Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

Cysteine as a system-specific substrate for transport system ASC in rat hepatocytes.

The rapid transport of $\text{L}$-cysteine into isolated rat hepatocytes escapes detectable inhibition by 2-(methylamino)-isobutyric acid at levels up to 50 mM. The system transporting cysteine instead is convincingly similar to the $\text{ASC}$ system described for the Ehrlich cell in structural and steric specificity and in pH sensitivity. The Na⁺-dependent uptake of 2-aminoisobutyric acid is almost evenly divided between Systems $\text{A}$ and $\text{ASC}$, showing better accommodation of its two α-methyl groups by $\text{ASC}$ than in the Ehrlich cell. The hepatocyte $\text{ASC}$ system tolerates Li⁺-for-Na⁺ substitution better than does System $\text{A}$, although the tolerance depends on amino acid structure. Adaptive regulation and insulin and glucagon stimulation were not seen under conditions producing these effects for System $\text{A}$.     

Regulatory characteristics of amino acid transport in newborn rat renal cortex cells

The uptake of α-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 °C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na⁺ during the preliminary incubation. Cycloheximide (50 μM) and purimycin (1 mM) as well as α-aminoisobutyric acid, glycine and proline (5 mM) in the pre- incubation buffer also abolish the effect, while actinomycin D (0.8 μM) partially inhibits the enhancement due to preliminary incubation. A kinetic examination of the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in efflux. There is no alteration in the apparent transport $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ but an increase in the $\text{V}$ for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.     

Calcium-independent and dependent steps in action of Clostridium perfringens enterotoxin on HeLa and Vero cells.

Purified enterotoxin (20–200 ng/ml) of $\text{Clostridium}$$\text{perfringens}$ rapidly induced bled and balloon formation on HeLa and Vero cells in the presence, but not the absence, of Ca²⁺. The action of the toxin involved two, sequential, temperature-dependent steps: The first was Ca²⁺-independent and included binding of toxin and the bound toxin after 30–60 sec could no longer be removed by washing. The second step was Ca²⁺-dependent and eventually led to bled and balloon formation. On adding Ca²⁺ to cells pretreated with toxin in Ca²⁺-free medium, bled and balloon formation started immediately. The ionophore A23187 mimicked the action of toxin. The effects of sucrose (0.2 M), trypsin-treatment of the cells and various pretreatments of the toxin on the action of enterotoxin were studied.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

Specificity of gentamicin accumulation by rat renal cortex.

The accumulation of gentamicin by rat renal cortex $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ was not inhibited by probenecid, tetraethylammonium, cephalosporins nor α-aminoisobutyric acid, but was significantly blocked by other aminoglycosides (neomycin, tobramycin and kanamycin). The data suggest that specific binding sites for aminoglycosides are present on the surface or in cells of the renal proximal tubule.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Glucose and fatty acid metabolism in McA-RH7777 hepatoma cells vs. rat primary hepatocytes: responsiveness to nutrient availability

The overabundance of dietary fats and simple carbohydrates contributes significantly to obesity and metabolic disorders associated with obesity. The liver balances glucose and lipid distribution, and disruption of this balance plays a key role in these metabolic syndromes. We investigated (1) how hepatocytes balance glucose and fatty acid metabolism when one or both nutrients are supplied in abundance and (2) whether rat hepatoma cells (McA-RH7777) reflect nutrient partitioning in a similar manner as compared with primary hepatocytes. Increasing media palmitate concentration increased fatty acid uptake, triglyceride synthesis and beta-oxidation. However, hepatoma cells had a 2-fold higher fatty acid uptake and a 2-fold lower fatty acid oxidation as compared with primary hepatocytes. McA-RH7777 cells did not synthesize significant amounts of glycogen and preferentially metabolized the glucose into lipids or into oxidation. In primary hepatocytes, the glucose was mostly spared from oxidation and instead partitioned into both de novo glycogen and lipid synthesis. Overall, lipid production was rapidly induced in response to either glucose or fatty acid excess and this may be one of the earliest indicators of metabolic syndrome development associated with nutrient excess.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells

Microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins. Previously, we demonstrated that the N-terminal 1,000 residues of apoB (apoB:1000) are necessary for the initiation of apoB-containing lipoprotein assembly in rat hepatoma McA-RH7777 cells and that these particles are phospholipid (PL) rich. To determine if the PL transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB:1000-containing lipoproteins, we employed microRNA-based short hairpin RNAs (miR-shRNAs) to silence Mttp gene expression in parental and apoB:1000-expressing McA-RH7777 cells. This approach led to 98% reduction in MTP protein levels in both cell types. Metabolic labeling studies demonstrated a drastic 90–95% decrease in the secretion of rat endogenous apoB100-containing lipoproteins in MTP-deficient McA-RH7777 cells compared with cells transfected with negative control miR-shRNA. A similar reduction was observed in the secretion of rat endogenous apoB48 under the experimental conditions employed. In contrast, MTP absence had no significant effect on the synthesis, lipidation, and secretion of human apoB:1000-containing particles. These results provide strong evidence in support of the concept that in McA-RH7777 cells, acquisition of PL by apoB:1000 and initiation of apoB-containing lipoprotein assembly, a process distinct from the conventional first-step assembly of HDL-sized apoB-containing particles, do not require MTP. This study indicates that, in hepatocytes, a factor(s) other than MTP mediates the formation of the PL-rich primordial apoB:1000-containing initiation complex.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Insulin stimulation of amino acid transport in isolated rat hepatocytes is independent of hormone internalization.

Isolated rat hepatocytes were used to investigate the relationship between the effect of insulin on amino acid transport and hormone internalization. As previously observed with fibroblastic cells, 10 mM methylamine inhibited the clustering and internalization of the hormone-receptor complex in hepatocytes. Direct measurement of ¹²⁵I-insulin binding indicated that methylamine did not decrease the binding capacity of the cells. When used at concentrations that did not affect the basal rate of α-aminoisobutyric acid transport, methylamine did not cause a specific decrease in the stimulation by insulin. The data indicate that the internalization of insulin is not required for the expression of its biological effect on amino acid transport.     

Amino acid transport in placental slices mechanism of increased accumulation by prolonged incubation

The accumulation of α-aminoisobutyric acid by placental slices is increased dramatically upon prior incubation of the slices in amino acid-free, buffered saline. This increase is inhibited by inhibitors of protein synthesis and is accompanied by an increased $\text{V}$ for the transport process. While alternative explanations are discussed, these data suggest that the incubation effect may be mediated through an increase in the number of available transport sites which are synthesized during the incubation period. Incubation with an amino acid mixture diminishes the increase as well as general protein synthesis, suggesting that a reduced availability of amino acids may initiate compensatory changes in the synthesis of cellular transport proteins.     

Amino acid influx across the mucosal border of the rat intestine in vivo

The lack of an in vivo method for measuring influx of amino acid into the mucosa has prevented a systematic comparison of characteristics of amino acid influx in vivo with prior in vitro studies. We developed and validated a technique for measuring amino acid influx in vivo. The mucosa is exposed briefly to labelled amino acid perfused luminally at a rapid rate and tissue uptake is measured. The brief exposure period insures that amino acid is confined to the segment. The rapid perfusion rate minimizes concentration of endogenous Na⁺ in the lumen and permits Na-dependency for α-aminoisobutyric acid influx to be demonstrated in vivo for the first time. We also demonstrated the inhibitory effect of K⁺ and competition by glycine on α-aminoisobutyric acid influx in vivo. The saturation kinetics for l-leucine in vivo and in vitro were compared under varying perfusion rates and with and without stirring with air. Under optimal conditions of agitation (rapid perfusion and bubbling with air), the apparent Michaelis constant ($\text{K}{}_{\mathrm{<mtext>t</mtext>}}$) is decreased to be almost equal to that determined under comparable influx conditions in vitro. These studies demonstrate no major difference between characteristics of amino acid transport under more physiologic in vivo conditions as compared with prior in vitro studies.     

Prostaglandin E2: A factor in the pathogenesis of cholera

Injection of cholera toxin $\text{in vivo}$ into loops of intestine in rats caused the production of an exudate. This was found to contain prostaglandin E₂ by assay on the rat stomach strip and by thin-layer chromatography. The amounts found ranged from 20 to 40 ng per loop of intestine. Introduction of 30 ng of prostaglandin E₂ into intestinal loops caused the production of an exudate similar in volume to that found after the introduction of cholera toxin. These results indicate that the exudate in cholera is caused by the action of prostaglandin liberated by the enterotoxin. It is suggested that an inhibitor of prostaglandin release could be added to the solutions used in treatment for the restoration of fluids and electrolytes, with the object of blocking the action of toxin still present in the intestinal lumen, thereby achieving a more rapid therapeutic result.     

S-Adenosylmethionine synthetase isozyme patterns from rat hepatoma induced by N-2-fluorenylacetamide

Isozyme patterns of $\text{S}$-adenosylmethionine synthetase have been measured with and without dimethylsulfoxide in hepatoma of rats induced by $\text{N}$-2-fluorenylacetamide. The isozymes of α- and β-types existing in normal rat liver gradually decreased with the progress of hepatocarcinoma, and the kidney type γ-enzyme appears along with disappearance of both α- and β-enzymes. The liver from rat fetus contains a greater part of γ-type enzyme.