (pHi) regulation in ovarian cells was studied in acid-loaded avian granulosa cells by monitoring the recovery of normal pHi using a trapped fluorescein derivative as an indicator. The resting pHi of freshly isolated granulosa cells from preovulatory follicles was 6.80 +/- 0.08 when the extracellular pH (pHo) and sodium concentration (Na+o) were 7.3 and 144 mmol/l respectively. While exposure of granulosa cells to high pHo (pHo greater than 7.45) medium shifted the pHi upward with time, incubation of the cells in low pHo (pH less than 6.80) buffer resulted in a significant decrease in pHi. In contrast, pHi remained constant when pHo was varied between the broad range of 6.8-7.4. When the cytoplasm was acidified by treatment with nigericin in choline+ buffer, both the magnitude and rate of recovery of normal pHi was suppressed significantly with decreasing pHo, but increased in high pHo medium. The recovery of pHi was dependent upon the concentration of extracellular sodium, in that the recovery rate and magnitude increased concomitantly with increases in Na+o concentrations, while the recovery was abolished when Na+o was completely replaced with choline+. In addition, the sodium ionophore monensin enhanced the recovery rate of normal pHi in a concentration-dependent manner. This action of monensin was observed only when sodium was present in the incubation medium, indicating that Na+o entry is important for the recovery of normal pHi. Monensin also evoked further cytoplasmic alkalinization in fully recovered cells, with a relative net effect dependent upon the level of Na+o present.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium ionophores increase intracellular pH in chicken granulosa cells.

Several hormone agonists exert their physiological actions by triggering an inositol phospholipid-Ca2+ signalling cascade and cytosolic alkalinization. Although calcium ionophores have been used extensively to probe the role of Ca2+ in the regulation of steroidogenesis in granulosa cells, the precise relationship between changes in intracellular Ca2+ (Ca2+i) and pH (pHi) is unclear. In the present study we have used a fluorescent pH indicator, 2'7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein, to examine the influence of two Ca2+ ionophores, ionomycin and 4-Bromo-A23187 (4-Br-A23187), on pHi in chicken granulosa cells. Chicken granulosa cells from the largest preovulatory follicle were incubated with Ca2+ ionophores (0-2 microM) and/or inhibitors of Na+/H+ antiport (amiloride, dimethylamiloride and ethylisopropyl amiloride; 0.5, 5 and 50 microM respectively) in the presence of Na+ (or choline+; 0-144 mM) and/or Ca2+ (0-10 mM). Ionomycin or 4-Br-A23187 elicited a rapid and sustained cytosolic alkalinization. The magnitude of increase in pHi was dependent on the concentration of the Ca2+ ionophore and the presence of extracellular Ca2+ but independent of extracellular Na+. Pretreatment of the cells with amiloride or its analogues failed to affect the increase in pHi induced by the Ca2+ ionophores significantly. These findings demonstrate that, in addition to their widely reported effects on Ca2+i redistribution in granulosa cells, 4-Br-A23187 and ionomycin cause Ca(2+)-dependent cytosolic alkalinization. This action of the Ca2+ ionophores is independent of the Na+/H+ antiport. Caution must be exercised in using Ca2+ ionophores as probes to define the role of Ca2+ in the regulation of granulosa cell function.

Use of flow cytometry to measure the interaction between Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice.

Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals. Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor. Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old). Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice. 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa. Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Calcium enhances Acanthamoeba polyphaga binding to extracellular matrix proteins.

PURPOSE: To characterize better the ameba-host interactions that may be involved with the pathogenesis of Acanthamoeba keratitis, the role of calcium (Ca2+) on the binding of Acanthamoeba polyphaga to extracellular matrix proteins was examined in vitro. METHODS: The binding of a metabolically labeled A. polyphaga (CDC:0187:1) isolate from a case of human keratitis to collagen type IV, laminin, and fibronectin was assessed through a range of calcium concentrations in the external fluid. Binding to collagen IV was studied in detail, with and without other divalent cations and calcium channel modulators. RESULTS: Calcium increased binding in a dose-dependent manner, with significant effects at 0.1 to 1.0 microM and near-maximal effects at 1 to 100 microM, depending upon the matrix protein. Magnesium alone had no effect on ameba binding to collagen IV but suppressed the action of calcium. Strontium enhanced ameba binding, with maximal effect at 100 microM. The calcium channel antagonists nifedipine and diltiazem-HCl and a calcium channel activator, Bay-K8644, had no effect on the action of calcium. However, the inorganic calcium antagonists, lanthanum and cobalt, suppressed the effect of calcium. CONCLUSION: Low concentrations of calcium enhance the adhesion of A. polyphaga to extracellular matrix proteins. It remains uncertain whether calcium acts intracellularly or at the cell surface.

Acanthamoeba binds to extracellular matrix proteins in vitro.

PURPOSE: To identify host-tissue amoeba interactions that may be important in the pathogenesis of Acanthamoeba keratitis, the ability of the opportunistic pathogen Acanthamoeba polyphaga to bind various components of the extracellular matrix (collagen type IV, laminin, or fibronectin) was examined in vitro. METHODS: A polyphaga, isolated from a case of human amoebic keratitis, was used in the studies. In the experiments, 96-well plates were coated with 0-, 5-, 10-, 20-, or 50-micrograms/ml solutions of the basal lamina proteins laminin or collagen type IV, the extracellular matrix protein fibronectin, or casein (control). Amoeba were metabolically labeled with 35[S]-methionine, and 1x 10(4) labeled amoeba in phosphate buffered saline (PBS) were seeded per well and allowed to bind for 20 min. After washing with PBS, bound amoeba were solubilized with 1% sodium dodecyl sulphate (SDS) and scintillation counting was used to determine the number of bound amoeba. RESULTS: Counts from casein and protein-free controls were not significantly different from each other (P > 0.05). There was a significant increase in the binding of 35[S]-labeled A. polyphaga to collagen IV, laminin, and fibronectin over controls (P < 0.0001) and the binding was concentration-dependent. The rank order of binding was collagen > or = laminin >> fibronectin. Alpha-methyl-mannopyranoside, but not fucose, inhibited binding of labeled A. polyphaga to collagen IV, laminin, and fibronectin in a concentration-dependent manner. CONCLUSION: In summary, the binding assays show that Acanthamoeba bind preferentially to collagen, laminin, and fibronectin, in that order, and that the adherence process is inhibited by mannose.

The effect of kaurenol on steroidogenesis and cyclic adenosine monophosphate production in rat granulosa cells.

The effect of kaurenol (ent-kaur-16-en-15 beta-ol) on steroidogenesis and cyclic AMP production was examined in rat granulosa cells in short-term incubations (6 h). Kaurenol alone significantly augmented the production of progesterone in time- and concentration-dependent manner but attenuated the accumulation of the progesterone metabolite 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P). The steroidogenic effect of kaurenol is due to the acute stimulation of pregnenolone production from endogenous cholesterol and an inhibition in 20 alpha-hydroxysteroid dehydrogenase which catalyzes the metabolism of progesterone to 20 alpha-OH-P. Kaurenol had no appreciable effect on conversion of exogenous pregnenolone to progesterone. Although kaurenol was without effect on basal cyclic AMP generation, it inhibited the actions of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and forskolin on the production of the cyclic nucleotide. Kaurenol also significantly attenuated the LH-, FSH- and forskolin-stimulated progesterone and 20 alpha-OH-P production in a concentration-dependent manner. Because kaurenol induced steroidogenesis without increased cyclic AMP accumulation, it is concluded that its action on basal steroidogenesis is mediated by a mechanism independent of the cyclic nucleotide. Kaurenol may serve as a useful tool for elucidating cyclic AMP-independent action(s) of hormones in intact tissue/cells.

Bovine serum albumin enhances calcium currents in chicken granulosa cells.

The effect of bovine serum albumin (BSA) on Ca2+ currents in chicken granulosa cells was examined using both the nystatin-perforated and the conventional whole cell patch clamp techniques. Under voltage-clamp conditions, depolarizing voltage steps evoked inward Ca2+ currents with both methods. The time- and voltage-dependence of Ca2+ currents measured with the perforated patch technique was similar to those obtained with conventional whole cell recording. Commercially prepared BSA and essentially fatty acid free BSA both rapidly enhanced the amplitude of Ca2+ currents. However, the fatty acid free BSA was more potent, and its potency was greatly reduced by incubation with saturating concentrations of oleic acid. These data show that BSA, a common constituent of incubation media, can influence ion channels in the plasma membrane of granulosa cells.

Progesterone stimulates fibronectin production by chicken granulosa cells in vitro.

Experiments were conducted in vitro to examine the effect of progesterone on fibronectin production by chicken ovarian granulosa cells. Granulosa cells isolated from the largest (F1; mature) and third-largest (F3; developing) preovulatory follicles as well as form a pool of immature small yellow follicles (SYF) of the domestic chicken ovary were incubated in serum-free Medium-199 and the amounts of fibronectin and progesterone produced were quantified by enzyme-linked immunosorbent assay and radioimmunoassay respectively. The amounts of basal fibronectin and progesterone produced by granulosa cells from F1, F3 and SYF follicles increased with advancing stages of follicular development. Thus, the quantity of basal fibronectin secreted by granulosa cells was directly proportional to the amount of progesterone produced by them. Exogenously supplied progesterone increased the amount of fibronectin secreted by F1 and F3 cells in a dose-dependent manner, but its effect on SYF cells was marginal. Cyanoketone (an inhibitor of progesterone synthesis) suppressed basal fibronectin production by F1 and F3 granulosa cells and its inhibitory action was reversed by exogenous progesterone. The progesterone antagonist RU 486 also attenuated basal fibronectin production by F1 and F3 granulosa cells, but only the highest concentration affected SYF cells. The inhibitory effect of RU 486 was diminished in the presence of exogenous progesterone. These data show that progesterone regulates fibronectin production by chicken granulosa cells. They suggest that in avian granulosa cells, endogenous progesterone can stimulate fibronectin synthesis in an intracrine or autocrine manner.

Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450SCC.

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450SCC (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F1) second (F2) and third (F3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450SCC generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F1 than in samples from F2 or F3. In cells, LH added simultaneously with the P-450SCC substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2-3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450SCC substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450SCC substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorogenic for the substrate P-450SCC enzyme. In granulosa cells that were preloaded with the P-450SCC substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.

Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves.

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

Characterization of brush border membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine.

This study was conducted to characterize enterocyte apical membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine. Duodenal, jejunal, and distal ileal segments were isolated from three 26-kg pigs and enterocyte brush border membrane, enriched between 19- and 24-fold in sucrase specific activity, was prepared by Mg(2+) precipitation and differential centrifugation. With P-nitrophenyl phosphate as substrate, the optimum pH for porcine brush border membrane-bound alkaline phosphatase activity was defined to be 10.5 for all three segments. At the optimal pH, the kinetics of membrane-bound alkaline phosphatase were determined for the three intestinal segments. The affinity of this enzyme (K(m), mM) in the jejunum (0.64 +/- 0.07) was four times greater than that in the duodenum (2.75 +/- 0.59) and the distal ileum (2.71 +/- 1.14). These results indicate that different isomers of membrane-bound alkaline phosphatase might have been expressed in different segments of porcine small intestine. The maximal specific activity (V(max), micromol/mg protein . min) of this enzyme was highest in the duodenal (7.74 +/- 0.95), intermediate in the jejunal (4.31 +/- 0.18), and lowest in the distal ileal (3.53 +/- 0.84) brush border membrane. Therefore, the maximal specific activity of brush border membrane-bound alkaline phosphatase along the intestinal longitudinal axis in growing pigs decreases from the duodenum toward the distal ileum.

Endothelial-like cells from the bovine placental cotyledon.

A cell-line was established from bovine placental cotyledon. When cultured in M199 with 10% fetal bovine serum, this cell-line had a doubling time of about 18 h. With immunohistochemistry, it was demonstrated that this cell-line expressed vimentin and angiotensin-converting enzyme (ACE). While both molecules are expressed in endothelial cells, ACE is usually considered to be a specific marker for endothelial cells. Furthermore, cells were shown to take up Dil-Ac-LDL (acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3'-tetramethylindo-carbocyanine perchlorate). This characteristic feature has been used to identify endothelial cells. Finally, when cultured on matrigel, this cell-line formed tube-like structures similar to those formed by endothelial cells. Tube-formation on matrigel is a physiological property specific to endothelial cells. In conclusion, these three lines of evidence strongly suggest that this cell-line is endothelial cell in nature. Further studies using an endothelial cell-line from bovine placenta may help to elucidate the cause of bovine placental retention, a major cause for economic loss in bovine industry. Furthermore, an endothelial cell-line could be an important tool in research areas such as tissue remodeling, angiogenesis, and cancer.

Estimation of apparent L-amino acid diffusion in porcine jejunal enterocyte brush border membrane vesicles.

There is an overlap of carrier-mediated L-amino acid transport and apparent simple diffusion when measured in intestinal brush border membrane vesicles. Using L-threonine and L-glutamine as representative amino acids, this study was undertaken to estimate apparent simple diffusion of L-amino acids and to establish the effective dosage of HgCl2 for completely blocking carrier-mediated L-amino acid transport in porcine jejunal enterocyte brush border membrane vesicles. Jejunal mucosa was scraped from three pigs weighing 26 kg. Enterocyte brush border membrane vesicles, with an average enrichment of 24-fold in sucrase specific activity, were prepared by Mg2+-precipitation and differential centrifugation. In vitro uptake was measured by the fast filtration manual procedure. HgCl2 blocked the carrier-mediated initial transport of L-threonine and L-glutamine under Na+-gradient condition in a dose-dependent manner. At the minimal concentration of 0.165 micromol HgCl2 mg(-1) protein, carrier-mediated L-threonine and L-glutamine transport was completely inhibited. The apparent L-threonine and L-glutamine diffusion was estimated to be 8.6+/-0.7 and 12.4+/-1.0% of the total uptake at the substrate concentrations of 5 microM (L-threonine) and 50 microM (L-glutamine). Therefore, the treatment of porcine brush border membrane vesicles with a minimum of 0.165 micromol HgCl2 mg(-1) protein completely blocks carrier-mediated L-amino acid transport and enables the direct estimation of apparent L-amino acid diffusion in enterocyte brush border membrane vesicles.

Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles.

With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1.9 and 9.4 mM), respectively. Substrate concentrations affected the duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.